Copyrightó2007by the Genetics Society of America
DOI:10.1534/genetics.107.075465
The in Silico Map-Based Cloning of Pi36,a Rice Coiled-Coil–Nucleotide-Binding Site–Leucine-Rich Repeat Gene That Confers Race-Speci?c
Resistance to the Blast Fungus
Xinqiong Liu,*,?Fei Lin,*Ling Wang*and Qinghua Pan*,1
*Laboratory of Plant Resistance and Genetics,College of Resources and Environmental Sciences,South China Agricultural University, Guangzhou,510642,China and?Key Biotechnology Laboratory of State Ethnic Affairs Commission,College of Life Science,
South-Central University for Nationalities,Wuhan,430074,China
Manuscript received May4,2007
Accepted for publication May15,2007
ABSTRACT
The indica rice variety Kasalath carries Pi36,a gene that determines resistance to Chinese isolates of rice
blast and that has been located to a17-kb interval on chromosome8.The genomic sequence of the reference
japonica variety Nipponbare was used for an in silico prediction of the resistance(R)gene content of the
interval and hence for the identi?cation of candidate gene(s)for Pi36.Three such sequences,which all had
both a nucleotide-binding site and a leucine-rich repeat motif,were present.The three candidate genes were
ampli?ed from the genomic DNA of a number of varieties by long-range PCR,and the resulting amplicons
were inserted into pCAMBIA1300and/or pYLTAC27vectors to determine sequence polymorphisms cor-
related to the resistance phenotype and to perform transgenic complementation tests.Constructs con-
taining each candidate gene were transformed into the blast-susceptible variety Q1063,which allowed the
identi?cation of Pi36-3as the functional gene,with the other two candidates being probable pseudogenes.
The Pi36-encoded protein is composed of1056amino acids,with a single substitution event(Asp to Ser)at
residue590associated with the resistant phenotype.Pi36is a single-copy gene in rice and is more closely
related to the barley powdery mildew resistance genes Mla1and Mla6than to the rice blast R genes Pita,Pib,
Pi9,and Piz-t.An RT–PCR analysis showed that Pi36is constitutively expressed in Kasalath.
T HE?lamentous ascomycete Magnaporthe grisea (Hebert)Barr,which is the causal agent of rice blast,remains the most important pathogen in most rice-producing(Oryza sativa L.)regions(O u1985).The use of resistance(R)genes in crop breeding programs has been, and will undoubtedly remain,the major means for its con-trol.However,because of the instability of the pathogen and a high level of variability in pathogenicity between isolates(O u1979;V alent and C humley1994),host re-sistance is typically only short-lived in disease-prone envi-ronments(K iyosawa1981;M ackill and B onman1992). The establishment of durable resistance requires the iso-lation of multiple R genes,as this simpli?es the process of R gene stacking into elite cultivars,via either marker-aided breeding or transgenesis.
The rice–rice blast interaction has long served as a model system to study plant–pathogen interactions(V alent 1990).Race-speci?c resistance closely follows the classi-cal gene-for-gene relationship(S ilue′et al.1992;J ia et al.2000).The isolation and subsequent characterization of R genes will help to unravel the molecular mechanisms underlying the interaction between host and pathogen. Although more than50rice R genes have been docu-mented to date(C hen et al.2005;L iu et al.2005),only6 (Pib,Pita,Pi9,Pid2,Pi2,and Pizt)have as yet been isolated (W ang et al.1999;B ryan et al.2000;Q u et al.2006;C hen et al.2006;Z hou et al.2006).The sequences of5of these (Pib,Pita,Pi9,Pi2,and Pizt)include both nucleotide-binding site(NBS)and leucine-rich repeat(LRR)domains, while Pid2encodes a receptor-like kinase.
Plants use R genes to detect the presence of pathogen, and then to induce a spectrum of defense responses. The interaction between R gene products and pathogen elicitors has been established by a variety of direct and indirect experimental evidence(J ia et al.2000;G u et al. 2005;D odds et al.2006).The commonest class of R gene encodes proteins containing an NBS–LRR domain(B ent 1996;H ammond-K osack and J ones1997;H ulbert et al. 2001).These have been classi?ed into two types on the basis of the presence/absence of an N-terminal TIR do-main.Genes in the TIR group are only known among the dicotyledonous species(M eyers et al.1999;P an et al.2000;
B ai et al.2002).The non-TIR group typically includes a coiled-coil(CC)domain at the N terminus.The NBS region is thought to be involved in signal transduction
Sequence data from this article have been deposited with the EMBL/ GenBank Data Libraries under accession no.DQ900896.
1Corresponding author:Laboratory of Plant Resistance and Genetics, College of Resources and Environmental Sciences,South China Agricul-tural University,Guangzhou,510642,China.
E-mail:panqh@https://www.doczj.com/doc/7818381481.html,
Genetics176:2541–2549(August2007)
cascades involving phosphorylation/dephosphorylation events with either ATP or GTP(T raut1994;D angl and J ones2001),whereas the CC domain may facilitate ho-modimerization of the proteins themselves or heterodi-merization with other proteins,generating interactions that lead to the repression of signaling(M offett et al. 2002;H wang and W illiamson2003).Several studies have identi?ed the LRR domain as the major determinant of recognition speci?city for the pathogen avirulence factor(s)(M eyers et al.1998).LRR-containing sequences are prone to adaptive evolution(P arniske et al.1997; M cdowell et al.1998;E llis et al.2000;S un et al.2001), and in particular,their insertions and deletions have been shown to be responsible for both R gene loss of function and recognition speci?city(A nderson et al. 1997;W ulff et al.2001).For example,particular loss-of-function alleles of the Arabidopsis thaliana genes RPS2 and RPM1differ from the effective wild type by only one amino acid residue in the LRR domain(B ent et al.1994;
G rant et al.1995).
The indica rice variety Kasalath(formerly coded as Q61)confers a stable and high level of partial resistance against Chinese isolates of rice blast.The resistance gene Pi36has recently been mapped to a location on chro-mosome8(L iu et al.2005).In this paper,we describe the positional cloning of Pi36gene based on a prior bio-informatics analysis,long-range PCR(LR–PCR),and an ef?cient transformation-competent arti?cial chromo-some(TAC)vector-based transformation technique.We believe that this approach should be widely applicable within rice and also other plant species.The cloned Pi36 gene represents an important resource for the develop-ment of durable resistance to rice blast,and along with other R genes its sequence should inform the molecular basis of disease resistance in plants.
MATERIALS AND METHODS
Candidate gene prediction:Candidates for Pi36were iden-ti?ed in silico from the output of the gene prediction programs FGENESH,RiceGAAS,and Gramene,using as a reference the Nipponbare sequence for the17-kb genomic region de?ned by the?anking markers CRG2and RM5647(Figure1A).To verify which,if any,of these are true candidates for Pi36,the sequence from the same genetic interval was derived from the blast-resistant variety Kasalath and two blast-susceptible varie-ties,Aichi Asahi and Lijiangxintuanheigu(LTH),using a PCR walking approach.The interval was divided into11overlapping ampli?able fragments,and sequence comparisons were per-formed between the alleles from the resistant and susceptible genotype at each candidate gene following a sequence assembly generated by DNAStar software(https://www.doczj.com/doc/7818381481.html,). Candidate gene cloning:Primer sets were designed to am-plifyeachcandidategene,includingtheirpromoterandterminator regions,using the above-mentioned gene annotation.Restriction sites to enable cloning were identi?ed from the Kasalath genomic sequence.Pi36candidates were ampli?ed from Kasalath genomic DNA by LR–PCR.The50-m l LR–PCR reactions contained2.5units TaKaRa LA T aq(TaKaRa,Dalian,China),13GC reaction buffer I,400m m dNTP,100-ng template,and0.2m m of each primer.The primer sequences,PCR conditions,and restriction enzymes used are listed in Table1.Puri?ed LR–PCR products of candidates Pi36-1and Pi36-2from three independent reactions were ligated into the Bam HI and Pst I sites of the vector pCAMBIA1300to form R36L1CAM and R36L2CAM,respectively.For the longest candidate,Pi36-3,the LR–PCR products were cloned into the Asc I site of TAC vector pYLTAC27to form R36L3TAC.To improve transformation ef?ciency,the Pi36-3insert was later recloned into the vector pCAMBIA1300AscI,in which an Asc I site was engineered into the multiple cloning sites.This construct was named R36L3CAM.The constructs were validated by restriction analysis and sequenced from both ends using the vector primers CAM1300F and CAM1300R.Details of the vectors,constructs and primers used are listed elsewhere(Table1;supplemental Table S1 at https://www.doczj.com/doc/7818381481.html,/supplemental/). Complementation analysis:Constructs containing each can-didate gene were transformed into Agrobacterium tumefaciens strain EHA105by electroporation(GenePulser Xcell,Bio-Rad, Hercules,CA).Clone stability was tested as per Q u et al. (2003),and stable constructs were transformed into the highly blast-susceptible rice variety Q1063,as described by H iei et al. (1994).Selfed T1and T2progeny were tested for reaction to blast infection with pathogen isolates CHL39and CHL273, using the spray method described elsewhere(P an et al.2003; L iu et al.2005).For these phenotyping tests,Kasalath and Q1063represented,respectively,the positive and negative controls.A number of resistant transgenic individuals were randomly selected and subjected both to PCR veri?cation for the presence of the transgene,using the gene-speci?c primers CRG4F and CRG4R(supplemental Table S1at http://www. https://www.doczj.com/doc/7818381481.html,/supplemental/),and to Southern hybridization analysis to estimate the transgene copy number.For the latter procedure, 3m g genomic DNA was digested to completion with Hin dIII;the products were separated by0.8%agarose gel electrophoresis and were then transferred to a nylon mem-brane(Hybond-N1,Amersham,Buckinghamshire,UK).A part of the HptII sequence,ampli?ed from the vector pCAM-BIA1300by primers HptF and HptR(see supplemental Table S1),was labeled with a-?32P by random primer labeling (TaKaRa)for use as a hybridization probe.Southern hybrid-ization was also used to infer the copy number of Pi36-like genes in rice.Genomic DNA of Kasalath and the blast-susceptible variety AS20-1was digested to completion with Eco RI,Kpn I,or Bam HI and probed with sequences ampli?ed from the59-untranslated region(UTR),39UTR,and a part of the largest intron(L-intron)of the Pi36gene(see supple-mental Table S1).
Gene expression analysis:Two-week-old seedlings of Kasalath and the blast-susceptible variety LTH were inoculated with path-ogen isolate CHL39and maintained in a greenhouse.Leaf sam-ples were collected at0,6,12,24,48,and72hpi.Total RNA was isolated using the TRIzol reagent(Invitrogen,Carlsbad,CA), following the manufacturer’s instructions.The assessment of gene expression levels was obtained in a two-step reverse transcription PCR(RT–PCR)process.The initial RT reaction used the SuperScript II reverse transcriptase kit(Invitrogen), following the manufacturer’s instructions.For the second PCR reaction,a0.5–2m l aliquot of the?rst reaction was used as template.Each experiment was performed in replicate.To enable discrimination between the various RT–PCR amplicons, the RT–PCR primers(see supplemental Table S1at http://www. https://www.doczj.com/doc/7818381481.html,/supplemental/and Figure4C)were designed from exonic sequence?anking the predicted Pi36introns,and genomic DNA was included as a negative control.Primers for rice actin(supplemental Table S1)were used as a positive RT–PCR control.Semiquantitative RT–PCR was performed with23, 26,29,32,and35cycles.
2542X.Liu et al.
Rapid ampli?cation of cDNA ends:The 59and 39end sequences of the cDNA were determined by rapid ampli?ca-tion of cDNA ends (RACE)using the GeneRacer kit (Invi-trogen),following the manufacturer’s instructions.We used total RNA extracted from the leaves of resistant (Kasalath)and susceptible (LTH and AS20-1)plants,harvested 24hpi.The same RACE primers were used for both the resistant and susceptible templates.The full-length cDNA was divided into three ampli?able fragments.The 59RACE was generated by a nested PCR using the primary primer set of GeneRacer 59primer and GSP1,and the second set of the GeneRacer 59nested primer and GSP2;similarly,the 39RACE was generated by the set GSP3and the GeneRacer 39primer,and the inter-mediate RT–PCR fragment was obtained with the set GSP4and GSP1(see supplemental Table S1at https://www.doczj.com/doc/7818381481.html,/supplemental/).The relative locations of all the gene-speci?c primers are shown in Figure 3C (note that the intermediate RT–PCR fragment overlaps both the 59and 39RACE frag-ments).The RACE products were ligated into the pGEM-T vector (Promega,Madison,WI),following the manufacturer’s instructions,and sequenced.
DNA and protein sequence analysis:DNA sequence simi-larity analysis was performed using software BLASTN and BLASTX (A ltschul et al .1997;https://www.doczj.com/doc/7818381481.html,/BLAST).The promoter and polyadenylation regions were ana-lyzed using TSSP and POLYAH,respectively (https://www.doczj.com/doc/7818381481.html,/berry.html).Genomic sequence comparisons were per-formed with pairwise BLAST (https://www.doczj.com/doc/7818381481.html,/BLAST/bl2seq/bl2.html),and protein sequence similarity analysis was performed with BLASTP (A ltschul et al .1997).Multiple sequence alignments were obtained with ProbCons (D o et al .2005),and based on these outputs,a phylogenetic tree was generated using the molecular evolutionary genetic analysis (MEGA)program (N ei and K umar 2000).Boot-strapping was used to provide a con?dence estimate for each branch point.The theoretical isoelectric point and protein molecular weight were computed using DNAStar software.The CC structure was predicted by COILS (https://www.doczj.com/doc/7818381481.html,/software/COILS_form.html).
RESULTS
Pi36candidate genes:The Pi36locus has been mapped
within a 17-kb interval (Figure 1A).To identify candi-dates for the gene,the Nipponbare version of the 17-kb interval was scanned by gene prediction software,re-vealing the three NBS–LRR-type sequences Pi36-1,Pi36-2,and Pi36-3.Both Pi36-1and Pi36-2were identi?ed by RiceGAAS (Figure 1B),while Pi36-3was identi?ed by both Gramene and FGENESH.The Pi36-3sequence includes both Pi36-1and Pi36-2.LR–PCR products,each representing one of the alleles of the three candidate sequences,were successfully generated;these had the anticipated lengths of 5.9,9.5,and 16.1kb.To exclude PCR artifact as a source of sequence variation,three independent LR–PCR products were cloned for each of the candidate genes from each of the templates,and these were independently sequenced.The allelic versions of each of the three candidate genes were then compared.The Kasalath alleles differ from those in the susceptible varieties by a number of base substitutions and small indels (data not shown).The level of nucleotide sequence
T A B L E 1
P r i m e r s e q u e n c e s u s e d f o r t h e a m p l i ?c a t i o n o f c a n d i d a t e g e n e s f o r P i 36b y l o n g -r a n g e P C R
C a n d i d a t e g e n e
P r i m e r
S e q u e n c e (59–39)
a
E x p e c t e d s i z e (k b )R e s t r i c t i o n s i t e b P C R c o n d i t i o n s c
V e c t o r
P i 36-1
R 36L 1F C G G G A T C C T C A A C A G A C T T G A C A T G C A C A T G C T G A T T 5.9
B a m H I
A
p C A M B I A 1300
R 36L 1R C G G G A T C C A A G A A T G T A A C G T G T G C C T C A G A C T C G G T G P i 36-2
R 36L 2F G C A G T C A C T G C A G G T C C T A C G A C A T G G A G G A T A T C A T C G A C G C C T T C 9.5
P s t I B p C A M B I A 1300R 36L 2R G T C A G G T C T G C A G C A T G T G G C C A G A C T C T G T T G G T G G A T T G A A G C P i 36-3
R 36L 3F G C T A G C A T G G C G C G C C C T T C G A C A C G C A A A C G T G C A C A C A G C C A C C T A T C 16.1
A s c I C
p Y L T A C 27/p C A M B I A 1300-A s c I
R 36L 3R
G C T T G C T A G G C G C G C C T G C G A T G C C A C T T C G C T C T T T G C C G A T C T G G T T G
a
N u c l e o t i d e s c o r r e s p o n d i n g t o a r e s t r i c t i o n e n z y m e r e c o g n i t i o n s i t e a r e u n d e r l i n e d .b
E n z y m e u s e d f o r c l o n i n g .c A l l P C R s i n c l u d e d a n i n i t i a l d e n a t u r a t i o n s t e p (94°/2m i n ),f o l l o w e d b y 30a m p l i ?c a t i o n c y c l e s u n d e r t h e c o n d i t i o n s i n d i c a t e d .A t t h e e n d o f t h e c y c l i n g p r o c e d u r e ,a ?n a l i n c u b a t i o n o f 72°/10m i n w a s g i v e n .A ,94°/30s e c ,65°/6.5m i n ;B ,94°/30s e c ,63.8°/10.5m i n ;C ,94°/30s e c ,62.8°,17m i n .
Rice Blast R Gene Pi36
2543
similarity between the alleles was97%(between Kasalath and either Nipponbare or Aichi Asahi)and98%(Kasalath vs.LTH).Gene prediction identi?ed the same set of three candidate genes in each of the three alternative versions of the Nipponbare17-kb segment. Complementation analysis of the candidate genes: The constructs containing each candidate gene were individually transformed into the highly susceptible variety Q1063.A total of259and39independent T0trans-formants,respectively,were generated using R36L1CAM and R36L2CAM.For the analysis of Pi36-3,24(R36L3TAC)and63(R36L3CAM)independent T0individuals were obtained.The pattern of segregation for rice blast resis-tance was observed among the T1progeny of each primary transgenic.All T1individuals derived from a T0parent carrying R36L1CAM or R36L2CAM were highly susceptible to blast isolates CHL39and CHL273(which are both avir-ulent on Kasalath and virulent on Q1063).T en of24tested T1families derived from a T0parent carrying R36L3TAC segregated resistant vs.susceptible in a ratio between1:3.5 and2.8:1,while the segregation ratio shown by33T1families derived from a T0parent carrying R36L3CAM varied from 1:3to4:1(data not shown,supplemental Figure S1at http:// https://www.doczj.com/doc/7818381481.html,/supplemental/).
To con?rm the presence and stable integration of the transgene Pi36-3,molecular assay was?rst conducted by Southern blot analysis.The results showed that all the resistant transgenic plants harbored the transgene of interest,most of which contained between one and three copies of the transgene Pi36-3,although few transgenic plants contained multiple copies(Figure2).To further verify steady inheritance of the transgene Pi36-3,two T2lines,LX182T2-2and LX182T2-6,whose progeny segregated3:1for resistance,were chosen for a cosegre-gation analysis between blast resistance and the presence of the marker CRG4,which lies within Pi36(Figure1A). Since resistance cosegregated perfectly with the presence of CRG4(Figure3),Pi36-3must represent a functional gene.
Structure of Pi36:Six RACE products from both the 59and39end of the genomic sequence of Kasalath were sequenced to identify the size and structure of Pi36.The 59and39RACE products as well as an intermediate RT–PCR fragment overlapped one another,thereby pro-viding complete coverage of the transcribed region (Figure4C).The size and structure of Pi36were deter-mined by comparing the full-length cDNA sequence with the genomic DNA sequence.Pi36contains a3171-bp coding region,interrupted by four introns(433,5069, 124,and259bp)and?anked by a65-bp59UTR and a 725-bp39UTR.A123-bp intron is present within the39 UTR(Figure4C).The size and structure of Pi36are rather different from those predicted by annotation
of
F igure2.—Southern blot analysis of blast resis-
tant transgenic plants.Genomic DNA was isolated
from resistant transformed and susceptible non-
transformed plants.A fragment of hptII was used
as a https://www.doczj.com/doc/7818381481.html,ne1,molecular weight marker
l Hin dIII;lane2,Kasalath(resistant);lane3,
Q1063(susceptible);lanes4–12,transgenic T1
progeny.
F igure1.—In silico map-based cloning of Pi36.(A)Physical
and genetic map surrounding the Pi36locus.The numbers
below the map represent distances in kilobases,as estimated
from the Nipponbare genome sequence.The numbers in pa-
rentheses represent the number of recombinants/gametes in
the mapping population previously reported(L iu et al.2005).
(B)Pi36candidate genes.Pi36-1and Pi36-2were predicted by
RiceGAAS and Pi36-3by both FGENESH and Gramene.The
shaded box represents the coding region,and the hatched
boxes represent predicted59promoter and39poly(A)re-
gions,respectively.The numbers above the map refer to loca-
tion on the reference Nipponbare genomic sequence.The
targets for the LR–PCR primers are indicated.
2544X.Liu et al.
the genomic sequence (Figure 4,B and C),which con-sists of 13introns and 14exons,with the translation stop codon at position 2,884,299.In the cDNA-derived structure,the stop codon is shifted 59by 2784bp.Both start codons,however,lie at position 2,872,609(Figure 4,A–C).
Sequence analysis of the Pi36-encoded protein:A comparison of the deduced amino acid sequence of the Pi36alleles from two susceptible japonica varieties ?LTH (pi36j1)and Nipponbare (pi36j2) and the resistant Kasalath identi?ed nine substitutions and two deletions among the alleles.In addition,six substitutions distin-guish the alleles Pi36and pi36i from the blast-susceptible indica variety AS20-1.A global analysis suggests that just one substitution at residue 590de?nes the functional Pi36gene.The deduced 1056-amino acid sequence of the Pi36-encoded protein has a molecular mass of 120kDa and a calculated isoelectric point of 6.61,and con-tains six conserved motifs typical of NBS proteins (Figure 5).The GMGGLGKTT sequence (beginning at residue 206)conforms to the kinase 1a (P loop)consensus,while IVIDDIWD (beginning at residue 286)and GSKILVTTRK (beginning at residue 310)correspond,respectively,to the kinase 2and kinase 3a consensus motifs (T raut 1994;G rant et al .1995).In addition,GVPLAIITIAS (beginning at residue 372)and LKNCLLYL (beginning at residue 427)correspond,respectively,to the con-served R gene NBS domains 2and 3consensus motifs (T raut 1994;G rant et al .1995).The ?nal conserved NBS motif VHD (beginning at residue 501)corre-sponds to the conserved MHD (methionine–histidine–aspartate)motif.The C-terminal region of the protein includes 17imperfect LRR repeats (residues 578–1056),composed of 15%leucine.The repeats,which are based on an L xx L xx L xx L x L consensus,vary in length between 22and 44amino acids.LRRs 14,15,16,and 17show little or no similarity to the LRR consensus.The primary structure of the LRR-containing domain is illus-trated in Figure 5.Finally,a COIL analysis (L upas et al .1991;https://www.doczj.com/doc/7818381481.html,/software/COIL_form.html)showed that a CC region is probably present (P .0.95)between amino acids 24and 52.The
CC
F igure 3.—Pi36gene complementation test and molecular analysis of the transgenic lines.(A)Resistance phenotypes of the Pi36gene donor cv.Kasalath and its receptor cv.Q1063as well as transgenic plants of two T 2lines segregated into 3R:1S against isolate CHL39.R,resistant;S,susceptible.(B)Cosegregation analysis of the resistance phenotype with the transgenes.The am-pli?ed fragment with the primer pair CRG4F and CRG4R was subsequently digested with Hae III,and the digested product was subjected to 1.5%agarose gel electrophoresis.M represents standard molecular weight marker
DL2000.
F igure 4.—The structure of Pi36,as deduced from its genomic and cDNA sequences.(A)The target gene region.The numbers above the map show genomic positions in the Nippon-bare genomic sequence.(B)Gene structure as deduced from the genomic DNA sequence.(C)Gene structure as deduced from the cDNA se-quence.The shaded box indicates an exon,and the line an intron.The positions of 59and 39UTR (hatched boxes),the translation start co-don (ATG),and the translation stop codon (TGA or TAG)are also shown.The annealing targets of the RACE and RT-PCR primers are indicated.(D)Structure of the Pi36-encoded protein,in which three tandem conserved domains are shown.
Rice Blast R Gene Pi362545
region contains three perfect h xx h xx h and one h xx h xxx motif (where h represents one of L,I,M,V,or F,and x is any residue).Overall the evidence suggests strongly that Pi36belongs to the CC–NBS–LRR family of R genes.Phylogenetic analysis of Pi36:Southern hybridiza-tion analysis was employed to estimate the copy number of Pi36-related genes in rice.Only a single hybridizing fragment was present in both resistant and susceptible genotypes,indicating that Pi36is a single-copy gene (supplemental Figure S2at https://www.doczj.com/doc/7818381481.html,/supplemental/).Interestingly,only a single copy is pre-sent in both reference sequences of varieties Nipponbare and 93-11by BLAST analysis,suggesting that Pi36is a single-copy gene in the rice genome.The evolutionary relationship between Pi36and twelve R genes related was assessed by a phylogenetic amino acid-based sequence analysis using ProbCons and MEGA (Figure 6).The de-gree of homology shared by these genes varies consider-ably,and two heterogeneous groups can be recognized,re?ecting an early divergence in the evolution of the R gene family.Cereal R genes are largely clustered into one of these groups,whereas those derived from the di-cotyledonous species fall into the other group.The cereal R genes were further classi?ed into eight distinct sub-groups:Mla1(I),Pi36(II),Mla6(III),Pita (IV),Pib (V),
Piz-t/Pi9(VI),Xa1(VII),and rp3(VIII)(Figure 6).Pi36appears to be more closely related to the barley powdery mildew R genes Mla1and Mla6than to the rice blast R genes Pita ,Pib ,Pi9,and Piz-t.Pi36,Mla1,and Mla6are well conserved in their NBS region,but diverge signi?cantly in the LRR region (data not shown).
Expression pattern of Pi36:Only a faint band of the expected size was detected when 23cycles were used,most likely due to the low expression level of the Pi36gene.However,a stronger band was observed when 29,32,and 35cycles were applied (Figure 7).The results revealed that no detectable differences in expression could be observed either in a time course postinocula-tion with blast pathogen,or between resistant and sus-ceptible hosts.Thus,the expression of Pi36is likely to be constitutive,and is not induced by blast infection.
DISCUSSION
An ef?cient cloning strategy for Pi36:Positional cloning is the accepted means to isolate genes where only phenotype and genomic location are known.The latter requires the prior generation of a high-resolution genetic map in the region surrounding the target.Generally,the map needs to be complemented
with
F igure 5.—Deduced amino acid sequence of the Pi36encoded protein.The seven conserved motifs forming the NBS region are underlined.Residue 590,the single amino acid substitution distinguishing the blast-resistant from the blast-susceptible form of the protein,is double underlined.The C-terminal LRR region is shown separately from the rest of the sequence.
2546X.Liu et al .
large insert(YAC or BAC)libraries.Where these require-ments are met,the target can be narrowed down to a single insert(or a contig)based on the presence of closely linked hybridizing markers(S ong et al.1995; Y oshimura et al.1998;W ang et al.1999;B ryan et al.2000; S un et al.2004;Q u et al.2006;C hen et al.2006).However, in rice,because its almost complete genome sequence is publicly available,much of this process can be performed in silico.With respect to Pi36,the necessary high-resolution genetic map had already been assembled (L iu et al.2005).Thus the identi?cation of candidates for Pi36could be reduced to a bioinformatics-based search of the relevant physical genomic segment.Valida-tion of the candidates was then achieved using a trans-genic complementation test.The process was accelerated by exploiting the capability of LR-PCR to amplify sequences too large for conventional PCR(F euillet et al.2003;H orvath et al.2003;S ong et al.2003).In the event,the three candidate genes were all recovered by LR–PCR,avoiding the need to subclone from a large insert library.
Insert size in the pCAMBIA1300vector is limited, making it dif?cult to clone sequences as large as10kb. However,the pLYTAC27TAC vector tolerates a much larger insert size(L iu et al.2002)and was successfully used to clone Pi36-3(.16kb).We were then able to transfer the target fragment into a modi?ed form of pCAMBIA1300for the complementation study.This general approach has proven to be an effective means of cloning large genes.
Pi36belongs to the CC–NBS–LRR family of R genes: Some580NBS-encoding genes have been identi?ed in the rice genome.Of these, 490belong to the CC–NBS–LRR family and101are thought to be pseudo-genes(B ai et al.2002;M onosi et al.2004).Of the three candidates for Pi36,one is a functional copy,while the other two are probably pseudogenes.Interestingly,the structure of Pi36deduced from the genomic sequence was rather different from that deduced from the cDNA sequence.There are13introns in the former,but only5 in the latter(Figure4).This resulted in removing stop codons from genomic positions2,884,229to2,881,515. That is,the stop codon in the latter one was shifted from the right?anking marker RM5647for2784bp(Figure 1).This is an additional evidence to suggest that the latter is the actual structure of Pi36,because of the occur-rence of recombination in this region within a stretch of6.4kb(L iu et al.2005).Our results can be considered in light of the hypothesis that automatic annotations commonly inserted introns to remove stop codons or frameshift mutations(M onosi et al.2004).Introns inter-rupting the NBS domain of R genes are more common in cereals than in dicotyledonous species(B ai et al. 2002).In rice,some six putative R genes(B ai et al.2002) and three characterized rice blast-resistance genes(Pi-ta, B ryan et al.2000;Pib,W ang et al.1999;Pi9,Q u et al. 2006)carry intron(s)in their NBS domain.One intron is present in the NBS region of Pi36(of length5069bp, and beginning at amino acid residue284;Figure4C), similar in size to that present in Pi9.Pi36therefore has a unique structure with respect to intron position and size when compared with other rice genes.Conserved splicing sites(gt and ag)were present at the intron/ exon junctions of introns1,2,3,and5,but at intron4, the splicing site was ag and ct(Figure4C).Introns
have
F igure7.—Expression patterns of
Pi36assayed by semiquantitative RT–
PCR.Two-week-old resistant Kasalath
and susceptible LTH seedlings were
inoculated with blast(isolate CHL39).
The expression of Pi36was assayed at
various time points postinoculation.Ge-
nomic DNA(gDNA)serves as a control
to distinguish PCR products from cDNA
and gDNA.The rice Actin1gene acted
as a positive
control.
F igure6.—Phylogenetic analysis of Pi36with other10R
genes.Multiple amino acid alignments were conducted using
ProbCons and a neighbor-joining phylogenetic tree was gen-
erated using MEGA.Numbers on branches indicate the per-
centage of1000bootstrap replicates which support the
adjacent node.The unit branch length is0.2nucleotide sub-
stitutions per site,as indicated by the bar.
Rice Blast R Gene Pi362547
been commonly detected in the59UTR region of R genes(V os et al.1998;W ang et al.1999;V an D er V ossen et al.2005)but seldom in the39UTR.Two39UTR introns are present in Pi9(Q u et al.2006),and one in Pi36.Whether these features of Pi36have any biological signi?cance has yet to be determined.
The LRR regions of rice NBS–LRR genes vary con-siderably in size and sequence,re?ecting substantial divergence in the R genes(B ai et al.2002).The LRRs occupy almost the entire C-terminal region of the CC–NBS–LRR proteins(M eyers et al.2003),but the repeats are mostly imperfect,with only few conforming to any consensus sequence(B ai et al.2002).In Pib,Pita,and Pi9, the regions are leucine-rich but have no clearly distin-guishable repetitive structure.A similar pattern pertains to the Pi36sequence,which encodes a136-residue non-LRR region at its C-terminus.Further research is needed to establish whether the sequences at the Pi36C-terminus play any role in the determination of speci?city with respect to particular pathogen isolates.
Evolutionary relationships between Pi36and other NBS–LRR R genes:R genes are involved in the disease resistance response in a wide variety of plant species. They share a common structure and therefore probably act via a common mechanism.In evolutionary terms, it is widely assumed that the R genes have a common origin(C aicedo et al.1999).The functional and evolu-tionary analysis of R genes is the focus of much current research.Pi36is a single-copy R gene,and hence could represent a useful model for such functional and evolu-tionary studies.At the protein level,the Pi36product most closely resembles the barley Mla1and Mla6pro-teins,and is less closely related to the rice bast Pib and Pita proteins.Multiple alignment of the amino acid se-quences of Pi36with10other R genes has demonstrated that nonconservative residue substitution was most fre-quent in the LRR domain and least in the NBS domain, supporting the widely held view that the LRR regions are subject to diversifying selection,and that they are responsible for speci?city(M c D owell et al.1998;M eyers et al.1998;S un et al.2001).
A single amino acid mutation is responsible for the resistance phenotype:Pathogen–plant coevolution op-erates by simultaneous selection for avirulence genes in the pathogen and resistance genes in the host(S tahl and
B ishop2000).The direct interaction between an NBS–LRR protein and a pathogen avirulence gene prod-uct was?rst shown for the rice Pita/Avr-Pita system(J ia et al.2000).The deduced Pita protein of a susceptible host differs from that of a resistant one by a single substitution of serine for alanine(B ryan et al.2000). Similarly,we have established that a single amino acid difference distinguishes the resistant and susceptible alleles of the Pi36product.In this case,the replacement of asparagine by serine determines blast resistance.A possible mechanistic explanation of the large biological effect of this small sequence difference could relate to the?nding that when Nectria haematococca mycelia invade host tissue,high levels of free asparagine and homoserine become readily accessible to the fungus,and this induces the expression of pel D,a known virulence factor(R ogers et al.2000).Thus serine and asparagines residues may be important for determining the resistance or resistance-related response.A more likely reason is that sequence variation at the active site affects molecular interactions and therefore changes function(H anzawa et al.2005). We are presently attempting the isolation of Avr-Pi36to enable the dissection of the interactions between the host and the pathogen.
We are grateful to Y Liu for the kind provision of the pYLTAC27 vector and to Robert Koebner for critical reading of the manuscript. This research has been supported by grants from the National973 project(2006CB1002006),the National863projects(2002AA2Z1002; 2006AA10A103;2006AA100101),the Innovation Research Team Project from the Ministry of Education of China(IRT0448),the Guangdong Provincial Natural Science Foundation(039254),and the Special Project for the Distinguished University Professor from the Department of Education of Guangdong Province.
LITERATURE CITED
A ltschul,S.F.,T.L.M adden,A.A.S chaffer,J.Z hang,Z.Z hang
et al.,1997Gapped BLAST and PSLBLAST:a new generation of protein database search programs.Nucleic Acids Res.25:3389–3402.
A nderson,P.A.,G.J.L awrence,B.C.M orrish,M.A.A yliffe,E.J.
F innegan et al.,1997Inactivation of the?ax rust resistance
gene M associated with loss of a repeated unit within the leucine-rich repeat coding region.Plant Cell9:641–651.
B ai,J.,L.A.P ennill,J.N ing,S.W.L ee,J.R amalingam et al.,
2002Diversity in nucleotide binding site-leucine-rich repeat genes in cereals.Genome Res.12:1871–1884.
B ent,A.F.,1996Plant disease resistance genes,function meets
structure.Plant Cell8:1757–1771.
B ent,A.F.,B.N.K unkel,D.D ahlbeck,K.L.B rown,R.S chmidt
et al.,1994RPS2of Arabidopsis thaliana:a leucine-rich repeat class of plant disease resistance genes.Science265:1856–1860.
B ryan,G.T.,K.S.W u,L.F arrall,Y.L.J ia,H.P.H ershey et al.,
2000A single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pi-ta.Plant Cell12:2033–2045.
C aicedo,A.L.,B.A.S chaal and B.N.K unkel,1999Diversity and
molecular evolution of the RPS2resistance gene in Arabidopsis https://www.doczj.com/doc/7818381481.html,A96:302–306.
C hen,S.,L.W ang,Z.Q.Q ue,R.Q.P an and Q.H.P an,2005Genetic
and physical mapping of Pi37(t),a new gene conferring resistance to rice blast in the famous cultivar St.No.1.Theor.Appl.Genet.
111:1563–1570.
C hen,X.W.,J.J.S hang,D.X.C hen,C.L.L ei,Y.Z ou et al.,2006A
B-lectin receptor kinase gene conferring rice blast resistance.
Plant J.46:794–804.
D angl,J.L.,and J.D.G.J ones,2001Plant pathogens and inte-
grated defence responses to infection.Nature411:826–833.
D o,C.B.,M.S.M ahabhashyam,M.B rudno and S.B atzoglou,
2005ProbCons:probabilistic consistency-based multiple se-quence alignment.Genome Res.15:330–340.
D odds,P.N.,G.J.L awrence,A.M.C atanzariti,T.T eh,C.I.W ang
et al.,2006Direct protein interaction underlies gene-for-gene speci?city and coevolution of the?ax resistance genes and?ax rust avirulence https://www.doczj.com/doc/7818381481.html,A103:8888–8893.
E llis,J.G.,P.D odds and T.P ryor,2000The generation of plant
disease resistance gene speci?cities.Trends Plant Sci.5:373–379.
F euillet,C.,S.T ravella,N.S tein,L.A lbar,A.N ublat et al.,
2003Map-based isolation of the leaf rust disease resistance gene Lr10from the hexaploid wheat(Triticum aestivum L.)https://www.doczj.com/doc/7818381481.html,A100:15253–15258.
2548X.Liu et al.
G rant,M.R.,L.G odiard,E.S traube,T.A sh?eld,J.L ewald et al.,
1995Structure of the Arabidopsis RPM1gene enabling dual speci?city disease resistance.Science269:843–846.
G u,K.,B.Y ang,D.T ian,L.W u,D.W ang et al.,2005R gene expres-
sion induced by a type-III effector triggers disease resistance in rice.Nature435:1122–1125.
H ammond-K osack,K.E.,and J.D.G.J ones,1997Plant disease resis-
tance genes.Annu.Rev.Plant Physiol.Plant Mol.Biol.48:575–607.
H anzawa,Y.,T.M oney and D.B radley,2005A single amino acid
converts a repressor to an activator of?owering.Proc.Natl.Acad.
https://www.doczj.com/doc/7818381481.html,A102:7748–7753.
H iei,Y.,S.O hta,T.K omari and T.K umashiro,1994Ef?cient
transformation of rice(Oryza sativa L.)mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.Plant J.6: 271–282.
H orvath,H.,N.R ostoks,R.B rueggeman,B.S teffensonm,D.V on
W ettstein et al.,2003Genetically engineered stem rust resis-tance in barley using the https://www.doczj.com/doc/7818381481.html,A 100:364–369.
H ulbert,S.H.,C.A.W ebb,S.M.S mith and Q.S un,2001Resistance
gene complexes,Evolution and utilization.Annu.Rev.Phytopa-thol.39:285–312.
H wang,C.,and V.M.W illiamson,2003Leucine-rich repeat-medi-
ated intramolecular interactions in nematode recognition and cell death signaling by the tomato resistance protein Mi.Plant J.34:585–593.
J ia,Y.L.,S.A.M cadams,G.T.B ryan,H.P.H ershey and B.V alent, 2000Direct interaction of resistance gene and avirulence gene products confers rice blast resistance.EMBO J.19:4004–4014. K iyosawa,S.,1981Gene analysis for blast resistance.Oryza18:196–203.
L iu,X.Q.,L.W ang,S.C hen,F.L in and Q.H.P an,2005Genetic and physical mapping of Pi36(t),a novel rice blast resistance gene lo-cated on rice chromosome8.Mol.Genet.Genomics274:394–401. L iu,Y.G.,H.L iu,L.C hen,W.Q iu,Q.Z hang et al.,2002Development of new transformation-competent arti?cial chromosome vectors and rice genomic libraries for ef?cient gene cloning.Gene282: 247–255.
L upas,A.,M.V andyke and J.S tock,1991Predicting coiled coils from protein sequences.Science252:1162–1164.
M ackill,D.J.,and J.M.B onman,1992Inheritance of blast resis-tance in near-isogenic lines of rice.Phytopathology82:746–749. M cdowell,J.M.,M.D handaydham,T.A.L ong,M.G.A arts,S.
G off et al.,1998Intragenic recombination and diversifying se-
lection contribute to the evolution of downy mildew resistance at the RPP8locus of Arabidopsis.Plant Cell10:1861–1874.
M eyers,B.C.,K.A.S hen,P.R ohani,B.S.G aut and R.W.M ichelmore, 1998Receptor-like genes in the major resistance locus of lettuce are subject to divergent selection.Plant Cell11:1833–1846.
M eyers,B.C.,A.W.D ickerman,R.W.M ichelmore,R.M.P echerer, S.S ivaramakrishnan et al.,1999Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding superfamily.Plant J.20:317–332.
M eyers,B.C.,A.K ozik,A.G riego,H.K uang and R.W.M ichelmore, 2003Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis.Plant Cell15:809–834.
M offett,M.,G.F arnham,J.P eart,and D. C.B aulcombe, 2002Interaction between domains of a plant NBS-LRR protein in disease resistance related cell death.EMBO J.21:4511–4519. M onosi,B.,R.J.W isser,L.P ennill and S.H.H ulbert,2004Full-genome analysis of resistance gene homologues in rice.Theor.
Appl.Genet.109:1434–1447.
N ei,M.,and S.K umar,2000Molecular Evolution and Phylogenetics.
Oxford University Press,Oxford.
O u,S.H.,1979Breeding rice for resistance to blast,a critical view, pp.79–137in Proceedings of the Rice Blast Workshop.International Rice Research Institute,Mantla,Philippines.
O u,S.H.,1985Blast,pp.109–201in Rice Diseases,https://www.doczj.com/doc/7818381481.html,mon-wealth Mycological Institute,Kew Surrey,UK.
P an,Q.,Y.S.L iu,O.B udai-H adrian,M.S ela,L.C armel-G oren et al.,2000Comparative genetics of nucleotide binding site-leucine rich repeat resistance gene homologues in the genomes of two dicotyledons,tomato and Arabidopsis.Genetics155: 309–322.P an,Q.H.,Z.H u,T.T anisaka and L.W ang,2003Fine mapping of the blast resistance gene Pi15,linked to Pii,on rice chromosome
9.Acta Bot.Sin.45:871–877.
P arniske,M.,K.E.H ammond-K osack,C.G olstein,C.M.T homas,
D.A.J ones et al.,1997Novel disease resistance speci?cities re-
sult from sequence exchange between tandemly repeated genes at the Cf-4/9locus of tomato.Cell91:821–832.
Q u,S.,G.C oaker,D.F rancis,B.Z hou and G.L.W ang,2003De-velopment of a new transformation-competent arti?cial chromo-some(TAC)vector and construction of tomato and rice TAC libraries.Mol.Breed.12:297–308.
Q u,S.,G.L iu,B.Z hou,M.B ellizzi,L.Z eng et al.,2006The broad-spectrum blast resistance gene Pi9encodes a nucleotide-binding site-leucine-rich repeat protein and is a member of a multigene family in rice.Genetics172:1901–1914.
R ogers,L.M.,Y.K.K im,W.G uo,L.G onza′lez-C andelas,D.L i et al., 2000Requirement for either a host-or pectin-induced pectate lyase for infection of Pisum sativum by Nectria hematococca.
https://www.doczj.com/doc/7818381481.html,A97:9813–9818.
S ilue′,D.,J.L.N otteghem and D.T harreau,1992Evidence for a gene-for-gene relationship in the Oryza sativa-Magnaporthe grisea pathosystem.Phytopathology82:577–580.
S ong,J.Q.,J.M.B radeen,S.K.N aess,J.A.R aasch,S.M.W ielgus et al.,2003Gene RB cloned from Solanum bulbocastanum confers broad spectrum resistance to potato late blight.Proc.Natl.Acad.
https://www.doczj.com/doc/7818381481.html,A100:9128–9133.
S ong,W.Y.,G.L.W ang,L.L.C hen,H.S.K im,L.Y.P i et al.,1995A receptor kinase-like protein encoded by the rice disease resis-tance gene,Xa21.Science270:1804–1806.
S tahl,E.A.,and J.G.B ishop,2000Plant-pathogen arms races at the molecular level.Curr.Opin.Plant Biol.3:299–304.
S un,Q.,N.C.C ollins,M.A yliffe,S.M.S mith,J.D rake et al., 2001Recombination between paralogues at the rp1rust resis-tance locus in maize.Genetics158:423–438.
S un,X.L.,Y.L.C ao,Z.F.Y ang,C.G.X u,X.H.L i et al.,2004Xa26,a gene conferring resistance to Xanthomonas oryzae pv.oryzae in rice, encodes an LRR receptor kinase-like protein.Plant J.37:517–527.
T raut,T.W.,1994The functions and consensus motifs of nine types of peptide segments that form different types of nucleotide binding sites.Eur.J.Biochem.229:9–19.
V alent,B.,1990Rice blast as a model system for plant pathology.
Phytopathology80:33–36.
V alent,B.,and F.G.C humley,1994Avirulence genes and mech-anism of genetic instability in the rice blast fungus,pp.111–134 in Rice Blast Disease,edited by R.S.Z eigler,S.A.L eong and P.S.
T eng.CABI,Wallingford,UK.
V an D er V ossen, E. A.,J.G ros, A.S ikkema,M.M uskens,
D.W outers et al.,2005The Rpi-blb2gene from Solanum bul-
bocastanum is an Mi-1gene homolog conferring broad-spectrum late blight resistance in potato.Plant J.44:208–222.
V os,P.,G.S imons,T.J esse,J.W ijbrandi,L.H einen et al.,1998The tomato Mi-1gene confers resistance to both root-knot nematodes and potato aphids.Nat.Biotechnol.16:1365–1369.
W ang,Z.X.,M.Y ano,U.Y amanouchi,M.I wamoto,L.M onna et al., 1999The Pib gene for rice blast resistance belongs to the nucle-otide binding and leucine-rich repeat class of plant disease resis-tance genes.Plant J.19:55–64.
W ulff,B.B.H.,C.M.T homas,M.S moker,M.G rant and J.D.G.
J ones,2001Domain swapping and gene shuf?ing identify se-quences required for induction of an Avr-dependent hypersensi-tive response by the tomato Cf-4and Cf-9proteins.Plant Cell13: 255–272.
Y oshimura,S.,U.Y amanouchi,Y.K atayose,S.T oki,Z.X.W ang et al.,1998Expression of Xa1,a bacterial blight-resistance gene in rice,is induced by bacterial inoculation.Proc.Natl.Acad.Sci.
USA95:1663–1668.
Z hou,B.,S.Q u,G.L iu,M.D olan,H.S akai et al.,2006The eight amino-acid differences within three leucine-rice repeats between Pi2and Piz-t resistance proteins determine the resistance speci-?city to Magnaporthe grisea.Mol.Plant-Microbe Interact.11:1216–1228.
Communicating editor:A.H.P aterson
Rice Blast R Gene Pi362549
网络克隆技术 在很多朋友都想学到网络克隆技术。但是网络上有那么几篇网络克隆的文章，并不是一那么准确。 所以这次小申子决定推出傻瓜教程系列第二弹.网络克隆篇。 本方法经过实验，绝对通过。而且只要网络好，系统没问题，绝对不会出现问题。 恩，好，不多说废话了，现在开始我们的教程。 首先.我们做好一张母盘.并将操作系统目录里的TEMP目录里的东西,以及临时文件夹全部清空.之后将母盘系统备份到另外一块硬盘上.首先确认备份的系统有一个比较大的分区.并确认能装下你的硬盘备份. 将准备好的另外一块硬盘挂在你做好的机器上.之后重新启动到DOS模式,启动GHOST.选择LOCAL→DISK→TO IMAGE.之后选择你的母盘,按回车 '
选择你你准备好的一个足够大的分区 给文件起个名字..按回车
它会问你压缩方式.选NO是不压缩.FAST是快速压缩.HIGH是高压缩率.由于我选的都是HIGH,而且从来没出过什么问题,在这里也推荐你使用.
因为FAT32格式分区最大只能识别2G的文件.所以每到达2G时候,会提示你会重新建立文件.
好了,文件建立完了,我们该建立GHOST服务器了.启动WINDOWS打开GHOSTSRV.EXE文件.我们讲在后面提供下载,也可以在GHOST企业版里找到.之后给服务器起一个名字.我起的名字是shenzi 选择克隆客户端 之后选择你备份到另外一个硬盘的备份文件. 点击接受客户的按扭
好了,现在服务器已经进入接收状态了. 之后到客户端,就是你准备要克地机器.在GHOST.EXE文件所在的目录里,创建一个WATTCP.CFG的文件.此文件是指定IP以及网关的.如果没有此文件GHOSTT会自动扫描DHCP服务器.不推荐使用DHCP服务器.所以我们用DOS的命令,EDIT建立一个WATTCP.CFG的文件吧. 在GHOST.EXE所在的目录里输入edit空格wattcp.cfg
一、组织总RNA的提取 相关试剂：T rizol；氯仿；苯酚；异丙醇；75%乙醇；RNase-free水 相关仪器：制冰机；液氮&研钵/生物样品研磨仪；高速离心机；移液器（1ml、200μl、100μl/50μl）；涡旋振荡仪；恒温金属浴。 相关耗材：解剖工具，冰盒，离心管，离心管架，吸头（1ml，200μl/300μl），一次性手套，实验手套。 实验步骤 1.取暂养草鱼，冰上放置一段时间，然后解剖，剪取肠道50~100mg，放入研钵中，加入 液氮迅速研磨，然后加入1ml 预冷TRIzol试剂，充分研磨至无颗粒物存在。 2.转移到离心管中，室温放置5min，使细胞充分裂解； 3.按1ml Trizol加入200μl氯仿，盖上盖子，迅速充分摇匀15s，然后室温放置3min； 4.4℃，,12000g 离心15min； 此时混合物分为三层，下层红色的苯酚氯仿层，中间层和上层无色水相；RNA存在于无色水相中； 5.小心吸取上清液，千万不要吸取中间界面，否则有DNA污染；转移至一个新的离心管， 加入等体积的异丙醇，轻轻混匀； 6.室温放置10min；4℃，,12000g 离心10min； 7.弃上清，加入1ml 75%乙醇洗涤；涡旋，悬浮沉淀；4℃，,12000g 离心5min； 8.弃上清；可以再次用75%乙醇洗涤沉淀； 9.弃上清；用移液器轻轻吸取管壁或管底的残余乙醇，注意不要吸取沉淀；室温放置5min 晾干沉淀；（RNA样品不要过于干燥，否则极难溶解） 10.沉淀中加入30μl RNase-free水，轻弹管壁，使RNA溶解。 RNA质量检测 相关试剂：溴酚蓝，TEB/TAE电泳缓冲液，溴乙锭（EB） 相关仪器：（超微量分光光度计，移液器（2.5μl 或2μl 规格，10μl规格），电子天平，电泳仪，电泳槽，凝胶成像仪，微波炉，制冰机） 相关耗材：（无菌无绒纸，吸头，离心管架，PCR管，PCR管架，锥形瓶，烧杯，一次性手套，实验手套，冰盒） （1）RNA纯度的检测：测定其OD260和OD280的值，根据其OD260/ OD280的比值，当其比值在1.9~2.1之间，说明提取的总RNA纯度比较高，没有蛋白质和基因组的污染。 （2）RNA完整性的检测：取2μlRNA，与2μl溴酚蓝混匀，用1%的琼脂糖进行凝胶电泳，20min后，在凝胶成像系统中观察效果。当28S与18S条带清晰，且亮度比大约是2:1时，5S条带若隐若现，而且没有其它条带时，说明完整性不错，可以用于下游逆转录实验。
（生物科技行业）功能基因的克隆及生物信息学分析
功能基因的克隆及其生物信息学分析 摘要：随着多种生物全基因组序列的获得，基因组研究正从结构基因组学（structuralgenomics）转向功能基因组学(functionalgenomics)的整体研究。功能基因组学利用结构基因组学研究获得的大量数据与信息评价基因功能(包括生化功能、细胞功能、发育功能、适应功能等)，其主要手段结合了高通量的大规模的实验方法、统计和计算机分析技术[1]，它代表了基因分析的新阶段，已成为21世纪国际生命科学研究的前沿。功能基因组学是利用基因组测序获得的信息和产物，发展和应用新的实验手段，通过在基因组或系统水平上全面分析基因的功能，使生物学研究从对单一基因或蛋白的研究转向多个基因或蛋白同时进行系统的研究，是在基因组静态的组成序列基础上转入对基因组动态的生物学功能学研究[2]。如何研究功能基因，也成为我们面临的一个课题，本文就克隆和生物信息学分析在研究功能基因方面的应用做一个简要的阐述。 关键词：功能基因、克隆、生物信息学分析。 1.功能基因的克隆 1.1图位克隆方法 图位克隆又称定位克隆，它是根据目标基因在染色体上确切位置，寻找与其紧密连锁的分子标记，筛选BCA克隆，通过染色体步移法逐步逼近目的基因区域，根据测序结果或用BAC、YAC克隆筛选cDNA表达文库寻找候选基因，得到候选基因后再确定目标基因。优点是无需掌握基因产物的任何信息，从突变体开始，逐步找到基因，最后证实该基因就是造成突变的原因。通过图位克隆许多
控制质量性状的单基因得以克隆，最近也有报道某些控制数量性状的主效基因（控制蕃茄果实大小的基因克隆[3]、控制水稻成熟后稻谷脱落基因克隆[4]以及小麦VRN2基因克隆[5]等）也通过图位克隆法获得。 1.2同源序列克隆目的基因 首先根据已知的基因序列设计PCR引物，在已知材料中扩增到该片段，并经克隆测序验证，利用放射性同位素标记或其他非同位素标记该PCR片段作为探针，与待研究材料的cDNA文库杂交，就可以获得该基因cDNA克隆，利用克隆进一步筛选基因组文库，挑选阳性克隆，亚克隆并测序，从中就可以筛选到该基因的完整序列。 1.3结合连锁和连锁不平衡的分析方法 结合连锁和连锁不平衡的分析方法是未知基因克隆研究领域发展的新方向[6]。(Linkagedisequilibrium,LD)。与连锁分析不同,连锁不平衡分析可以利用自然群体中历史发生的重组事件。历史上发生的重组使连锁的标记渐渐分布到不同的同源染色体上,这样就只有相隔很近的标记才能不被重组掉,从而形成大小不同的单倍型片段(Haplotypeblock)。这样经过很多世代的重组,只有相隔很近的基因,才能仍处在相同的原始单倍型片段上,基因间的连锁不平衡才能依然存在。所以基于连锁不平衡分析,可以实现目的基因的精细定位。林木大多为自由授粉的异交物种,所以连锁不平衡程度很低,林木基因组中的LD可能会仅局限于非常小的区域,这就为目的基因的精细定位提供了可能,结合SNP检测技术,科学家甚至可以将效应位点直接与单个的核苷酸突变关联起来,进行数量性状寡核苷酸
基因克隆及转基因 一、基因克隆及转基因过程 1、设计引物 软件是https://www.doczj.com/doc/7818381481.html,sergene.v7.1，用到里面的PrimerSelect和EditSeq。 一般原则：1、长度：18-25； 2、GC含量：40-60％，正反向引物相差不要大于5%； 3、Tm值：55以上（到65），实在不行50以上也可以，正反向引物相差不要大 于5； 4、3’端结尾最好是GC，其次是T，不要A； 5、正反向引物连续配对数小于4； 6、在NCBI上的Primer Blast上看引物特异性如何； （如果克隆的话不能满足条件也没办法。） 不是必须条件，但可以考虑：多个基因设计引物时，可尽量使Tm值相似，方便PCR。 步骤： 一、打开PrimerSelect和EditSeq。 二、在EditSeq中输入你的序列。 引物有一对F和R 1、对于F是从5’到3’，在序列的前部分选择长度为18-25bp的碱基，如果你是要验证就随便选，如果你是要克隆就在最开始选，不符合原则就只能在你选的后边增或减碱基。 2、将选择的F引物输入到PrimerSelect中，在File中选择Enter New Primer，复制，OK，然后可以看到引物的情况，看看长度、Tm、GC含量是不是符合标准，不符合就继续选。 3、对于R是从3’到5’，选中序列，在EditSeq的Goodies中选择第一个“反向互补”，此时序列已反向互补，按照前面F的方法搜索R的引物。、 4、注意你想要的目的带的大小，比如序列是1000bp，你想PCR出来800大小的目的带，那就要看看F和R之间的长度在你想要的范围内。可以将R反向互补，在正向的序列中搜索R在的位置，就是在EditSeq中选择Search，点击第一个Find，开始搜寻。 5、搜索完引物在PrimerSelec中的Report中选择前两个查看二聚体情况。 6、在NCBI上的Primer Blast上看引物特异性如何。 7、因为是克隆，所以引物要有酶切位点，酶切位点的加入主要考虑所用到的表达载体，在NEBcutter网站中输入总序列查看可用的酶切位点。在引物上游加入酶切位点，注意加入时载体的表达的方向，前面的酶切位点在引物F上，后面的酶切位点在引物R上。一般在引物上游还要加上两个保护碱基。 2、提取醋栗DNA 3、PCR扩增与目的基因回收 PCR先找合适的退火温度，找到后回收时就可以多PCR几管，一般我们用20ul的体系，PCR5管就可以回收，就是琼脂糖凝胶回收，将目的基因用刀片切下来，用试剂盒回收。回收完可以再跑电泳检测一遍。 PCR： 20ul体系：灭菌水13.8ul，若模板为质粒灭菌水14.3ul； 2.5mMdNTP2.0ul；
实验一大肠杆菌感受态细胞的制备及转化 [实验原理]（供参考，试剂盒的Solution SS成分未知） 细菌处于容易吸收外源DNA的状态叫感受态。转化是指质粒DNA 或以它为载体构建的重组子导入细菌的过程。其原理是：在0℃下的CaCl2低渗溶液中，细菌细胞膨胀成球形。转化缓冲液中的DNA形成不易被DNA酶所降解的羟基—钙磷酸复合物，此复合物粘附于细菌细胞表面。42℃短时间热处理（热休克），可以促进细胞吸收DNA复合物。将处理后的细菌放置在非选择性培养液中保温一段时间，促使在转化过程中获得的新的表型(如Amp抗性) 得到表达。然后再涂布于含有氨苄青霉素的选择性平板上，37℃培养过夜，这样即可得到转化菌落。[仪器、材料与试剂] (一)仪器1．小型高速离心机2．恒温摇床3．恒温箱4．‐20℃冰箱5．恒温水浴器 (二)材料1．氨苄青霉素2．大肠杆菌DH5a3．pUC194．1.5mL 离心管5．枪头、枪6．试管、培养皿 (三)试剂1．快速感受态细菌制备试剂盒（申能博彩公司产品）2．LB 培养液在950mL去离子水中加入：胰蛋白胨(tryptone) 10g酵母提取物(yeast extract) 5g NaCl 10g 摇动容器直至溶质完全溶解，用Na0H调节pH至7.0，加入去离子水至总体积为1L，121℃湿热灭菌20min。 3．氨苄青霉素(Amp)，用无菌水配制成100mg／mL 溶液，置‐20℃冰箱保存。 [实验步骤]
1．从大肠杆菌DH5a平板上挑取一个单菌落接于2mL LB培养液的试管中，37℃振荡培养过夜。 2．取50mL菌液转接到一个含有5mL LB培养液锥形瓶中，37℃振荡培养2小时。以下步骤按修改后的试剂盒说明书进行。 3．用灭菌的枪头取0.5mL的大肠杆菌培养物于1.5mL灭菌离心管中，冰上放置3分钟后，加入0.5mL预冷的Solution SS。在冰上小心地用1mL 枪头将细胞悬浮起来。注意：1mL的取液器设定在500mL。悬浮细胞要轻，防止细胞进入枪内。 4．将上述细胞分装于1.5mL离心管(离心管要在放在冰上预冷) 中，每管0.1mL。细胞可以立即使用或储存。 5．将感受态细胞迅速转移到‐20℃或更低的低温冰中。注意：在转移过程中要防止温度升高，解决的办法之一是在塑料袋里装上低温冰块，将细胞迅速转移到塑料里，将整个塑料袋放到低温冰箱内。 转化：1．新鲜制备的或‐20℃下保存的100mL感受态细胞，置于冰上，完全解冰后轻轻地将细胞均匀悬浮。 2．加入5mL pUC19质粒，DNA浓度为10pg/mL，轻轻混匀。 3．冰上放置30分钟。 4．42℃水浴热激60秒。 5．冰上放置2分钟。 6．加400mL LB培养液，37℃ 250转／分振荡培养30分钟。 7．室温下4000rpm离心5分钟，用枪头吸掉400mL上清液，用剩余的培养液将细胞悬浮。
基因克隆载体上的各种常用蛋白标签 蛋白标签(proteintag)是指利用DNA体外重组技术，与目的蛋白一起融合表达的一种多肽或者蛋白，以便于目的蛋白的表达、检测、示踪和纯化等。随着技术的不断发展，研究人员相继开发出了具有各种不同功能的蛋白标签。目前，这些蛋白标签已在基础研究和商业化产品生产等方面得到了广泛的应用。 美国GeneCopoeia(复能基因)为客户提供50多种蛋白标签，可以满足客户的不同需求，包括各种最新型的标签，如：SNAP-Tag?、Halo Tag?、AviTag?、Sumo等;也提供齐全的各种常用标签，如eGFP、His、Flag等等标签。 以下是部分蛋白标签的特性介绍，更加详细的介绍可在查询产品的结果列表里面看到各种推荐的蛋白标签和载体。 TrxHIS His6是指六个组氨酸残基组成的融合标签，可插入在目的蛋白的C末端或N末端。当某一个标签的使用，一是能构成表位利于纯化和检测;二是构成独特的结构特征(结合配体)利于纯化。组氨酸残基侧链与固态的镍有强烈的吸引力，可用于固定化金属螯合层析(IMAC)，对重组蛋白进行分离纯化。使用His-tag有下面优点： 标签的量小，只有~0.84KD，而GST和蛋白A分别为~26KD和~30KD，一般不影响目标蛋白的功能; His标签融合蛋白可以在非离子型表面活性剂存在的条件下或变性条件下纯化，前者在纯化疏水性强的蛋白得到应用，后者在纯化包涵体蛋白时特别有用，用高浓度的变性剂溶解后通过金属螯和去除杂蛋白，使复性不受其它蛋白的干扰，或进行金属螯和亲和层析复性; His标签融合蛋白也被用于蛋白质-蛋白质、蛋白质-DNA相互作用研究; His标签免疫原性相对较低，可将纯化的蛋白直接注射动物进行免疫并制备抗体。 可应用于多种表达系统，纯化的条件温和; 可以和其它的亲和标签一起构建双亲和标签。 Flag标签蛋白 Flag标签蛋白为编码8个氨基酸的亲水性多肽(DYKDDDDK)，同时载体中构建的Kozak序列使得带有FLAG的融合蛋白在真核表达系统中表达效率更高。FLAG作为标签蛋白，其融合表达目的蛋白后具有以下优点： FLAG作为融合表达标签，其通常不会与目的蛋白相互作用并且通常不会影响目的蛋白的功能、性质，这样就有利用研究人员对融合蛋白进行下游研究。 融合FLAG的目的蛋白，可以直接通过FLAG进行亲和层析，此层析为非变性纯化，可以纯化有活性的融合蛋白，并且纯化效率高。 FLAG作为标签蛋白，其可以被抗FLAG的抗体识别，这样就方便通过Western Blot、ELISA等方法对含有FLAG的融合蛋白进行检测、鉴定。
4植物基因克隆的策略与方法 基因的克隆确实是利用体外重组技术,将特定的基因和其它DNA顺序插入到载体分子中。基因克隆的要紧目标是识不、分离特异基因并获得基因的完整的全序列,确定染色体定位,阐明基因的生化功能,明确其对特定性状的遗传操纵关系。通过几十年的努力由于植物发育,生理生化,分子遗传等学科的迅速进展,使人们把握了大量有关植物优良性状基因的生物学和遗传学知识,再运用先进的酶学和生物学技术差不多克隆出了与植物抗病、抗虫、抗除草剂、抗逆,育性、高蛋白质及与植物发育有关的许多基因。我们实验室对天麻抗真菌蛋白基因作了功能克隆的研究(舒群芳等,1995;舒群芳等,19 97),为了克隆植物基因也探讨了其它克隆方法,本文论述基因克隆的策略、方法及取得的一些进展。 1功能克隆(functional Cloning) 功能克隆确实是按照性状的差不多生化特性这一功能信息,在鉴定和已知基因的功能后克隆(Collis,1995)。其具体作法是:在纯化相应的编码蛋白后构建cDNA文库或基因组文库,DNA文库中基因的选择按照情形要紧可用二种方法进行,(1)将纯化的蛋白质进行氨基酸测序,据此合成寡核苷酸探针 从cDNA库或基因组文库中选择编码基因,(2)将相应的编码蛋白制成相应抗体探针,从cDNA入载体表达库中选择相应克隆。功能克隆是一种经典的基因克隆策略,专门多基因的分离利用这种策略。 Hain等从葡萄中克隆了两个编码白藜芦醇合成的二苯乙烯合成酶基因(Vst1和Vst2),葡萄中抗菌化合物白藜芦醇的存在,能够提升对灰质葡萄孢(B otrytis cinerce)的抗性,在烟草和其它一些植物中无二苯乙烯合成酶,因此克隆该基因通过转基因后,对有些植物产生对灰质葡萄孢的抗性专门有意义(H ain等,1985)。Kondo等1989年对编码水稻巯基蛋白酶抑制剂的基因组DN A做了克隆和序列分析(Kondo等,1989)。周兆斓等构建了水稻cDNA文库,分离了编码水稻巯基蛋白酶抑制剂的cDNA(周兆斓等,1996)。植物蛋白酶抑制剂是一类天然的抗虫物质,它可抑制摄食害虫对蛋白质的消化,使害虫因 缺乏所需氨基酸而导致非正常发育或死亡。胡天华等人从烟草中分离出流行于我国的黄瓜花叶病毒(Cucumber Mosaic virus)(CMV),并克隆了编码该
MaxDOS 网刻服务端2.01 2009元旦修正版下载(修正杀软报毒问题),及新版网刻教程。 管理提醒: 本帖被Max 执行加亮操作(2008-08-17) MaxDOS 网刻服务端 2.0 2009元旦版 网刻服务端升级为 MAXNGS 2.0 修正网刻服务端同一时间内DHCP只能提供一个IP地址,第二台无汉获得IP地址. 修正DHCP分配 IP时,一台机器分配了两个IP.修正TFTP不能同时传送多个进程. 增加多网段功能,当使用大网段时,可设置超过多少台机器自动跳跃至下一网段,理论支持无 限台客户端,增加方案功能,当首次设置完毕后,方案将自动被保存,下次使用无需再次设置. MaxDOS 网刻服务端(MaxNGS) 2.0版下载地址: 电信下载 https://www.doczj.com/doc/7818381481.html,/soft/MaxNGSrv2.rar 网通下载 https://www.doczj.com/doc/7818381481.html,/soft/MaxNGSrv2.rar 注意事项： １、我们在网刻前，首先要做的是把要网刻的源分区或硬盘做成一个GHOST镜像，如果有不会做全盘G HOST镜像的，请参见https://www.doczj.com/doc/7818381481.html,/bbs/read.php?tid=26435 ２、前提客户机必须先安装好MaxDOS 7客户端或者需要有PXE、U盘、光盘版MaxDOS 7可当引导使用，服务器可以安装或不安装都可以，网刻前请关闭局域网中的其它DHCP服务端设备，以免造成IP分配错误。(注: 如果可以请尽量不要使用和内网现有的IP同一地址段) ３、具备以上条件后，我们开始进行正式的网络克隆，请先下载MaxDOS网刻的专用服务端，下载地址H ttp://https://www.doczj.com/doc/7818381481.html,/soft/7ngsrv.rar下载完毕后解压出7ngsrv.rar的压缩包到任意位置(注：必须解压，不可直接运行压缩中的程序)，然后打开MAXNGS.EXE 网刻服务端主程序。 MaxDOS 7网刻服务端使用教程
超级一键网克使用方法 进行网克需要的条件： 一键网克软件：超级一键网克，当然其他的网克软件也可以。 一台已经装好系统的机器（操作系统98以上即可，推荐XP）用作服务器， 一台已经装好全部软件并且配置好的机器作为母盘，当然，如果你只对系统盘进行网克的话，这台机器也可以兼做服务器，但是如果你想全盘网克的话，那就需要另外一台机器做服务器了，道理嘛，因为做全盘镜像的时候镜像文件当然不可能放在源盘上，而必须放在另外一块硬盘上，就好象你左手永远不可能摸到左手的手背一样，呵呵。 其它客户机，数量255台以下，当然多于255台也可以，只是下面的IP地址和子网掩码要重新配置，大家参看有关资料。不过我想大家一次网克也不可能会有那么多台机器的，呵呵。 注意事项：首先，这些机器间物理网络要保持畅通（废话！）。另外，如果你的网吧是杂牌军的话，那就不要试了，因为每台机器的配置如果不一样的话，所装的驱动也不一样，否则网克后可能有太多的不可预知的后果，经常蓝屏肯定是家常便饭了，当然也有可能根本就启动不了。以上说的配置主要是显卡和主板（如果你的声卡是集成的话）。硬盘无所谓，但要注意，目标盘的容量一定要大于母盘。当然了，最好是配置完全一样，这样基本上不会出什么问题。
当然，如果你的机器配置不一样的话，也可以进行网克，大家可以把雨林木风ghost版里面的ghost文件提取出来作为镜像，这样也比一台一台装系统要快得多了。不过那应该是分区网克，具体方法你学会了全盘网克那自然不在话下。下面开工。 第一步，制作镜像，如果只制作系统盘的镜像，那么在单机操作就行了。具体操作我想大家都会。下面讲的是制作全盘镜像，当然你不必拆开机器挂两个硬盘了，网克嘛！ 1、配置tftpd32 解压超级一键网克到服务器的任意盘，比如E盘，双击TFTPD32.EXE，关闭防火墙，或者在应用规则里设为允许访问网络。最好关掉，否则下面你要点若干次允许。点DHCP server标签，这里有几个地方要填一下，当前目录和server不要填，程序自动判断。IP地址池随便填，但要注意不要跟服务器的IP地址冲突，当然要符
基因克隆的步骤 一、RNA的提取 1. 提取RNA前将洗净干燥的瓷研钵放入-70℃预冷10 min； 2. 从液氮罐中取出样品组织放入预冷的研钵中，加液氮淹没后立即研磨，边研磨边加液氮，整个过程都 不要使液氮挥干； 3. 趁冷，将样品粉末50-80 mg加入1.5 mL离心管后再加入1 mL Trizol，样品体积应不超过所使用Trizol 体积的10%，然后按照Trizol试剂盒说明操作； 4. 室温静止5-15 min。对于某些蛋白、多糖或脂含量很高的细胞或组织Trizol裂解后可能会有不溶物或 油脂状漂浮物，需12000g、4℃离心10 min，然后吸取澄清的Trizol裂解产物至一新的离心管中；5. 每mL Trizol中加0.2 mL氯仿。小心盖上离心管，剧烈地涡旋振荡15 sec，室温（24℃）静置3-5 min； （必需步骤） 6. 12000 g、4℃离心15 min，此时混合物分成三部分：底层为苯酚-氯仿层，中间层，上层水相层，RNA 完全存在水相中； 7. 把小于80%的水相层（每mL Trizol约可吸0.5-0.55 mL）转移至一新的离心管中，并弃去下面的有机 相（小心避免吸到中间层）。往水相中加入异丙醇来沉淀RNA，每使用1 mL Trizol，便加500 uL的异丙醇，颠倒混匀，室温下孵育样品10 min； 8. 4℃、12000 g离心样本10 min弃去上清，每mL Trizol加入1 mL 75%乙醇，涡旋混匀，室温沉淀10 min 后，7500 g、4℃离心5 min，弃上清。再用离心机甩一下（5000rpm，离心1 s）； 9. 小心吸走乙醇，短暂地在室温下置2-5 min，让RNA团风干，不要用离心干燥装置或真空干燥装置， 因为过度干燥会导致很难用水重新溶解RNA，然后在55-60℃温浴10 min，然后-70℃保存。[RNA]（ng/uL）=A260×40×稀释倍数 [DNA]（ng/uL）=A260×50×稀释倍数 纯DNA：OD260/OD280≈1.8（＞1.9，表明有RNA污染；＜1.6，表明有蛋白质、酚等污染） 纯RNA：1.7＜OD260/OD280＜2.0（＜1.7时表明有蛋白质或酚污染；＞2.0时表明可能有异硫氰酸残存） 二、M-MLV RT反转录 1. oligo dT 1 uL + dNTP（ 2.5 mM）4 uL + 1-5 ug total RNA + 水= 12 uL Heat mixture to 65℃ for 5 min and quick chill on ice； 2. 上述12 uL液体+ 5×Buffer 4 uL + 0.1 M DTT 2 uL + RNase OUT 1 uL Mix contents of the tube，incubate at 37℃for 2 min 3. 上述19 uL液体加M-MLV RT 1 uL
机房管理——网络克隆大比拼 2005年第4期《电化教育咨讯》文/陈杰 经常在一些电脑杂志上看到介绍单机情况下如何系统克隆的软件和操作，虽然比单独安装要快，但是如果面对标准配置为64台电子教室，这也并不是什么好办法，因为要重复64次相同操作。那么有没有什么好的办法可以让我们可以在网络环境下进行克隆呢？ 一、网络克隆 Netghost Ghost 这个软件想必大家已经很熟悉，但会网络Ghost 的人却很少。Ghost 分两种，一种以Ghost6.5/7.0/7.5命名的是企业版；还有一种以Ghost2002/2003命名的是个人版。笔者下面要介绍的网络Ghost 就是企业版的。 1、软件准备 (1Ghost7.0客户端和服务器端； (2网卡驱动程序for dos 。 一个64台电脑的网络电子教室，使用的电脑配置全部相同。那我们可以用其中的一台电脑装好系统之后，再装好所有的应用软件。用Ghost 对C 盘做镜像，并把这个镜像文件存放到电子教室的服务器上面。 2、服务器端设置(win98或2000 启动ghostsvr.exe （1）设置服务器名称，如sever 。 （2）镜像名称处选择好已经copy 到服务器的Ghost 文件的路径。 （3）选择分区
3、客户端的设置(dos 要准备好3个文件 （1）网卡的驱动程序 （2） ghost.exe。 （3）编辑一个wattcpv.cfg 文本文件（为了以后避免IP 地址冲突设定） IP=192.168.0.XX 指定客户端 IP地址 NETMASK=255.255.255.0 子网掩码 GATEWAY=196.198.0.1 网关 最后把这3个文件放到同一个目录下面，运行X:\ghost\ghost.exe,然后就直接进入Ghost 的界面了。 这时你会发现multicasting 的选项已经亮了，选择它后，会发现弹出一个窗口，这里会显示你当前电脑的IP ，并要求输入服务器的名称，然后就会直接读到服务器的ghost 文件了。这个时候需要对主机执行开始命令，客户机就开始恢复。在服务器端更多选项里填入连接的机器数, 客户机就自动开始恢复，之后就同单机版一样了。 二、三茗网络对拷软件 1、软件准备 （1）网卡驱动程序for dos （2） nc.exe 这就是网络对拷的主程序 2、发送机端(dos
功能基因的克隆及其生物信息学分析 摘要：随着多种生物全基因组序列的获得，基因组研究正从结构基因组学（structural genomics）转向功能基因组学(functional genomics)的整体研究。功能基因组学利用结构基因组学研究获得的大量数据与信息评价基因功能(包括生化功能、细胞功能、发育功能、适应功能等)，其主要手段结合了高通量的大规模的实验方法、统计和计算机分析技术[1]，它代表了基因分析的新阶段，已成为21世纪国际生命科学研究的前沿。功能基因组学是利用基因组测序获得的信息和产物，发展和应用新的实验手段，通过在基因组或系统水平上全面分析基因的功能，使生物学研究从对单一基因或蛋白的研究转向多个基因或蛋白同时进行系统的研究，是在基因组静态的组成序列基础上转入对基因组动态的生物学功能学研究[2]。如何研究功能基因，也成为我们面临的一个课题，本文就克隆和生物信息学分析在研究功能基因方面的应用做一个简要的阐述。 关键词：功能基因、克隆、生物信息学分析。 1.功能基因的克隆 1.1 图位克隆方法 图位克隆又称定位克隆，它是根据目标基因在染色体上确切位置，寻找与其紧密连锁的分子标记，筛选BCA克隆，通过染色体步移法逐步逼近目的基因区域，根据测序结果或用BAC、YAC克隆筛选cDNA表达文库寻找候选基因，得到候选基因后再确定目标基因。优点是无需掌握基因产物的任何信息，从突变体开始，逐步找到基因，最后证实该基因就是造成突变的原因。通过图位克隆许多控制质量性状的单基因得以克隆，最近也有报道某些控制数量性状的主效基因（控制蕃茄果实大小的基因克隆[3]、控制水稻成熟后稻谷脱落基因克隆[4]以及小麦VRN2 基因克隆[5]等）也通过图位克隆法获得。
网络克隆系统操作说明(v2.0) -- 2012.02.17 jason 说明：本文仅供网友参考。 一、概述 1、网络克隆原理 所谓网络克隆，简单的讲就是通过网络把需要备份/恢复镜像的客户端连接到服务器，主要利用网络启动+ GHOST技术把镜像上传到服务器或下载到客户端。我们现在是应用工具MAXDOS通过网卡启动为客户端PC安装系统镜像，它是基于C/S的网络模式，主要利用了PXE技术、ghost的远程克隆功能、DHCP技术和TFTP技术，在这里不作深入描述。 2、实际应用的环境 新厂房采用了更高级的网络设备，使得网络环境有较大的改善，局域网线路传输速率可以达到1Gbps，这为实现网络克隆，提高电脑系统安装的便捷性和效率创造了条件。 3、实现方案 如下图所示，在局域网内通过网络为客户端安装系统镜像，通过第三方工具MAXDOS 构成C/S模式的网路。准备好需要安装的系统镜像后，在服务端设置好方案和网络配置，启动ghost服务端监听客户端请求；只需在客户端设置电脑为从网络启动，待服务端监听到客户端后，点击“发送”键开始为客户端安装系统镜像。如果采用多点传送方式，服务端每次可为多台电脑同时安装系统镜像。
二、操作流程 参考上图中的人工操作部分。 服务端： 1、制作/准备系统镜像。一般在安装镜像之前，镜像管理人员会把做好的镜像存放在服务 器上，操作人员可以忽略这一步； 2、在服务端打开MAXDOS。MAXDOS是一系列工具的合集，不需要进行安装，进入MaxDOS 目录，双击运行“MaxNGS.exe”，如下图所示：
3、设置启动方案。 步骤一：如上图，点击右上角的“方案设置”按钮，然后设置方案名称（方案名称可随
网管必学：诚龙网维全自动PXE网刻工具图文教程 在我们维护大批电脑时，常常会遇到批量安装操作系统，尤其是网吧；如果网吧规模达到几十台，按照原来的传统安装方式，恐怕要安装个几天；随着电脑技术的发展，网络批量克隆安装系统已经越来越成熟，今天，我们将以诚龙网络克隆工具为例，一起来学习下它的使用方式。 所需工具： 1、诚龙网维全自动PXE网刻工具11.0 2、已经安装好系统后备份的ghost镜像文件 使用步骤： 一、网克服务器的设置 1、断开本网络与其它网络的连接（保证在网络内没有DHCP服务器）。 由于该工具中含有DHCP服务器，所以局域网内必须关闭相关的DHCP服务器，在网吧中，一般只有路由器带有DHCP功能，所以在网络克隆的时候，最好首先关闭路由器，以免客户机在寻找IP地址时出错。 (小知识：DHCP服务器通俗点说，就是能够自动分配IP地址的服务器，网吧中的路由器一般都带有DHCP自动IP地址分配功能) 2、清除服务器端ip地址。 因为该工具会自动设置你计算机的IP地址，所以一般我们建议将网克服务器的IP地址设为自动获取，这样相对来说，不容易出现IP地址分配出错的情况。 3、运行“诚龙网维全自动PXE网刻工具11.0.exe”，进行必要的设置后，点击--开始网刻。如下图中描述，单击浏览选择系统镜像后，在上面选择网克的类型，一般来说，我们只单纯的克隆C盘以实现系统重新安装；当然，在全新安装网吧系统时，我们就可以选择全盘网络克隆了； 软件界面 设置完毕后，我们单击这里的“开始网克”，出现以下提示时，单击"确定"，服务器就准备完毕了；
Ghost网克服务器 二、客户机的设置 现在的主板中，基本上都已经集成了网络启动功能，因此，我们只需要在BIOS中，将第一启动设置为网卡启动就可以了； （小知识：有关主板如何设置网络启动，可以在主板说明书中找到；大家可以参考下说明书；如有任何问题，您也可以到：ask 来提问，我们将尽量详细为您解答；） 下面，我们将以老式REL8139网卡为例，说明如何进行网卡设置： Realtek RTL 8139网卡设置 按下电源开关，系统开始自检，当自检完硬盘、光驱后，出现以下提示： Realtek RTL 8139 (A/B/C)/RTL8130 Boot Agent Press Shift-F10 to configue…… 此信息默认为停留3秒钟，此时，按下SHIFT--F10进入网卡配置菜单，共有四个选择：1．Network Boot Protocol (PXE RPL)按空格改变网络引导协议 2．Boot order (Rom Disable禁止BOOR ROM引导 Int 18h先从BIOS设置中的次序引导 Int19h先从BOOT ROM引导 PnP/BEV从BBS引导) 3．Show config Message (Enable Disable)启动时是否显示SHIFT—F10 4．Show Message time (3 seconds 4seconds 5seconds 8seconds 10seconds) 启动时shift—f10提示信息停留的时间。 新网卡的Boot order 选项为禁止BOOT引导，所以，所有新网卡必须进入设置程序，将其设为INT18 或INT19设置完毕后，按F4保存退出。 故障1：有些主板与PXE BOOTROM不兼容，不出现SHIFT—F10提示，或者出现E—28提示BIOS结构与BOOT ROM不一致。此时便无法从芯片引导无盘WIN98。 解决方法是：更新BOOT ROM版本。另外，此类主板主要是AMI版本的BIOS，在AWORD BIOS版本上的主板则很少出现。 片刻后工作站从服务器上获取IP地址出现，并下载所需文件，等待服务器传输ghost文件。
图位克隆基因研究进展 宋成标 摘要图位克隆是在不清楚基因产物结构和功能的情况下，根据基因在染色体上都有稳定的基因座实现的。随着各种分子标记技术和高质量基因组文库构建技术的发展，图位克隆现已经成为分离生物体基因的一种常规技术。本文主要概述了图位克隆的一般步骤，包括目的基因的初步定位、精细定位和遗传做图、染色体步行和登陆及利用功能互补实验鉴定目的基因。最后，对图位克隆技术存在的局限和发展前景作了初步的分析。 关键词图位克隆, 分子标记, 精细定位, 基因组文库 Abstract Map-based cloning is based on the functional genes have their particular gene locus on chromosomes,when we know about the structure and function of gene products unclearly.With the rapid development of molecular marker technologies and constructing high quality genomic library technologies, map-based cloning had already become a common bio—technique for gene isolation. This article summarized mainly the processes of the map-based cloning in principle,including first-pass mapping of candidate gene、fine scale-mapping and building genetic map、chromosome walking or landing and finally complement experiment for identifing candidate gene. Finally the problems and the prospects in the map-based cloning are analyzed Keywords Map-based cloning, Molecular marker, Fine maping, Genomic library 从遗传学观点来看，基因克隆有两条途径：正向遗传学途径和反向遗传学途径。正向遗传学途径指的是通过被克隆基因的产物或表型突变去进行，如传统的功能克隆及近年来迅速发展的表型克隆；反向遗传学途径是根据被克隆的目的基因在染色体上都有稳定的位置来实现的。由于在多数情况下，我们并不清楚基因产物的结构和功能，很难通过正向遗传学途径克隆基因，而反向遗传学途径则显示了较好的前景。其中可以利用的主要有三种方法，分别是转座子标签法、随机突变体筛选法和图位克隆法。转座子标签法中受转座子的种类、转座频率及有些植物存在内源转座子等的影响，随机突变体筛选法则随机性较大且不能控制失活基因的种类和数量等，限制了它们的应用。图位克隆（map-based cloning）又称为定位克隆（positional cloning），1986年首先由剑桥大学的Coulson 等提出，用该方法分离基因是根据目的基因在染色体上的位置进行的，无需预先知道基因的DNA序列，也无需预先知道其表达产物的有关信息。它是通过分析突变位点与已知分子标记的连锁关系来确定突变表型的遗传基础。随着模式物种（拟南芥、水稻）全基因组测序的完成，各种分子标记技术的发展促进了高密度分子标记连锁图谱的建立和各种数据库的完善。图位克隆技术越来越成熟，已经成为分离生物基因的一种常规方法。本文将对图位克隆技术的相关策略作一介绍。 1图位克隆的策略 自1992年图位克隆技术首次在拟南芥中克隆到ABI3(Girauda et al., 1992)基因和F AD3 (Arondel et al., 1992)基因以来，图位克隆技术在其它相关技术快速发展的支持下迅速发展起来。它是依据功能基因在生物基因组中都有相对稳定的基因座，在利用分子标记技术对目的基因进行精细定位的基础上，用与目的基因紧密连锁的分子标记筛选已构建的DNA文库（如Cosmid、YAC、BAC等文库），构建出目的基因区域的遗传图谱和物理图谱，再利用此物理图谱通过染色体步行、跳跃或登陆的方式获得含有目的基因的克隆，最后通过遗传转化和功能互补实验来验证所获得的目的基因（图1）。 初步定位（First-pass maping）-------构建遗传图谱（constructing genetic map）-----精细定位（fine maping）---------构建物理图谱( constructing physical map)------染色体步移、登陆（chromosomal walking、landing）-------确定侯选基因（Consider candidate genes）----遗传互补验证目的基因（Through genetic complementation (transformation) to identify candidate gene）（请帮我画一个简易图表，把内容填进去） 图1 图位克隆的主要步骤 Figure 1 Key steps in map-based cloning process
微机室批量克隆系统方法（要求电脑配置要相同） 一：1、在主机上用EasyMacToIPGet_v0.4(x86)采集所有电脑的MAC地址和对应的IP，并根据实际情况把相应的电脑改成要求的计算机名和IP，最后生成MacToIP.ini文件。 2、在一台电脑上先做好系统，装好驱动，并在C盘装好各类应用软件，IP地址设为自动获取。 3、把EasyMacToIPSet_v0.4(x86).exe和事先准备好的MacToIP.ini放到C盘根目录下的X86文件夹内。并把EasyMacToIPSet_v0.4(x86)发送到桌面快捷方式，文件名改为EasyMacToIPSet .lnk。 4、在桌面新建一文本文档，内容为 C: CD C:\Users\Administrator\AppData\Roaming\Microsoft\Windows\Start Menu\Programs\Startup del EasyMacToIPSet.lnk exit 另保存为DEL.bat到c:\X86文件夹内。 5、在开始-运行里输入gpedit.msc ，进入组策略，双击计算机配置-windows设置-脚本（启动/关机），双击关机，在关机属性里点添加，在添加脚本窗口点浏览找到c:\X86\del.bat，确定。退出。 6、安装冰点还原，重启后设好密码，再次重启后把冰点还原设为重启两次后冻结。 7、重启并利用ghost软件做成镜像文件。 8、用GHOST镜像浏览器打开生成的镜像，找到桌面上的EasyMacToIPSet.lnk，移动到C:\Users\Administrator\AppData\Roaming\Microsoft\Windows\Start Menu\Programs\Startup文件夹，关闭GHOST镜像浏览器。 二：1、把做好的镜像拷到主机上，和万能网络克隆工具PXEGHOA 6.1A 绿色版放入同一个文件夹内。 2、运行PXEGHOA_x86.exe，输入要克隆的电脑数，确定等待...... 3、客户机开机并设为从网络启动。 4、所有客户机进入网络克隆中......，完成后自动重启进入系统，EasyMacToIPSet_v0.4(x86)将自动根据之前准备好的计算机名和IP地址进行修改。 5、把电脑重启两次（可通过红蜘蛛电子教室进行操作统一操作），所有的电脑就完全设置好了，并且还原精灵也开始了对系统的保护。
DNA结构与复制中的相关计算的三种常用方法 一、特值法： 先按照碱基比例假设DNA片段中碱基总数为100或200等整百数，再根据碱基互补配对原则（A－T，C－G）图解分析求解。 例：一个DNA分子中，G和C之和占全部碱基数的46％，又知在该DNA分子的一条链中，A和C分别占碱基数的28％和22％，则该DNA分子的另一条链中A和C分别占碱基数的（）。 A．28％、22％B．22％、28％C．23％、27％D．26％、24％ 【解析】假设DNA每条链的碱基数为100，依题意得：（图略） ∵甲链: A=28, C=22,G+C=46, ∴甲中G=24, T=100-28-46=26。则乙中A=26，C=24。故选D。 练习：分析某生物的双链DNA，发现腺嘌呤与胸腺嘧啶之和占全部碱基的64％，其中一条链上的腺嘌呤占该链全部碱基的30％，则对应链中腺嘌呤占整个DNA分子碱基的比例是（） A．17％B．32％C．34％D．50％
二、首尾法： 根据DNA复制的过程与特点可以知道：一DNA分子复制n次后，将得到2n个DNA分子，其中保留原来母链的DNA 数目为2个。在处理与此相关的计算题过程中，我们只需要考虑开始和结尾的差异就可以顺利求解，笔者习惯于称之为首尾法。 例：假如一个DNA分子含有1000个碱基对（P元素只是32P）,将这个DNA分子放在只含31P的脱氧核苷酸的培养液中让其复制两次,则子代DNA分子的相对分子量平均比原来( )。 A.减少1500 B.增加1500 C. 增加1000 D.减少1000 【解析】每个碱基对应一个脱氧核苷酸，含1个磷酸基，即1个磷原子。复制两次后形成4个DNA分子，8条单链。其中两条含32P，6条含31P，因而相对分子量减少6000，4 个DNA平均减少1500。故选A。 练习：已知14N-DNA和15N-DNA的相对分子量分别为a和b。现让一杂合DNA分子在含14N的培养基上连续繁殖两代，则其子代DNA的平均相对分子量为（） A.(3a+b)/4 B.(a+3b)/4 C.(7a+b)/8 D.(a+7b)/8 三、公式法： 基于DNA的半保留复制，我们可以归纳出公式：X=m(2n-1)。