CN104928303A - Function and application of arabidopsis thaliana POL2A gene in reduction division recombination - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering and particularly relates to a function and application of an arabidopsis thaliana POL2A gene in reduction division recombination. The application comprises the following steps of using sequence characteristics of coding POL2A protein and the authentication of the new function of the gene in reduction division, i.e. using a fluorescent label site system to analyze the influence of POL2A to reduction division recombination frequency and distribution, and providing a fluorescent label-containing genetic material; providing the function of the gene of analyzing how complete mutation of the gene causes embryo death in reduction division, and providing the technology of a series of cell biology and the like. The important function of POL2A in reduction division recombination is proved in an eukaryote for the first time; the function is used, the modern biotechnology method is used, and the recombination frequency of a generic material in a reduction division process is controlled, so that aggregation of excellent characters is quickened, separation of the excellent characters in a hybrid offspring is delayed, and the aim of propelling and improving breeding study level is fulfilled.

Description

The function of Arabidopis thaliana POL2A gene in meiotic recombination and application thereof

Technical field

The invention belongs to gene engineering technology field, be specifically related to plant pOL2Athe function of gene in meiotic recombination, and utilize the function of this gene, changing the application in crop breeding process, be difficult to the interval occurring to exchange under especially changing natural condition, more effectively can accelerate breeding efficiency.

Background technology

Reduction division is the necessary vital process of eukaryote sexual propagation, and in reduction division process, DNA replication dna once, cell fission twice, makes to be separated from the pair of homologous karyomit(e) of father and mother respectively; Nonhomologous chromosome is independent assortment then, thereby produce that there is multiple genetic make up, that chromosome number reduces by half gamete, the zygote formed after gametogamy maintains chromosomal number in parent, thus ensure that the stability of genetic material, and species are multiplied.Homologous chromosomes identification and pairing, joint conference and exchange are the critical events in meiosis prophase, and these events normally orderly completing ensure that the correct formation of bivalent and the clean cut separation of homologous chromosomes.Therefore, homologous chromosomes interaction in early stage is one of the study hotspot in domestic and international plants ' reproduction development field, also belongs to the basic research work of frontier nature.The twice DNA synthesis occurred in this process: before reduction division the horizontal DNA replication dna of the full-length genome of S phase with repair (Double Strand Break Repair with the modulated DNA double splitting of chain being reassembled as object, DSB), although few compared with the former in resultant quantity, but this process is equally to ensureing the integrity that genetic information is transmitted, and the normal generation of restructuring is most important.

Reduction division core event is the interaction between homologous chromosomes, and the interaction of homologous chromosomes comprises the pairing of homologous chromosomes, joint conference, restructuring and is separated.Wherein, recombinate and occur between homologous chromosomes, instead of occur between sister strand in reduction division process, the regrouping process occurred in this process and mitotic division is different.In eukaryote maiotic restructuring by sPO11the double-strand break of mediation is initial, has found 3 in Arabidopis thaliana sPO11homologous gene, namely atSPO11-1, atSPO11-2, atSPO11-3.According to existing, atSPO11-1with atSPO11-2that the initial institute of meiotic recombination is required, atSpo11-3relevant (Grelon et al., 2001 are repaired to DNA; Yin et al., 2002).After the double-strand break that SPO11 mediates is formed, at the double-strand two ends of fracture through processing the single stranded DNA tail that all can produce 3 ' end, the recombinase that RAD51 is relevant is combined with this single stranded DNA and forms nucleic acid-protein fiber, this structure helps the identification of homologous region between homologous chromosomes and invades double-strand, the gene of RAD51 family has played important function in this process, has 3 in Arabidopis thaliana rAD51family gene ( atrad51, atrad51cwith atxrcc3) can Abnormal Circumstances In Meiosis be caused, nourishing and growing normally but abortion of these three mutant, the double-strand break that abortion all causes due to SPO11 in reduction division process can not be repaired, and causes a large amount of DNA fragment (Bleuyard et al., 2004; Li et al., 2004; Li et al., 2005).In order to ensure chromosomal accurate separation, each karyomit(e) at least needs an exchange (this exchange becomes and must exchange), but the distribution exchanging site is not random, multiple exchange sites on same karyomit(e) also have spaced, and the number of intersection plays an important role to correct separation of homologous chromosomes in reduction division process with distribution.This phenomenon relates to again another phenomenon---interferes, namely after an exchange produces, near this exchange, another possibility exchanged of generation can reduce, and Here it is interferes responsive type to exchange (type I COs), two albumen MSH4/5 of main dependence and bacterium MutS albumen homology; There is another kind of point other exchange at random simultaneously, be called and interfere non-sensitive type to exchange (type II COs), mainly rely on formation heterodimeric thing restriction endonuclease MUS81 and MMS4/EME1 subunit (Ma, 2006); Also has non-chromosome cross-pathway (NCO) in addition.

Meiotic recombination process mainly comprise DNA double splitting of chain, chain end processing, chain invasion, chain synthesis and dissociate, formed exchange and non-exchange.Wherein, DNA synthesis is a requisite link in meiotic recombination.Existing research has been found that the significant components of some participation semiconservative DNA replication processes is also for reduction division is required.A gene that take part in replication initiation in people and yeast saccharomyces cerevisiae cDC45also be S phase DNA replication dna institute required (Stevens et al. 2004) before reduction division in Arabidopis thaliana; It is same as the initial required minichromosome maintenance protein 2-7(Mini-chromosome maintenance of DNA replication dna, mCMhomologous gene 2-7) mCM8also knownly in people and Xenopus laevis take part in DNA replication dna (Maiorano et al. 2005; Volkening et al. 2005), nearest report indicates mCM8be similarly the reduction division institute required (Crismani et al. 2013) of Arabidopis thaliana; The research of the archaeal dna polymerase δ being responsible for lagging strand synthesis in DNA replication dna process is shown, the yeast saccharomyces cerevisiae sudden change physical efficiency having lacked most end four residues completes normal mitotic division, but but result in the remarkable decline of meiotic recombination frequency, infer that reason may be take part in reduction division DNA double splitting of chain in yeast to repair (Maloisel et al. 2004); Replication factor C that responsible PCNA is loaded ( rFC1) research find that this gene participates in depending on the formation of the CO of the interference responsive type approach of MSH4, infer that the synthesis of DNA lagging strand plays an important role (Wang et al. 2012) in meiotic homologous regrouping process DNA double splitting of chain reparation.Therefore by finding the non-lethal mutation body of the mitotic division process that do not affect being the required archaeal dna polymerase ε of DNA replication dna equally, we attempt the function that researching DNA polysaccharase ε may exist in reduction division.

Archaeal dna polymerase epsilon(POL ε) be one conservative in eukaryote, and the polysaccharase containing many subunits.It has the basic function of archaeal dna polymerase except as one of three polysaccharases: be responsible for the synthesis of DNA and chromosomally copy.Polysaccharase epsilon also participates in multiple important cell processes, comprises DNA damage reparation, DNA restructuring and suitable adjustment cell cycle progression.And research work in recent years shows, polysaccharase epsilon also participates in Chromatin Remodeling and epigenetic regulation.

Archaeal dna polymerase epsilon, for the formation of replication fork in DNA replication dna process, carries out and stablizes have keying action (Pursell et al. 2008).In yeast, archaeal dna polymerase epsilon comprises a catalytic subunit and three adjustment subunits, and this structure is relatively conservative in other biological.In yeast, the maximum subunit POL2 of archaeal dna polymerase epsilon, is the synthase of catalytic subunit, takes over the synthesis that archaeal dna polymerase alpha completes leading chain DNA in DNA replication dna process.

In Arabidopis thaliana, pOL2Athe catalytic subunit of genes encoding archaeal dna polymerase epsilon.Research finds Arabidopis thaliana pOL2Anon-lethal mutation body surface existing to dormin (ABA) tetchiness, show that archaeal dna polymerase epsilon works in ABA signal transduction; And the somatocyte Homologous recombination frequency of this mutant has the rising of 60 times, also show DNA damage and repair susceptibility enhancing, the activation of gene silencing site, the change of histone modification level etc., demonstrate archaeal dna polymerase epsilon in homologous recombination and epigenetic regulation, play an important role (Yin et al. 2009).

Arabidopis thaliana pOL2Amutant in also show the prolongation of DNA replication dna process S phase, have scientist to point out this double bond may to be caused to rupture increase (Jenik et al. 2009) that (DSB) formed.And one of producing cause of DSB is exactly the cross exchanged that Meiosis produces, the mode of topmost reparation DSB is exactly homologous recombination, there are some researches show that archaeal dna polymerase epsilon participates in homologous recombination process, therefore the effect of archaeal dna polymerase epsilon in homologous recombination and mechanism need further research.

In fission yeast, the catalytic subunit Cdc20(of archaeal dna polymerase epsilon its in the mankind and yeast, there is extensive homology) and Dos2, Rik1, Mms19 form a silencing complex, in heterochromatin, regulate RNA polymerase activity is that DNA replication dna and heterochromatin assembling institute are necessary.Heterochromatic assembling needs to carry out in the process of S phase, and research shows in plant and animal, and heterochromatic silence needs DNA replication dna and archaeal dna polymerase epsilon (Li et al. 2011).

The central role of archaeal dna polymerase epsilon is to participate in DNA synthesis and chromosomally copies, and ensure that the formation of replication fork and stablizes.And it plays an important role in heterochromatin formation, gene silencing, homologous recombination, DNA damage reparation etc., correctly the carrying out of these processes is absolutely necessary for eukaryote Tempo of Meiosis, what ensure that Eukaryotic genome and genetic information can be stable entails filial generation, therefore archaeal dna polymerase epsilon is participated in the research of meiotic homologous recombination mechanism, contribute to resolving plant meiotic homologous regrouping process further, for crop breeding is provided fundamental basis.At present, relative dna polysaccharase epsilon is study hotspot in homologous recombination and epigenetic regulation, its concrete regulatory mechanism, and the conservative property between different plant species all needs to be studied further.

Summary of the invention

The object of the invention is to by the function of research Arabidopis thaliana POL2A in meiotic recombination, and utilize its functional performance, learn a skill in conjunction with modern biotechnology, on purpose the recombination frequency of directed change crop, to shorten the process of Crop Genetic Breeding.

The present invention utilizes known Arabidopis thaliana to participate in encoding DNA polymerase epsilon(POL ε) gene of maximum catalytic subunit pOL2A(AT1G08260).This full length gene 15949bp(SEQ ID NO.1), cDNA coding region sequence long 6486bp(SEQ ID NO.2), this genes encoding 2161 amino acid (SEQ ID NO.3).By comprising the comparison of people, mouse, rat, fruit bat, nematode, yeast 6 species POL2 aminoacid sequences to Arabidopis thaliana and other, determine pOL2Asome conserved domains (Figure 1B), hold C to hold from N and be respectively: nitrogen terminal domains, 3 '-5 ' 5 prime excision enzyme activity structural domain, 5 '-3 ' polymerase activity structural domain, central catalytic active domain, PCNA binding domains and Zinc finger domain.

The present invention also provides pol2a-1, pol2a-2with pol2a-3the primer of three mutant gene type qualifications, as shown in SEQ ID NO.4-SEQ ID NO.13, is specially: mutant pol2a-1genotype identification primer (genome band): sEQ ID NO.4 and SEQ ID NO.5,mutant pol2a-1genotype identification primer (T-DNA inserts band): sEQ ID NO.6 and SEQ ID NO.7,mutant pol2a-2point mutation sequencing primer: sEQ ID NO.8 and SEQ ID NO.9,mutant pol2a-3genotype identification primer (genome band): sEQ ID NO.10 and SEQ ID NO.11,mutant pol2a-3genotype identification primer (T-DNA inserts band): sEQ ID NO.12 and SEQ ID NO.13.

Three above-mentioned mutant: pol2a-1, pol2a-2, pol2a-3,being thered is provided by Arabidopis thaliana Biological Resource Center (ABRC, the U.S.), is Arabidopis thaliana pOL2Athree mutant materials, be respectively: SALK_096341C, CS6947 and SALK_124217.Wherein pol2a-1with pol2a-2mutational site exists pOL2An holds, and has all been positioned at 3 '-5 ' conservative region after 5 prime excision enzyme activity structural domain is that nitrogen distal process becomes and the non-lethal weak mutant (Fig. 2 A) that isozygotys. pol2a-3be the T-DNA insertion mutation of a mutated site on carbon teminal PCNA binding domains, genotype identification, less than the material that isozygotys, is isozygoty caused by embryonic death.That is: two kinds of non-lethal weak mutant that isozygoty: T-DNA insertion mutation body pol2a-1(insertion point is positioned at the 12nd exon) and Arabidopis thaliana pOL2Athe Point mutont of gene pol2a-2(mutational site is positioned at the 469th amino acid); A kind of T-DNA insertion mutation body of the embryonic death that isozygotys pol2a-3(insertion point is positioned at the 32nd intron).

The invention still further relates to Arabidopis thaliana pOL2Athe weak mutant of gene pol2a-1with pol2a-2plant phenotype and male meiosis abnormal, specifically by observation and the cytologic experiment of plant, as mature flower powder Alexandria red colouring, the dyeing of tetrad magenta and exhibition sheet method are observed the experiments such as mutant pollen mother cells process and are determined pol2a-1with pol2a-2abnormal Circumstances In Meiosis phenotype.

Specifically, pol2a-1with pol2a-2the phenotype of plant, shows as compared with wild-type: nourish and grow and do not have significant difference, but syngenesis phase mutant compares wild-type shows difference in obvious fertility: fruit pod is short; Petal is tall and thin, and filigree short (Fig. 2), flower pesticide pollen amount significantly reduces (Fig. 3 A). pol2a-1with pol2a-2the male meiosis phenotype of plant, showed as: from leptotene stage to pachytene stage compared with wild-type, and mutant and wild-type do not have notable difference.Diplotene stage, early stage joint conference removed, and form the cellular form similar to zygotene stage, so far mutant does not find any obvious exception yet.Synaptonemal complex completely dissolve after stain colour solid starts to concentrate, and forms form diplotene stage of classical " parallel wire---cross knot " between homologous chromosomes.But at majority pol2a-1with pol2a-2in cell, concentrated interchromosomal seems to there is more contact, makes karyomit(e) as the knitting wool be carelessly wound around, is difficult to recognize homologous chromosomes.After wild type chromosomal enters into the diakinesis stage, the bivalent of 5 pairs of homologous chromosomess formation of the clear symmetry of visible form, and in some cell of mutant, although can judge to have entered the diakinesis stage from concentrated depth, but interchromosomal still exists the contact except cross knot, complete 5 can not be formed to independent bivalent; Structure asymmetrical " quasibivalent " is formed in some cell; Also have in some cells and can not form bivalent---denumerable karyomit(e) (to) number is more than 5, may monomer be there is in hint.To I in mid-term, the contact between the most endocellular chromosome of mutant is still visible, causes being separated asynchronous by homologous chromosomes after spindle fibre tractive, occurs obvious chromosome bridge and fragment to later stage I.In earlier stage II, the chromosome fragment be scattered everywhere in visible cell.Mid-term II sepn process in still produce a large amount of chromosome fragment, cause latter stage II to be formed more than the nucleus of 4, after reduction division, produce many splits, finally to grow for abortive pollen grain (Fig. 3).

The present invention is also provided and is detected by Real Time PCR pol2a-1with pol2a-2in in reduction division process pOL2Athe method of genetic expression.Concrete steps are: get the inflorescence that mutant is not bloomed, and with TRIzol method extracting paddy rice RNA, and use DNaseI DNA digestion, and then reverse transcription forms cDNA, carries out quantitative fluorescent PCR analyze with it for template.Fluorescence quantification PCR primer nucleotide sequence as shown in SEQ ID NO.14 ~ SEQ ID NO.27, wherein, Real Time PCR internal reference primer: sEQ ID NO.14 and SEQ ID NO.15,real Time PCR detects pol2a-1with pol2a-2in in reduction division process pOL2Athe PCR primer of genetic expression: sEQ ID NO.16 ~ SEQ ID NO.27(i.e. P1-P 6).Detected result shows pol2a-1actual is on a molecular scale one pOL2Athe weak sudden change that transcriptional level have dropped; And pol2a-2transcriptional level compare wild-type and significantly increase, can infer that it is a point mutation (Fig. 4) causing POL2A function incompleteness.

The present invention also provides pol2a-1/pol2a-3trans zygote, RNAi interfere pOL2express transgenic line and pOL2A, pOL2Btwo two vegetable materials isozygotied of copy.The male meiosis of each material and the phenotype (Fig. 5) of pollen granule activity are provided simultaneously.Wherein, utilization is provided dMC1the special interference of Meiosis of promoters driven pOL2 _rNAicarrier construction and transgenic line.Be specially: find to exist in Arabidopis thaliana by sequence alignment pOL2Bgene with pOL2Asimilarity reaches 84%, considers pOL2A, POL2Btwo genes may functional redundancy in reduction division, have selected one section of nearly N end duplicate about 400bp base sequence in two genes and is building up to as target sequence and contains dMC1in the RNAi carrier of promotor, carry out Agrobacterium-mediated Transformation, thus obtain the special POL2 afunction mutant material of Meiosis, amplimer nucleotides sequence be classified as SEQ ID NO.28 and sEQ ID NO.29.

The present invention also provides the experiment by fluorescence in situ hybridization, utilizes digoxigenin labeled to be positioned at arabidopsis gene group chromosome long arm near-end grain end BAC F1N21, thus determines pol2a-1the method of interaction between chromosomes situation in mutant.And provide pol2a-1the changing conditions (Fig. 6) of this BAC signal in mutant, and then be provided in pol2a-1the interactional evidence of nonhomologous chromosome is there is in mutant.

The present invention also provides one to utilize immunofluorescence experiment to observe mutant pol2a-1the method that in mutant, can synaptonemal complex correctly be formed.Exist particular by ASY1 and ZYP1 albumen pol2a-1in mutant, location and wild-type compare, and result demonstrates pOL2Aaffect homologous chromosomes pairing but not joint conference (Fig. 7).

The present invention also provides one to utilize immunofluorescence experiment to observe mutant pol2a-1the location of γ-H2AX and RAD51 and change in mutant, thus determine whether POL2A sudden change have impact on the method (Fig. 8) of meiotic recombination process double center chain fracture restoration, and result shows pol2a-1the formation of mutant double-strand break is normal, but double-strand break reparation there occurs retardation, thus infers pOL2Asudden change may cause the reduction of DNA combined coefficient.

The present invention also provide a kind of by statistics wild-type and pol2a-1mutant at the initial rear different time points reduction division different times cell number of reduction division, thus is analyzed and is obtained mutant pollen mother cells process and occur retardation, and DSB remediation efficiency reduces the method (Fig. 8 C) of this inference.

The present invention also provides one to utilize hereditary path analysis pOL2Athe method of mechanism of action in Arabidopis thaliana reduction division process.Specifically by by some key genes of upstream and downstream in reduction division unknown mutation body and known reduction division process (as sPO11, RAD51, MUS81deng) hybridization of mutant, analyze the reduction division phenotype of two material that isozygotys and the similarities and differences (Fig. 9) of the parental phenotypes that isozygotys in F2 colony, can from hereditary angle judges unknown gene the link of function.The invention provides simultaneously pol2a-1mutant respectively with spo11-1-1, rad51-3, rfc1-2, rck-1, mus81the reduction division phenotype of the vegetable material that mutant hybridization obtains and each double-mutant, concrete phenotypic results is: 1. pol2a-1spo11-1-1two mutant phenotype is similar to spo11-1-1, explanation pOL2Agenetics position be positioned at sPO11-1the downstream of effect; 2. pol2a-1rad51-3two sudden change has similar rad51-3single prominent phenotype, illustrates equally pOL2Agenetics position be positioned at rAD51downstream; 3. pol2a-1rfc1-2two sudden change, does not find that obvious phenotype increases the weight of trend, close with rfc1-2, hint pOL2Aparticipate in the DNA that in type I restructuring approach, DSB repairs and synthesize link.4. pol2a-1rck-1double-mutant occurs similar pol2a-1middle nonhomologous chromosome interacts, explanation pOL2Aparticipating in DNA synthesis that DSB in type I restructuring repairs will be early than rCKthe link of expansion D-loop; 5. pol2a-1mus81-1there is the nonhomologous chromosome winding phenotype more serious than two kinds of single mutants in double-mutant, shows pOL2Afunctional independence in mUS81the type II restructuring approach participated in, and act on type I restructuring approach.

The present invention also provides a kind of method of immunofluorescence that utilizes to observe MLH1 albumen at diakinesis stage wild-type and mutant pol2a-1middle location, and then infer pOL2Awhether affect the method for type I regrouping process.Concrete observations is pol2a-1middle MLH1 protein localization reduces, and illustrates which reducing type I recombinates (Figure 10).

The present invention also provides one to utilize fluorescence labels site system (Fluorescent Tag Loci System, FTL), and the pollen granule number combined by the fluorescence that statistics offspring tetrad is different, can count the recombination frequency between three kinds of fluorescent marks, thus research pOL2Aaffect the method (Figure 10) of meiotic recombination frequency or distribution mechanism.

Based on foregoing, the invention provides the method determining POL2A concrete function in meiotic recombination, plant Arabidopsis thaliana is example in mode, and concrete steps are as follows:

(1) because POL2A is the required gene of mitotic division, disappearance causes embryonic death completely, and conventional route is difficult to study its function in reduction division.A kind of approach obtains corresponding weak mutant; Need structure one by the RNAi carrier of the promoters driven of reduction division specifically expressing in addition, and obtain positive transgenic plant;

(2) use the regular growth biology techniques described in literary composition, analyze the activity of pollen granule, the dyeing of quadrantal magenta and exhibition sheet method and observe mutant pollen mother cells process etc., determine the maiotic concrete link of this effect gene;

(3) by chromosome fluorescence in-situ hybridization, the interaction event that POL2A gene institute affects homologous chromosomes is analyzed, as matched.Immunofluorescence experiment analyzes location and the change of the various albumen in the reduction division process that may participate in mutant, determines the function of this gene in reduction division further;

(4) by the hybridization between multimutation body, analyze this gene and do mutually from intergenic heredity in different genetic recombination passage, as sPO11, RAD51, MUS81deng, after two material that isozygotys compares with the reduction division phenotype of respective single mutant, determine the position of this gene in path;

(5) fluorescence labels site system (Fluorescent Tag Loci System is utilized, FTL), by the pollen granule number that the fluorescence that statistics offspring tetrad is different combines, calculate the recombination frequency between several fluorescent mark, thus analyze this effect gene meiotic recombination frequency or distribution situation;

(6) according to existing result of study, in conjunction with the concrete biochemical function of this known gene, the concrete function of this gene in reduction division is comprehensively drawn.

The present invention, by research, determines the function of Arabidopis thaliana POL2A in meiotic recombination.

In the inventive method:

Obtain corresponding weak mutant described in step (1), be aforesaid pol2a-1, pol2a-2, pol2a-3.

Described in step (1), structure one is by the RNAi carrier of the promoters driven of reduction division specifically expressing, and obtains positive transgenic plant; Comprise: vitro detection pol2a-1with pol2a-2in in reduction division process pOL2Agenetic expression; Utilize dMC1the special interference of Meiosis of promoters driven pOL2 ^rNAicarrier construction and transgenic line.

Step uses regular growth biology techniques described in (2), analyze the activity of pollen granule, the dyeing of quadrantal magenta and exhibition sheet method and observe mutant pollen mother cells process, determine the maiotic concrete link of this effect gene, comprise Arabidopis thaliana pOL2Athe weak mutant of gene pol2a-1with pol2a-2plant phenotype and male meiosis abnormal, specifically by observation and the cytologic experiment of plant, determine pol2a-1with pol2a-2abnormal Circumstances In Meiosis phenotype.

The operation of step (3) comprising: the experiment utilizing fluorescence in situ hybridization, determines pol2a-1interaction between chromosomes situation in mutant; Immunofluorescence experiment is utilized to observe mutant pol2a-1in mutant, can synaptonemal complex correctly be formed; Immunofluorescence experiment is utilized to observe mutant pol2a-1the location of γ-H2AX and RAD51 and change in mutant, thus determine whether POL2A sudden change have impact on meiotic recombination process double center chain fracture restoration; The method of immunofluorescence is utilized to observe MLH1 albumen at diakinesis stage wild-type and mutant pol2a-1middle location, and then determine pOL2Awhether responsive type regrouping process is interfered in impact.

The operation of step (4) comprising: utilize hereditary path analysis pOL2Amechanism of action in Arabidopis thaliana reduction division process, especially by the hybridization by some key gene mutant of upstream and downstream in reduction division unknown mutation body and known reduction division process, analyze the reduction division phenotype of two material that isozygotys and the similarities and differences of the parental phenotypes that isozygotys in F2 colony.

According to foregoing, the present invention summarizes archaeal dna polymerase epsilon and participates in reduction division type I(and interfere responsive type to exchange) the concrete mode of regrouping process.Specifically as shown in figure 11: in wild-type, after forming double bond fracture by SPO11 shearing in reduction division process, between homologous chromosomes, strand invasion is mediated under the effect of the recombinase that RAD51 is correlated with, DSB reparation is carried out in type I regrouping process, wherein be responsible for the synthesis of a complete leading strand by POL ε, carried out the synthesis of the some Okazaki fragments of lagging strand by POL δ after, connect into complete chain by DNA ligase.And in POL ε mutant (pol2a-1, pol2a-2), due to POL ε afunction or weaken, carrying out type I regrouping process be, if two are interfered responsive exchange site more close, then replace POL ε to carry out short chain DNA synthesis by POL δ, complete DSB and repair (Figure 11 C); And if it is far away to exchange site gathering, POL δ cannot complete the synthesis of long segment, then can be completed DSB and be repaired by type II (interfering non-sensitive type to exchange) path, at this time need the DNA fragmentation synthesized will be relatively short, can have been replaced by POL δ (Figure 11 D); ? pOL εalso also exist in mutant and correctly can not identify homologous chromosomes, after DNB is formed, DNA single chain intrudes between nonhomologous chromosome, to cause in mutant interactional situation between nonhomologous chromosome, correctly can not complete DSB to repair, thus result in fertility reduction (Figure 11 E).

In the present invention, relate to other genes some and albumen: sPO11-1the effect of mediation cutting double-strand, formation DSB; rAD51be positioned at sPO11-1downstream, can in conjunction with strand mediation strand invasion; rFC1participate in the formation depending on the COs of the interference responsive type approach of MSH4, in DNA double splitting of chain reparation, participate in the synthesis of DNA lagging strand; rCKbe considered to yeast genes in Arabidopis thaliana mER3homologous gene, mER3as a helicase, the of service in yeast understands that its function is that expansion D-loop makes to produce the formation that more heteroduplex promotes dHJ, and the responsive restructuring approach of interference relied on by ZMM is required; mUS81mediate the restructuring of the non-sensitive type II type of interference; ASY1 and ZYP is lateral member and the axial member that Arabidopis thaliana participates in being formed synaptonemal complex respectively, and in wild-type, the Chromatin condensation that ASY1 participates in starting leptotene stage becomes the chromosomal process of visible wire, locates altogether with becoming the chromosome segment of fine rule; Overlap completely to ASY1 zygotene stage with karyomit(e), ZYP1 accumulates between the homologous chromosomes of pairing, and impel two fine rule homologous chromosomess of pairing to form the thick line of joint conference, ZYP1 locates altogether with becoming the chromosome segment of thick line; To the complete joint conference of pachytene chromosome, ZYP1 signal overlaps completely with karyomit(e), and ASY1 dissociates from karyomit(e) and enters kytoplasm, becomes disperse shape (Fig. 7 A); γ-H2AX is a kind of form that H2AX has been phosphorylated rear, is positioned on the nucleosome near double-strand break position, as a kind of signal for locating, plays a part to recruit and repairs complex body enforcement reparation double-strand break.

Arabidopis thaliana in the present invention pOL2Agene is very conservative in eukaryote, it is all a single copy gene, show that its function in reduction division is relatively conservative, according to the function of Arabidopis thaliana POL2A provided by the invention in reduction division, biotechnology can be passed through, accelerate the polymerization of good character in farm crop crossover process, shorten the seed selection process of farm crop good species.Concrete steps are as follows:

(1) at farm crop (as paddy rice, corn etc.), find pOL2Ahomologous gene, build containing reduction division specific promoter dMC1the RNAi interference vector driven;

(2) obtain corresponding RNAi transgenic line by genetic transformation, after Screening and Identification, preserve (this transgenic line is exactly the sterile line of artificial creation).By hybridizing with other strains, follow-up POL2A can change the feature of restructuring distribution drawn game portion frequency, the cold-zone of recombinating under normal condition can be allowed to exchange, reach the polymerization accelerating good character, thus subtract the process of breeding and planting;

(3) this gene of overexpression may increase recombination frequency, and therefore the transfer-gen plant of overexpression increases the output of crop possibly.

Existing research only shows that archaeal dna polymerase ε (POL2A) has critical function in mitotic division, and its function in eukaryote reduction division is set forth by the present invention.The present invention demonstrates the critical function of POL2A at meiotic recombination first time in eukaryote.The process that process due to variety of crops seed selection is actual is genetic material restructuring, optimize and select.Use modern biotechnology ways and means, in conjunction with the feature that POL2A gene pairs recombination frequency and distribution change, by controlling the recombination frequency of genetic material in reduction division process, reach the polymerization accelerating good character, slow down the separation of good character in hybrid generation, reach the level advancing and improve breeding research, serve the needs of human being's production life better.

Accompanying drawing explanation

Fig. 1. pOL2Amutated site and sequential analysis.

Fig. 2. pol2a-1, pol2a-2mutant plants form and fertility.

Fig. 3. pol2a-1, pol2a-2mutant plants male meiosis phenotype.

Fig. 4. pOL2Amutant expression analysis.

Fig. 5. wild-type, pol2a-1, pol2a-1pol2b, POL _rNAi chromosome morphology and flower pesticide fertility in transgenic line and trans zygote.

Fig. 6 .BAC FISH indicates pol2a-1in mutant, nonhomologous chromosome interacts.

Fig. 7. pachytene stage ASY1 and ZYP1 in wild-type and pol2a-1in location.

Fig. 8. in wild-type and mutant meiosis prophase I to mid-term I each period the change to 13 o'clock proportions in 10 o'clock.

Fig. 9. mutant genetics analysis.

Figure 10. the impact that POL2A interferes restructuring.

Figure 11. POL ε participates in reduction division type I(and interferes responsive type to exchange) the model of regrouping process.

Figure 12. the observation figure of A type and G type Homo~logous exchange and schematic diagram during fluorescence labels site system I2 combines.

Figure 13. the fluorescence tetrad pollen system of meiotic recombination frequency.

Embodiment

Following examples for illustration of the present invention, but are not used for limiting the scope of the invention; The experimental technique of unreceipted actual conditions, all conveniently condition in embodiment.

the plantation of embodiment 1 Arabidopis thaliana, hybridization, conversion and genotype identification

1. the plantation of Arabidopis thaliana

(75% ethanol, 30s after the sterilization of Arabidopis thaliana seed-coat; Aseptic washing three times, 60s/ time); Solid 1/2 MS substratum is seeded under aseptic condition.Cultivate in illumination box (22 DEG C, 16h illumination/8h is dark); The intermediate house (Fig. 2) that (grows two panels true leaf) after about 10 days.

2. the hybridization of Arabidopis thaliana

First plant good for genotype identification is removed the flower showed money or valuables one carries unintentionally, spend hero by three of outermost.One, to two days later, observes the column cap of eunuchs, if column cap presents the suede hair of white, and has glistening liquid materials to occur, proves that now column cap is applicable to pollination.The pollen of male parent is spread upon on column cap, can see that column cap has yellow powdery substance, to be on the safe side, pollination within second day, can be repeated once, should water a plant immediately after pollination enough, be conducive to plant recovery.Can see that after three days fruit pod extends, fruit pod base portion such as to darken at the phenomenon.After the flavescence of fruit pod, single fruit pod sowing.

In order to study pOL2Athe function played in meiotic recombination path, by inciting somebody to action pOL2Athe hybridization of some key genes (Fig. 9) of upstream and downstream in mutant and reduction division process, analyzes the reduction division phenotype of two material that isozygotys and the similarities and differences of the parental phenotypes that isozygotys in F2 colony, can from hereditary angle judges unknown gene the link of function.

3. the conversion of Arabidopis thaliana

Arabidopis thaliana entered the florescence after transplanting in about two to three weeks, treated that it is just bloomed, and when forming three to four fruit pods, namely can be used for transforming.Transform and cut off fruit pod and open flower the day before yesterday, stay and just show money or valuables one carries unintentionally and young tender bud.Conversion process is as follows: the purulence bacillus plate streaking with object plasmid of reincarnate is separated single bacterium colony, choose single bacterium colony in 5 ml YEB substratum (Rif 125 μ g/ml, Kan 50 μ g/ml or Amp 100 μ g/ml) in, 28 DEG C, 220 revs/min of overnight incubation; Then 1:100 is inoculated in the Erlenmeyer flask of 500 ml YEB, 28 DEG C, and 220 revs/min are continued shaking culture 12 about h is OD600 to 1.0-1.2 to stand density; Room temperature, 5000 g are centrifugal, and 15 min collect thalline; Be about 0.8 by conversion medium (0.5 × MS macroelement, the sucrose of 5%, 0.03% Silwet L-77,0.01 mg/L 6-BA, pH 5.8) suspended bacteria volume density to OD600; Conversion medium containing Agrobacterium is poured in 100 ml beakers, the Arabidopis thaliana of just having bloomed is inverted whole inflorescence is all immersed in conversion medium together with the blade of lotus throne base portion, maintain 3 min; Take out Arabidopis thaliana, lain on one's side and be placed on clean plastic pallet, and recover 24 h with the moisturizing of plastic film covering lucifuge; Vertically placed by Arabidopis thaliana, after watering, be normally cultured under being placed on light after growing 5 fruit pods and stop watering, after waiting seed maturity, sowing is sub, removal of impurities, 37 DEG C of oven dryings screenings after a week.

4. the genotype identification of Arabidopis thaliana

Leaves genomic DNA extracts: get the fresh lotus throne leaf of 1-2 sheet Arabidopis thaliana, in centrifuge tube, grind homogenate.Add 400 μ l extraction buffers, concuss.12,000g, after centrifugal 2 min of room temperature, moves to supernatant liquor in another centrifuge tube, adds isopyknic phenol and chloroform, concuss.To leave standstill after 2min 12,000g, centrifugal 10 min.Get upper liquid in a new centrifuge tube, add the Virahol of 70% supernatant volume, mix gently.After-20 DEG C of sedimentation 1h, 12,000g, centrifugal 10 min of room temperature.Abandon supernatant, 75% ethanol washes precipitation once.65 DEG C of baking oven 5min are dried.30-50 μ l distilled water, 4 DEG C of dissolvings are spent the night.

Search the mutant information of genes involved from http://signal.salk.edu/cgi_bin/tdnaexpress/, find interested mutant, order mutant seeds from www.biotech.wisc.edu/Arabidopsis/.Insert information according to the mutant T-DNA that http://signal.salk.edu/cgi_bin/tdnaexpress/ website provides, analyze insertion point and direction.Be method by PCR, qualification T-DNA inserts situation and whether Mutants homozygous.

5. by ripe plant phenotype observation analysis mutant fertility

pol2a-1with pol2a-2mutants homozygous vegetative growth phase lotus throne number of sheets amount, plant height and wild-type Col-0 difference are not obvious; The floral organ type that reproductive growth produces and quantity do not change. pol2a-1isozygoty material under standard temperature, 16 h light/8 h dark culture condition, under stressful environmental, compares the obvious early blossoming of wild-type.Trickle observation pol2a-2visible and the wild-type of plant has minor differences.Difference table is present: 1. lotus throne leaf epidermal hair is not obvious, and vein is shallow; 2. stem is thin.Different in this, syngenesis phase mutant compares wild-type and shows difference in obvious fertility: fruit pod is short; Petal is tall and thin, and filigree is short, and flower pesticide pollen amount significantly reduces (Fig. 2).

the Alexandria red colouring of embodiment 2 mature pollen

Get the inflorescence of Arabidopis thaliana, Kano stationary liquid room temperature is fixedly spent the night.Peel off the flower opened, get flower pesticide jaundice and still uncracked flower, immerse the PCR tubule that the red dye liquor in Alexandria is housed, dyeing time is depending on room temperature (RT>30 DEG C, T<30min; 15 DEG C of <RT<30 DEG C, 2h<T<4h; RT<10 DEG C, T<12h).After having dyeed, strip out flower pesticide, after removing impurity, add a small amount of dye liquor, cover plate, compressing tablet gently, after microscopy, nail varnish mounting ,-20 DEG C of preservations.

The red formula for dye liquor in Alexandria: 95% ethanol 10mL, malachite green (ethanolic soln of 1%) 1 mL, distilled water 50 mL, glycerine 25 mL, phenol 5mL, Chloral Hydrate 0.5g, 1% C.I. 42685 5 mL, 1% orange G 0.5 mL, Glacial acetic acid 1 m L (ALEXANDE., 1969).Cumulative volume constant volume is 100 mL, lucifuge 4 DEG C preservation (please add with said sequence when joining dye liquor, fully mix, otherwise be easy to occur precipitation).

Mutant mature flower powder fertility is observed by Alexandria red colouring.Except mutant flower pesticide is less, more obviously can produce about 500 to dye cherry normal pollen granule different from wild-type, and mutant pollen granule quantity obviously reduces, and produce and be coloured to deep green or brown abortive pollen grain in a large number, wherein pol2a-2abortive pollen ratio higher than pol2a-1, illustrate that male fertile significantly declines (Fig. 3 A) in mutant.

embodiment 3 analysis of tetrad

Get Arabidopis thaliana inflorescence Kano room temperature fixedly to spend the night, tap water 2-3 time, peel off the flower of the flower pesticide of band jaundice, 1-2 the flower getting inflorescence outermost, on slide, drips a small drops of water, after stripping out flower pesticide, removing dregs, then be caught broken flower pesticide gently with tweezers after dripping a water, or with needle point, pollen mother cell is extruded from coyote hole, and be coated on slide, natural air drying, drips a magenta, dyes after 2-3 minute, add cover glass, microscopy.

Different with the normal tetrad of wild-type, visible a large amount of many splits (Fig. 3 B) in mutant, wherein pol2a-1with pol2a-2the ratio producing many splits is respectively: 36% and 49.6%(Fig. 3 C).Right pol2a-1the abnormal tetrad of middle generation is classified, and we find that majority exists with abnormal ratio tetrad and five separate form, and both respectively account for about four one-tenth; Seven splits can be observed at most.The existence of many splits has implied the exception of mutant male meiosis process.

embodiment 4 is opened up sheet method and is observed mutant pollen mother cells process exception situation

Observe the method Primary Reference of the Arabidopis thaliana pollen mother cells process article of Ross by exhibition sheet method, slightly change, concrete grammar following (Ross and Fransz et al., 1996).Get the inflorescence of Arabidopis thaliana, Kano stationary liquid (Glacial acetic acid: dehydrated alcohol=1:3, V/V) room temperature is fixedly spent the night.Abandon the flower pesticide of jaundice, by remaining flower pesticide enzymolysis (enzymolysis solution: 0.3% cellulase+0.3% pectinase+0.5% cytohelicase, prepare with 0.01M citrate buffer solution, pH=4.5,-20 DEG C of Refrigerator stores after tubule packing), 37 DEG C, after 80-90min, 0.01M citrate buffer solution cleaning flower 3-5 time, for subsequent use.Slide drips a clear water, add the flower that 3-5 is in Meiosis, strip out flower pesticide, after dregs except removing flower pesticide, retain about 10 μ l water, flower pesticide is crushed with tip tweezers, pollen mother cell is discharged, and slide is put in the thermal station of 45 DEG C, on slide only remaining water membrane time, drip 60% Glacial acetic acid 10 μ l to moisture film district, after 30 seconds, drip 10 μ l 60% Glacial acetic acid again to moisture film district, and blow and beat solution with rifle head, after 1-3min, one-20 DEG C pre-cold Kano stationary liquids are dripped directly over moisture film district, extend piece, and slide is dried in thermal station.Often open slide and drip 6-8 μ l DAPI, after cover plate, lucifuge dyeing 10min, microscopy, takes pictures.

Observe pol2a-1with pol2a-2the form in pollen mother cells each period.From leptotene stage to pachytene stage, mutant and wild-type do not have notable difference.Diplotene stage, early stage joint conference removed, and formed the cellular form similar to zygotene stage, so far mutant visible any exception not yet.Synaptonemal complex completely dissolve after stain colour solid starts to concentrate, and forms form diplotene stage of classical " parallel wire---cross knot " between homologous chromosomes.But at majority pol2a-1with pol2a-2in cell, find that mutant cells began to interact to the visible obviously nonhomologous chromosome of I in mid-term from diplotene stage, produce fragment after isolation.Finally after reduction division, produce many splits, final growth is abortive pollen grain (Fig. 3 D).

embodiment 5 real-time quantitative fluorescence PCR detects expression conditions in mutant

1. the extraction of RNA: get Arabidopis thaliana col-0, pol2a-1with pol2a-2the inflorescence of not blooming of plant, powdery is ground to form with after liquid nitrogen freezing in mortar, add the EP pipe filling 1 ml TRIZOL reagent (Promega), after abundant vibration, room temperature places 5 minutes, in 4 DEG C, after the centrifugal 10min of 12000 rpm, supernatant liquor moves in new EP pipe, adds that 200 μ l chloroforms blend together emulsus, and room temperature leaves standstill 5 minutes, after the centrifugal 10min of 12000 rpm, supernatant liquor moves in new EP pipe, adds two volumes isopropanol precipitating RNA, dissolve rear 37 DEG C with DNaseI(Fermentas) digest degrade residual DNA 1 hour.Then on spectrophotometer, rna content is measured.

2. reverse transcription: according to the PrimeScript of TaKaRa company ^tMreverse Transcriptase specification sheets operates: add rice total RNA 5 μ l (5 μ g) in centrifuge tube, Oligo (dT) ₁₇primer(50 μm of ol/L) 1 μ l, dNTP(10 mmol/L) 1 μ l, and add to 10 μ l with distilled water; Brief centrifugation makes liquid concentrate at the bottom of pipe, and 65 DEG C of insulation 5 min, rapidly at cooled on ice 2 more than min.5 × PrimeScript is added in said mixture ^tMbuffer 4 μ l, RNase inhibitor (40 U/ μ l) 0.5 μ l, PrimeScript ^tMreverse Transcriptase (200 U/ μ l) 0.5 μ l, distilled water adds to 20 μ l, and the centrifugal liquid that makes concentrates at the bottom of pipe, and 42 DEG C of insulation 1 h, 70 DEG C of deactivation 15 min, cooled on ice, obtains Arabidopis thaliana cDNA.

3. Real Time PCR

Because this full length gene cDNA 6486bp(SEQ ID is NO.2), in order to detect accurately, we mutated site front end, the previous exon of mutated site, across sudden change, mutated site after an exon and mutated site rear end have chosen 6 Position Design primers (Fig. 4 A) altogether.By analyze we to find in pol2a-1 POL2A transcriptional level before on position with wild-type indifference, but significantly to lower after on position; Pol2a-2 transcription level compares wild-type significantly to be increased (Fig. 4 B).This result consistent with the result that the transcript profile that we analyze pol2a-1 and pol2a-2 flower development the 9th phase flower pesticide obtains (Fig. 4 C).The pol2a-1 actual weak sudden change being a POL2A transcriptional level and have dropped on a molecular scale that we analyze is described; Pol2a-2 is then a point mutation causing POL2A function incompleteness.

embodiment 6 vector construction and transgenic plant screening

1. the clone of cDNA and structure

According to the sequence information provided in Arabidopis thaliana database information in Tair, design primer, amplified the cDNA of goal gene by KOD plus DNA polymerase, be connected on pEASY-T1 Simple carrier after PCR primer is purified, verified by order-checking.

2. transgenic plant screening

The above-mentioned whole vector GV3101 successfully constructed, transforms object plant after checking, obtains positive transgenic plant, and analyze it by screening.

The 1/2MS substratum of screening culture medium to be concentration be 25 mg/L Totomycin.

embodiment 7 Fluorescence in situ hybridization (FISH)

By with Arabidopis thaliana total genomic dna for template, the tumor-necrosis factor glycoproteins of kinetochore 180bp is cloned, and is loaded on pGEM-T carrier, by order-checking carry out verifying (Kato and Lamb et al., 2004).The sequence of telomere also by after as above method clone, is loaded into pAtT4 carrier and is undertaken verifying (Richards et al. 1988) by order-checking.The method (Cuadrado et al. 1994) of the carrier reference Cuadrado of 5SrDNA and 45S rDNA.BAC clone (F19K16 and F1N21) orders in TAIR.Because telomere and kinetochore are tumor-necrosis factor glycoproteinss, signal is stronger, therefore is directly used by the probe of synthesis with fluorescence.5SrDNA and 45S rDNA and BAC is by passing through after nick translation test kit synthesising probing needle, and Anti-DIG-rhodamine Fab fragments is as the two anti-digoxin signals be used in detection probes.Concrete grammar, with reference to the article of Han2009, is slightly amended as follows (Han and Gao et al., 2009).Carry out pre-treatment to the slide that the way by karyomit(e) exhibition sheet makes, the deionized formamide 100 μ l of 70% often opens slide, covers sealed membrane, wet box, 95 DEG C of sex change 5-10min.Immerse 3min in the ethanol of 70% of-20 DEG C of precoolings immediately, then immerse 100% ethanol 3min, natural air drying.Proceed as follows under lucifuge condition, dilute kinetochore (1:200) or telomere (1:50) probe with hybridization solution, the probe after 20 μ l dilute often opens slide, and after covered, wet box, lucifuge, hatch 2-6h for 37 DEG C, 2 × SSC cleans 3 times, each 3min.Lucifuge, after natural air drying, 8 μ lDAPI redye, and observe after 10min.The probe way of 5SrDNA with 45S rDNA and BAC is different from kinetochore and telomere.Karyomit(e) exhibition sheet is after sex change pre-treatment natural air drying, and by PCR instrument, to the probe sex change of dilution, (5SrDNA and 45S rDNA hybridization solution 1:40 dilutes; BAC hybridization solution 1:20 dilutes), after 85 DEG C of 5min, immerse iceberg mixture immediately, after cooling, 10 μ l often open slide, and after covered, 37 DEG C of wet boxes hatch 8-12h, and 2 × SSC cleans 3 times, each 3min.Following operation needs lucifuge, and by hybridization solution dilution two anti-(1:10), 10 μ l often open slide, and after covered, the wet colour developing of 37 DEG C, box 2h, 2 × SSC clean 3 times, each 3min, lucifuge, and after natural air drying, 8 μ lDAPI redye, and observe after 10min.If background is deep, suitably can reduces the concentration of probe and strengthen the intensity of cleaning.Probe signals is too weak, can extend the time of slide sex change and improve the concentration of probe.Observed by Zeiss fluorescent microscope, taken pictures by the software Zeiss Axio Imager A2 carried, camera model is AxioCam MRc Rev. 3 FireWire (D).

We marked by digoxin and are positioned at arabidopsis gene group chromosome long arm near-end grain end BAC F1N21, by the method for fluorescence in situ hybridization, observe the change of F1N21 signal in mutant.4 F1N21 signals are always had in wild-type meiotic cell---pair of homologous karyomit(e), and often couple of homologous chromosomes Inner is respectively containing a pair sister chromatid; But because the sister chromosome fibroin (conhesin) that just gets adhered after DNA replication dna completes is held together, so under fluorescent microscope, a pair sister chromosome only visible signal before not being separated.In wild-type meiotic cell, leptotene stage and F1N21 region karyomit(e) also unpaired zygotene stage visible two signals; Pachytene stage homologous chromosomes pairing, joint conference, only a surplus signal; After diplotene stage to mid-term I according to restructuring occur position whether in F1N21 accessory signal quantity between 1 and 2---recombinable site can only see a signal close to F1N21, far then visible two signals; Adhesin albumen dissociates from sister chromatid in subtrahend first division process, makes from later stage I to II in mid-term, and visible 4 signals are in pairs with chromosomal change; Sister Chromosome Separation Correlative after later stage II, four signals lay respectively at four Ge He districts.

? pol2a-1in, leptotene stage to zygotene stage, the change of F1N21 signal was similar to wild-type.But in the pachytene stage, 22.6 %'s pol2a-1two signals (Fig. 6) can be observed in cell (n=31); At I in mid-term, 53.1% pol2a-1cell (n=32) obviously visible two signals not on a bivalent (Fig. 6), illustrate in mutant comprise F1N21 region karyomit(e) near there occurs and the abnormal interaction such as the joint conference of nonhomologous chromosome.

embodiment 8 immunofluorescence

Prepare before experiment: slide glass is soaked overnight in washing lotion (70 ml dehydrated alcohol+2 ml dense HCl+28 ml H2O), wears gloves, uses tap water 30min.For subsequent use in distilled water rinse 3 times, 95% ethanol rinse, twice rear immersion dehydrated alcohol.Slide is after spirit lamp burns, and naturally cooling, after masking foil parcel, 180 DEG C are dried 3h.After slide glass cooling, add 6 ul poly-l-lysines (1mg/ml) in slide one end.With cover glass (chloroform soak after 30min 180 DEG C dry 5h) while contact drop, carry out smear.The formation of " iris " should be observed during smear.If (do not form moisture film, then show that slide glass does not have wash clean, must heavily wash.) put into 45 DEG C after smear hot plate on dried overnight (the shortest can 4h), then put into section box, treated slide glass should use in one week.Cover glass: after soaking 15min with chloroform before using, stink cupboard is air-dry, for subsequent use.

Enzymolysis solution: 3% cellulase+3% pectinase+0.5% cytohelicase, dissolves with the citrate buffer solution of 0.01M, pH=4.5, tubule packing ,-20 DEG C of preservations.Stationary liquid: 1 × PBS solution+0.1% Triton X-100 of 2% paraformaldehyde, pH=7.4,4 DEG C of preservations, were finished in one week.Scavenging solution: Wash buffer I: 1 × PBS+1%Triton X-100, Wash buffer II:1 × PBS+0.1%Tween 20; Confining liquid: Block buffer:1 × PBS+5%BSA+0.1%Tween 20+1mMEDTA.

Flower before 8 phases of getting Arabidopis thaliana ,-0.1MPa, fix at the bottom of flower that 15min(fixes should sink to bottle under room temperature, venting of repeatedly bleeding can improve fixed efficiency) after, with citrate buffer solution rinsing 3 times, each 3min.Flower is proceeded to enzymolysis solution, 37 DEG C, enzymolysis 120min, citrate buffer solution rinsing 3 times, each 3min, preserve the good flower of enzymolysis for 4 DEG C.Flower pesticide is stripped out in little culture dish, the flower pesticide of removing jaundice, after flower pesticide being transferred to slide glass with pipettor, retain 10 μ l water, be caught broken flower pesticide with pincet, pollen mother cell is discharged, covered, pushing down cornerwise two angles of cover glass respectively with thumb and forefinger makes slide glass and cover glass relative displacement not occur, glass rod with gentle beat cover glass, karyomit(e) is discharged from cell.Slide is immersed in liquid nitrogen after 1min, take out slide, provoke cover glass rapidly with double-edged razor blade, slide glass natural air drying.

Slide glass is immersed wash buffer I, room temperature, 60min.Slide glass is immersed block buffer, 37 DEG C of 30min.Block buffer dilutes primary antibodie, often open slide drip 200 μ l dilute after primary antibodie, sealed membrane mounting, 4 DEG C spend the night (ASY1 and RAD51 Dilution ratio is 1:100, ZYP1 Dilution ratio be 1:200, tubulin Dilution ratio be 1:400).Slide glass is immersed block buffer, room temperature, 15min, wash 3 times.Slide glass is immersed block buffer, room temperature 60min.Block buffer dilutes two and resists, often open slide drip 200 μ l dilute after two to resist, sealed membrane mounting, 37 DEG C, 60min (antibody in the green skies press 1:300 and diluted, and the antibody dilution ratio of invitrogen is 1:500).Slide glass is immersed washing buffer II, room temperature, 15min, wash 3-6 time.Blot unnecessary water with thieving paper, often open slide and drip 8 μ l DAPI, microscopy after lucifuge dyeing 10min.

Arabidopis thaliana synaptonemal complex forms primarily of lateral member ASY1 and axial member ZYP1.In order to verify the abnormal chromosome effect in mutant, we observe the location of ASY1 and ZYP1 in mutant before and after joint conference by the method for immunofluorescence.In wild-type, the Chromatin condensation that ASY1 participates in starting leptotene stage becomes the chromosomal process of visible wire, locates altogether with becoming the chromosome segment of fine rule; Overlap completely to ASY1 zygotene stage with karyomit(e), ZYP1 accumulates between the homologous chromosomes of pairing, and impel two fine rule homologous chromosomess of pairing to form the thick line of joint conference, ZYP1 locates altogether with becoming the chromosome segment of thick line; To the complete joint conference of pachytene chromosome, ZYP1 signal overlaps completely with karyomit(e), and ASY1 dissociates from karyomit(e) and enters kytoplasm, becomes disperse shape; On karyomit(e), ZYP1 signal disintegrates along with synaptonemal complex after the pachytene stage and dissociates.Observe mutant pol2a-1the dynamic change of leptotene stage, zygotene stage and pachytene stage two kinds of albumen, does not find their loading and dissociates and the difference (Fig. 7) of wild-type.

In order to check pOL2Awhether sudden change can affect the DNA synthesis of meiotic recombination process double center chain fracture restoration (DSBR) link.We adopt the method for immunofluorescence to observe location and the change of γ-H2AX.γ-H2AX is a kind of form that H2AX has been phosphorylated rear, is positioned on the nucleosome near double-strand break position, as a kind of signal for locating, plays a part to recruit and repairs complex body enforcement reparation double-strand break.Therefore DSB can be indicated with γ-H2AX.DSB is formed from fine rule late period is under the effect of SPO11-1, arrives peak, along with the carrying out of repairing, repair completely to thick line later stage DSB to the early stage DSB quantity of amphitene.γ-H2AX signal is synchronous with it.? pol2a-1zygotene stage, γ-H2AX about has 185 ± 16 (n=32) individual signal, signal indifference (Fig. 3-12) individual with 189 ± 26 in wild-type (n=39); After entering the pachytene stage, in wild-type signal rapid drawdown to 57 ± 12 (n=55), but pol2a-1in still residual 113 ± 27 (n=19) signal as seen.Simultaneously, we also have detected the location of RAD51 in these two periods.RAD51 is a recombinase, and the strand that topmost function is considered to wherein one end after double-strand break in reduction division is combined, and the homologous chromosomal segments near the invasion of mediation strand produces heteroduplex, thus exercises the effect of follow-up reparation.Traditional viewpoint thinks that RAD51 take part in the reparation of all DSB, thus RAD51 number of signals and γ-H2AX similar, be later than again the appearance of the latter and dissociate.RAD51 signal in wild-type and pol2a-1amphitene to can be observed 188 ± 24 (n=15) and 187 ± 11 (n=14) period, there is no significant difference; And in pachytene stage wild-type, be only left 51 ± 14 (n=70), and still reach the signaling point of 87 ± 15 (n=5) as seen in mutant.Describe mutant pol2a-1in: the formation of double-strand break is normal; But double-strand break reparation there occurs retardation.Reason may be pOL2Asudden change causes the reduction (Fig. 8) of DNA combined coefficient.

In Arabidopis thaliana diakinesis stage cell, MLH1 overlaps with cross knots most in cytological structure and indicates the quantity of type I CO.Therefore by observing pol2a-1the number of signals of middle diakinesis stage MLH1 and understanding pOL2Amiddle type I approach CO number, in result display mutant, the corresponding wild-type of MLH1 signal reduces, explanation pol2a-1the restructuring approach (Figure 10 D, E) of type I is decreased in mutant.

the detection of embodiment 9 pollen mother cell Homologous recombination frequency

We analyze the frequency of pollen mother cell homologous recombination mainly based on help (Francis and Lam et al., 2007 of the visible system of a set of pollen; Berchowitz and Copenhaver, 2008).Ultimate principle is as follows: first build fluorescent mark strain (FTL, fluorescent-tagged line), a series ofly drives different fluorescent protein expression by postmeiotic specific expression promoter LAT52 by building qrtthe transfer-gen plant of background, screening is positioned the different fluorescently-labeled transfer-gen plant of the chromosomal band of same, and (transfer-gen plant is with different fluorescence protein genes, and fluorescence protein gene specifically expressing in tetrad, therefore, the tetrad combined with different fluorescence can be observed. qrtbackground the maiotic product of Arabidopis thaliana pollen mother cell---tetrad is enclosed in together, can not separate, can conveniently observe and add up).Because homologous chromosomes in reduction division process is separated, nonhomologous chromosome independent assortment, marker gene directly can be observed and be obtained in pollen.If occurring between mark to exchange or do not have exchange to observe obtains.For the I3 that we use, No. three karyomit(e)s there are three different loci in this strain respectively with CFP, YFP and RFP fluorescent mark, as shown in (Figure 12), the reduction division process sister strand of this plant of heterozygosis is all with fluorescent mark, and homologous chromosomes does not mark, if do not exchange between this section homologous chromosomes, as shown in A type, have two pollen granules to have three kinds of fluorescence in same tetrad, two other does not have fluorescence; If there are four line double exchanges of G type, then can form only redly with an one only bluish pollen in same tetrad, and one with red and green pollen, and one with blue and green pollen.Combining because different exchanges always has in 12, by adding up the number of the pollen granule of the different combination of offspring, the recombination frequency between these three fluorescent marks can be calculated, the physical location between these marks can be known by technology such as Tail-PCR.

atpol2a-1can produce the active pollen grain of about 2/3, this makes us can by based on the frequency of the visual analysis system Analysis for CO s of pollen.By inciting somebody to action atpol2a-1isozygoty with fluorescence sites qrtthe detection system of background hybridizes, by change (Figure 13 B) the quantitative predication mutant recombination frequency of fluorescence in observation band fluorescence tetrad pollen.Wherein I2a, I2b, I3a, I5c and I5d are 5 regions (Figure 13 A) be positioned on No. 2, No. 3 and No. 5 chromosome arms defined by the different fluorescin in both sides.Observe these area discovers except I3a, pol2a-1in other section recombination frequency significantly increase, and to observe pol2a-1in lack meiotic recombination frequency in the tetrad of a pollen granule, find that I2b, I3a and I5d section and wild-type do not have difference; I2a section recombination frequency significantly increases; And recombination frequency significantly declines in I5c.Therefore pOL2Asudden change changes meiotic recombination frequency (Figure 10 A, B, C) really.

<110> Fudan University

The function of <120> Arabidopis thaliana POL2A gene in meiotic recombination and application thereof

<130> 001

<160> 29

<170> PatentIn version 3.3

 

<210> 1

<211> 15949

<212> DNA

<213> archaeal dna polymerase epsilon(AT1G08260) full-length gene 1198bp

 

<400> 1

atgagcggag ataatcgaag acgggatcga aaggatacgc ggtggtcgaa gaagcctaag 60

 

gtagtgaaca ctgcggaaga tgaactcgaa tctaaactcg gatttgggct tttctctgaa 120

 

ggtgaaaccc gacttgggtg gttactcaca ttctcatcgg taagttctcc gacactctaa 180

 

tctggactca accttgttaa ttgagagtat ttgagtaggt ttcggattcg attatatgtg 240

 

aatttctgta aattaaatat aagcattggc gactgaacca tacaatgaaa ctccattagt 300

 

gcgagaattt aggattctag gtaatgtcaa agtactagta tttactgatt ggataatcta 360

 

ctacgttttg cagtcatctt gggaagatcg cgatactggg aaagtctata gttgtgtgga 420

 

cctttacttt gttactcagg tgagctgcta taattatggt cattactcat tactatgtca 480

 

gcttcgtata agttgatgaa cgattatatt tttgttgttg catttttcta aatagttaac 540

 

tgagttgagg atttttttca ggacggcttt tctttcaaga caaagtacaa gttccgtcct 600

 

tacttttatg cagccacgaa agtatgatag tttctctctg gacattatcg gaattctcct 660

 

tgaatcattc atgtattttc attttatatg tcaataaact gcaatgccaa attacaggac 720

 

aaaatggaat tggaactcga agcctacttg agacgccgat atgaaagaca agttgctgat 780

 

attgagattg ttgaaaaaga agatcttgat cttgtaagtc agtaaagcat actatgatta 840

 

ttattagttt aagtctgact tcctcattgt gtgatgtagt tgactagtgt cttccaccag 900

 

ttgattaatg ttgtttctct actcatatca gaaatatttg ctacaacctt tagttgttat 960

 

cctcttcttg taacccaaaa ctgaacattt gtagtaaata gtccaaaagt agtcttgaat 1020

 

ccattaggtg aaatcaatag gcgtttgcta gtatgtaaca tgggctgtag atgttaaagt 1080

 

tagttttctt taagtgttta ttgattgtcc tttagtgcaa ttgtatgcaa gatatgaagg 1140

 

tttcagtact ttttagttat caattcgctt aggatgtctt tgtagctttt ttctgtgata 1200

 

cacaattttg agttccttta ttttatgttc atgagcagaa aaatcatcta tctggcctgc 1260

 

aaaaaaaata cctaaaaata tcatttgata ctgtgcaaca attgatggaa gtcaagcggg 1320

 

atttgttaca tattgtcgaa cgaaaccagg caaaatttga tgcacttgaa gcatacgaat 1380

 

ccatattggc tggaaaacgg taagacatgt aattttgttc ttttaaatca cttaagagtt 1440

 

ttgttgattt tcaattttta cctcttaacc taagttgttt cttagttatt tttcaagttt 1500

 

atttttaagg gtttcagatg tcctgatcat ttttatcttt attcttggaa cgcagagaac 1560

 

aacgtcctca agattgctta gactctatcg tggacctccg cgagtacgat gtaccttatc 1620

 

atgttcgatt tgcaattgat aatggtatct tgtttagtca aaccagaatt tctgtttagt 1680

 

cctcaaaatt gtcaaaatca tgatgttttc ttggtgtcca gatgtgcgga gtgggcaatg 1740

 

gtacaatgtc agtatttcca gcaccgatgt catattggag aagaggacag atcttcttca 1800

 

acgcgcagaa gtccgtgttt gtgccttcga tatagagaca acaaagcttc cgttgaagtt 1860

 

tcctgatgct gaatatgatc aaattatgat gatatcctac atggtcgatg ggcaaggttt 1920

 

tctaattatt aacagagagg taactgctgt atacatttgt ctgtattgga tctgatattg 1980

 

taatgatgat tttttacatg gtggatgggg attgttaatt cattaattta gagaacctac 2040

 

tatatggcct tgctttgcca tttttgtaga aatgatccat ataagattat cccgcttgtt 2100

 

taattgattt cgtatgatgt aactttaaga ctgtttttgg actagcataa cattttgcta 2160

 

taatttaaaa cataaattct gatatgttaa ccgttttttg ttgcatatat catctctgtt 2220

 

tgatatctca gctctatgct ttccttatat attttgtact actgcaatct tgtttgatta 2280

 

ttaatgtagc atctttgcac ttaattcagt gtgttggcga agatgtagaa gatctggaat 2340

 

atacaccaaa accagaattt gaagggtatt tcaaagttac taatgttaaa aatgaggtaa 2400

 

aatacttaag ctcctgattt gaatataaaa tcagaaacat tgatgtgcag gtgagttatt 2460

 

tcttggttat ttcttgtagg ttgaactcct ccaaagatgg ttttaccata tgcaagagtt 2520

 

gaaacccggt atttatgtta catacaacgg tgactttttt gactggccat ttatagaacg 2580

 

gagagcatct catcatggga taaaaatgaa tgaggtatgc taaagtttta ttcgttgtgg 2640

 

ttttgaatta attttatctg tctgcatgac taaacggata taatatatct acttggaaag 2700

 

tgtatataaa agtttgtgac attattcgct gacattctat agtggccttg tgagtgtgcc 2760

 

tctgcataaa gtagaatagt gacgctgctc aagtacttcc tagactgtgt actgaattat 2820

 

cttgcacgac gatgcttact tttacatatt attgtttcct tgtggaagat aaattttcat 2880

 

ccttcaaaat attcagtgac ttgtgttttg catttcaata ctgaagtctt ctgagagttg 2940

 

gcaaggtgct gccactcatt ctcccggaac ttggttctct tctcagttct gtttaaacct 3000

 

atctttcttg ctttttcatt taaagcctag ttcgttgcat cttactcgtc acttcctatc 3060

 

ctatttctac tatcttccac tggagcataa tggtcaactg taattccata attggctggt 3120

 

gtgttttgtt tctttccctt gtggttcagg aattgggatt ccgttgtgat cagaatcaag 3180

 

gtgaatgtcg agctaaattc gcttgtcatt tggattgttt tgcttgggtt aaacgtgata 3240

 

gctatcttcc tcagggaagc catggtctga aggttcattc tacttctata tctgagcgat 3300

 

tatttatctt ttgtggtcat ataatatttt gttaagaaag tcatattatg attttgatgc 3360

 

ttgtttctta attaaatgcc aggctgttac aaaggcaaaa ctaggttatg atccactgga 3420

 

agtgaaccca gaggacatgg ttcggtttgc gatggaaaag ccccaggtat tgacgagaag 3480

 

ttaatcctgc tatttctgta aaacatgttt ttttaaccat tagagctggt tgttagatac 3540

 

tagaatgggc tagagttaca aactgtcgat gataagtgat caagtgattc aactatggct 3600

 

ctttatgggt tgcataaatg ttcttctgtt catctttaat tcattgtctt atcatcagta 3660

 

cacaaaattt agaagcggac atagaattca ggcaattaat ggtgtcaact gtagattttt 3720

 

ctagtctctc tgtagtgctt gaaatatttg tatgggtgct tatatcttat ttctattcaa 3780

 

tgctgggaat catctcgcag acaatggcct cctactctgt ctctgatgct gttgcaactt 3840

 

attacctata catgacatat gtcaatccat tcattttttc tctcgcaact atcatcccaa 3900

 

tggtccctga tgaggtttta cgtaaaggga gtggcaccct ttgtgaaatg cttctgatgg 3960

 

ttgaggtatc aacctattta ccacaaggtt tttagcaatc tttactgtgt gacacattct 4020

 

attaattgga agttttccat tcagcacagg cttacaaggc aaatgttgta tgtcccaaca 4080

 

aaaatcaagc tgaccctgag aaattctacc aaaatcaact tcttgaaagt gagacatata 4140

 

ttggtggtca tgtagagtgc cttgaaagtg gtgttttcag atctgacatt ccaaccagtt 4200

 

ttaaactcga ttcatcggca taccaggcaa tttatctgct ttatatttat tcactataat 4260

 

gtttgttgat ttgtttaatt gattgctgag cattactttt tctgatagag tttcaattca 4320

 

attgtcactg atttgtctgt agtttcctct gaagtgctgc agtttatagt aaaatctatg 4380

 

tagtctgtaa tgaatgtgtg aagcaaaatg cttcccattc ttgtgtgcat gattgtatca 4440

 

tagctaatta atgcacctat ctgatatttg acgctttgat tgccagcaac tgatcgataa 4500

 

ccttggtcgt gatctggaat atgctatcac tgtagaagga aaaatgagaa tggattcaat 4560

 

ttccaattat gatgaagtga aggacgaaat caaggagaag gtaatgaatt tctcttctga 4620

 

ataaaaatat tgatttaatg tttgttccct tgtttcttgt cagcatgttg ttactcttgt 4680

 

cgctttgtat ggctgacact gcatacacat tcatactgaa agatttttct tatacactca 4740

 

tacatgctta gctcgtatct atgagcctta tctcatgtag agtaatccca ttagggcttg 4800

 

tggaagaaca ttagttagtc cttagactac tgtttgagcc tccggaggaa atctgtctgt 4860

 

caagtatcct ctatcatagt tgttatcaac tatcatctgc atgcttggat attttaaagc 4920

 

agaatttata acctgatcgt cttgtgaatt atctcccttt atctttgatt cgggttgaag 4980

 

tcttagttct gtcgtggttt ggcggtttta ttgattttgt agcttgagaa gttacgagat 5040

 

gaccctatac gggaagaggg gcctcttatt tatcaccttg atgtcgcagc catgtatcca 5100

 

aatatcatct taacaaacag gcttcaggta catctaatct ctgtttgcat gtttctaact 5160

 

gatttaggga ttatatgttg tagcatgcga ttgtaaatca atatctacca aacatcagct 5220

 

aattggtgat gttccatata caaaataatc acaaccgtga cttttacgat tgtgaacccc 5280

 

ctagatatat tatgttaagc gaaagtttcc tctaaattct cagccaccat caatagttac 5340

 

ggatgagatt tgcacagcat gtgatttcaa ccgcccggga aagacctgtc ttagaaaact 5400

 

tgaatgggtt tggcgtggtg tgacattcat gggaaagaaa aggttcttaa catatatatc 5460

 

attcctaact ttggatttaa accttgttgg tatagtctta agttctcaac tttgtttcaa 5520

 

atggtttgca gtgattatta ccatttgaag aagcagattg agtctgaatt tgttgatgct 5580

 

ggtgcaaata taatgtcttc aaaatcgttc cttgatctgc caaaggtgga tcaacaatct 5640

 

aagctaaaag aacgactaaa aaaatattgt caaaaggtac tctctctcca ccgtagtaat 5700

 

attttcccca caatgtcaat atataatggc aagttttctg ctattggtag gcttataaac 5760

 

gagtacttga caagccaatt actgaagttc gtgaagctgg gatatgcatg agagaaaacc 5820

 

ctttctatgt ggatactgtt cggaggtgcg tttcttgatt aagactctat ttgtcgttta 5880

 

gtcttttggg aagcatctct cttgggtgaa catacaactg ctgataatga aacatttaga 5940

 

caagtactaa ttactttttc aaattgcttt ttatttggtg gtctttgtat gttattaaag 6000

 

ctttcgagat aggaggtatg aatacaaaac acttaacaag gtctggaagg gaaaattgtc 6060

 

agaggccaag gcaagtggta attcaatcaa gatccaggaa gcacaggtaa gttatcgttt 6120

 

atacttgctc atatatatga ttttcagaat ttcttttaga tggttattga cattaaaatt 6180

 

tggaacagga catggtagtg gtttacgatt ccttgcagct agctcataag tgtatactta 6240

 

attccttcta tggatatgtc atgcgaaagt gagtcctctc ttacaaagag cctggaattt 6300

 

ggtgttatta tcattgtctg ttccaattat gttgacccaa agctttactt aaaactaatt 6360

 

ccaggggtgc aagatggtat tccatggaaa tggctggtgt agttacctat actggagcca 6420

 

aaatcataca gaatgcccgt ttgcttattg agcgaattgg gaaaccactt gagttggata 6480

 

cagatggtat atggtgttgt ctccctggat cttttcctga gaactttact tttaaaacaa 6540

 

tgtaagattt tgtaatctcc atgatttaca tcattacatg tatatgttag gaatttcctt 6600

 

gttctgtttt ttttttttaa tatatagcat ttcactctaa ggtatttcca agaggcgttt 6660

 

tatttccttg tttaattaag gtggattaca ttttcttgtg gttctagaga tgttaataaa 6720

 

ccatcaatgg ttttcattat tgcagagaca tgaagaaact caccatctcg tatccttgtg 6780

 

ttatgcttaa tgttgatgtg gcaaagaata atacaaatga tcaatatcag gtttgccggc 6840

 

tattccttac ttaaaagtag cttctgtgtt cagtcttaaa taagcagatg atataacaag 6900

 

cccttttgtg gttctgaaag cattttgcct tgtgttcagt ttctgttttt actcttaatt 6960

 

ttggttttgg aacagactct tgtagatcct gtacgcaaga catataaatc acatagtgaa 7020

 

tgctcaattg aatttgaagt agatggaccg tacaaggtat gcttaaacgc tttgaatttt 7080

 

tgacatgtgc tgctctttaa ttgctgcaca aaaaaatatt tgtaaccctc tccattcagg 7140

 

caatgattat tcctgcatcg aaagaagaag gaatcttaat caagaagcgt tatgctgttt 7200

 

tcaatcatga tggaacctta gcagaactca aggggtttga gatcaagcgt agaggggagc 7260

 

tgaaactcat taaagttttc caggtaccct tcccttttct tgtgttgtaa tgattctgat 7320

 

tcataaaaaa atgtttcttt atttattttt aggctcaatg ttattgagtg tatttcgcca 7380

 

gatccactta tactagtaac tctttctagc agcagtttta aggttctaat tctgacgttt 7440

 

ttgtcataat aattctctgt tagggaagat tccaatggtt ctagattgct tgttcaggtg 7500

 

gccatttagc aattattgag agctcatctt ctagctccat tgttcgtaaa agaaagaaat 7560

 

gataaataaa atttttggta tctgacaaaa tgcatgtaac attttttttt aaaatctctg 7620

 

tcatatacat tggattcttg tgtttatgga ctgtgtaatg tagtaagacg ttcataattt 7680

 

gttagacaca agcatcccaa gaaaataggc agacttttac ttgatgaata tgtgtctctg 7740

 

ttgaaaaatt agaggtcagc ttagtttatg aagctgtcag gcgctgaaag aaatcatatt 7800

 

ttctttttct ttcctttccc tacaggccga gctttttgac aaatttctcc atggatcaac 7860

 

tcttgaagag tgttattctg ctgttgcagc ggttgctgat cgatggcttg acctactcga 7920

 

tgtatgtgaa atatcttgtt tcttgttctc ttttagttga ttatttcttt tacttagcag 7980

 

gaagactttt catacaggtg ctgctaaatt ctcttatttg cagttgggtc atttatctca 8040

 

taggttttct ttcacatttt ctccagaatc aaggaaaaga tattgccgat agtgaactgc 8100

 

tagattatat atcagaatca agcacaatga gcaaatcgtt agcagattat ggtgagcaaa 8160

 

agtcatgtgc agtgaccaca gcaaaacgcc tggctgaatt tcttggtgtt acaatggtca 8220

 

aagataaagg actacgttgc cagtacattg ttgcttgtga accaaaggta catttctttt 8280

 

aaacttgaaa tttctcgaca cttttgtgtt ttttcctcct ttgacatctg ctcaaggagt 8340

 

actagaagtt ggttactttg aaaggtatct ccaacccgaa catagcaggg aaaaccaaag 8400

 

agttaggtct catgtctgat tcacatatct tctttcatcc ttactgatat atttattagt 8460

 

acaagtaaca cataagtgga tgttgtacta acaatcttta ctgttgaagt tcttagttag 8520

 

gaacactgct gatatgtaca tgttccctta gttaagaatg ttgctcacat ctaaaaagta 8580

 

gttactgtcc gttaagcatc tttctcagct agtctagtat agtgtctcca tcacactgct 8640

 

tattagtaac tttttactgt tacgacttaa caacttttca tgattagggt acaccggtaa 8700

 

gcgaacgtgc tgttcctgtt gccatattta caacaaatcc gggtaaacat tctgttattt 8760

 

ggtgtgatgt attgttgcat tcttaacgct catatgtgat gtaaatcttc aaaattaaca 8820

 

gaagttatga agtttcattt gcgaaaatgg tgcaagacat catcagatgt cggaattcgt 8880

 

ctgattattg actggtcgta ttacaagcag cgccttagtt cagctatcca aaaagttatt 8940

 

accattcctg ctgcaatgca gaaggttcat tattactatt cttatccttt ttttaaaaag 9000

 

atttccttag tgtaattttt atatatatct aacgacttgt atgacctcta taggtggcaa 9060

 

atcccgtgcc tagggtgctt catcctgact ggctccataa aaaggtccgt gagaaagacg 9120

 

acaaatttcg ccagagaaaa ctagttgata tgttcagctc agcaaataaa gatgttgtgt 9180

 

tggacacaga tcttccggtg actaaagata atgtggaaga tattgaggat ttttgcaaag 9240

 

aaaataggcc ttccgtcaaa gggccaaagc caattgcgcg ttcctatgaa gtcaataaaa 9300

 

aacagtctga atgtgagcag caagagagct gggatacaga gttccatgat atctcatttc 9360

 

agaacattga caagagcgta aactaccaag gatggcttga actgaaaaag agaaaatgga 9420

 

aagtaactct agagaagaag aaaaaacgaa ggtaggtttt cataatggta tttgttcacc 9480

 

tgaagtttat tatacagcaa gacggttttg atttggaatg atatatagtt ccatataaca 9540

 

tctattgttt ttacaagtgt ctgaacttgt tgactgtatc ccttcgtttt gtaggttggg 9600

 

tgatctaaga tcttcaaacc aggttgatac tcacgagata aatcaaaaag tcggtcaggg 9660

 

aagaggaggt gtgggttcat actttaggag gcctgaagaa gctttgacta gttcacattg 9720

 

gcaggtgtgc acagtgacct atttgtattt atatatttgg ctattttttt gcaaccatat 9780

 

aaagctgtct ttccgacctt ttcagataat acagctggtc ccaagtccac agagcggaca 9840

 

attttttgct tgggtggttg tggaagggtt gatgcttaag atcccattgt cgatcccaag 9900

 

ggtgttttac attaattcca aagttcctat agatgaatat ttccaaggaa agtgtgtcaa 9960

 

caagattctt cctcatggaa ggccttgtta tagtttgact gaggcatgtg ttttaacctc 10020

 

tatacatctt tccccttgta ttccttctag ttagttacaa ttttctcgtt tctttacagg 10080

 

tcaagattca agaagatcaa tttaaaaaag aaagtaaaaa gcgtgcagct cttcttgcag 10140

 

accctggagt tgaggtgagg tttttataaa gtagaaaaag aaattgccat cacatgcctt 10200

 

gactatttaa aaacttttct tagtctagtt aactattatt tatgtgattt ctggtaccat 10260

 

tttgttttag tgagtagaca acttgtgtat ggtgtctggg agtacatgat atctgcctat 10320

 

ctgagtttga atttgtctgt tgtaaaactt acttcatcaa gtaccctgtg taaggaaaac 10380

 

taaatggagt tcacaagtga agggtagcta aattcatctc agttatgtgc gtctgtgatg 10440

 

gctctccatg ctgtcttgtt tgttaatttt attaaaatgt gaatgggatc caacgacttg 10500

 

taagcttggt gggagttatg cttttgttct aatgcttact gagacactag ccagataatc 10560

 

accaggcgaa cttgttggtt atcatgcatg ttgttacctt catttttcat aaagggaaat 10620

 

accatgttca tgcttggtag tctcatattt gaccatggca gggcatatat gaaactaagg 10680

 

tgcctctgga gtttagtgct atatgtcaga ttggatgtgt atgcaaaatt gataacaaag 10740

 

ccaagcaccg taacacccaa gatggatggg aagttggtga acttcacatg aaaactacaa 10800

 

ctgagtgtca ctacttgaag cgttcaattc cacttgttta cttgtataac aggtagagct 10860

 

ctctaagtaa catgactatc ccttttctga tagaaaccag ttctacttca tggagtttaa 10920

 

gttgaaggaa ggttttgatt gtaaacttat aataggcatt tcttagtcac aatgttagag 10980

 

tgtttacatt tctacgtttc tagtcatatt attctaagaa ccataatagg gtttctgtcg 11040

 

cttttcaaat tagtcccttt tacattttga actttttttc ttcttctttg ggatttatat 11100

 

ggtttcaatt atccaagccg gaatatctta tctcatggtt tacttgtatg cagcacctca 11160

 

acagggcgtg ctatatatgt tttatattgt catgtatcga agcttatgtc tgccgttgta 11220

 

gttgatcctt ttaacggcaa tgaattatta ccatctgcct tagagagaca gtttagagat 11280

 

agttgcctag aattatcact tgattcattg tcttgggatg gtatccgttt ccaggtaagc 11340

 

tctaaattca tcgcatacaa cccctgcagt aacagtcctg cattttttga gcttttgtga 11400

 

atgctaataa tcaggtgcac tacgttgacc atcccgaagc tgccaagaag ataatacaaa 11460

 

gagcaataag tgaatacagg taactcattt ttctgattat agaagtgtag ccagtttaat 11520

 

gtttccttga catgttgaat cccatagtgt ttctattgct gatcagttgc tcatgctttt 11580

 

tcctgtttaa ttttttcctt tgtgaactag ttggttactt cctaattttt tttttttgac 11640

 

attcagttgg ttacttccta atgaaataaa ttctatacgt taattggcaa aaaaaagaaa 11700

 

aaaaaaagaa gtgattgtct ggtgtgatca tggcggattt tggacttttt ttgagaggtc 11760

 

atgtcaggtc atgccactct ggtttgactt gttccatttt taccttttct tgcagagaag 11820

 

aaaattgtgg accaactgtt gctgtcattg aatgccctga ctttaccttc atgaaggaag 11880

 

gtattaaggc cctagatgat ttcccatgtg tgaggattcc cttcaacgac gatgacaata 11940

 

gttatcaggt tccttgacta ataaaccaat tggctacttt ttctcctttt gtcatgatag 12000

 

acgattattt gataagaaat atgtatgtgg gtttggttgc aatgcgtagc ctgtctcctg 12060

 

gcaacgtcca gctgcaaaaa tagcaatgtt ccgttgtgct gcagcatttc agtggttgga 12120

 

tcggaggatt acccaatcaa gatacgccca tgtaggttat gtttctttag tggatatata 12180

 

gttcctcata tcaatacttt tttgcagtta ttgacatttc ctgtacaact gactaggtgc 12240

 

ctctggggaa ttttggactt gattggctaa cattcacaat agatatcttc ctatccaggg 12300

 

cactccgtga ccagcaacag gttctttctt cgaattttcg tctactttct aatactttta 12360

 

atagttccaa tgttacgcca tatgctttct ttttctttct ttttttcctc gctctctttt 12420

 

tttctgtttg cctttttgct tttgctctgt gtgtgcatct tcactgattg tgattttttc 12480

 

tttttttcct tttcttttag aagtgcatat tatgtattta gaaagagaag agcagctttt 12540

 

ccatatcact gaattttctt ttgtataaca ggtcctttgg gtgtcagata atggtgttcc 12600

 

agaccttgga ggtataaaca atgaagaggc attctttgcc gatgaggtat gtataacagg 12660

 

tttttttttg ccttttggtt tctctggact ttgtcatgat cttctcccat ctccatgttt 12720

 

tttttttttt gtgctactgt cgaggtgata aactgacatg cttctttgac acaggtgcaa 12780

 

cagacaagtt tagtattccc tggagcgtat agaaaagtgt ctgtggagtt aaaggtcata 12840

 

aaaaaaaaat tacctaatca tcagtgcttt tttggatgtt gttctaatta acatgcaatg 12900

 

tgcagatcca taacttggct gttaatgccc ttctgaaaag caacctggtg aatgaaatgg 12960

 

aaggaggcgg ttttatggga tttgaacagg atgtgaatcc tagaggaatt aattcaaacg 13020

 

ataacaccag ttttgatgag accactggat gtgctcaagc atttcgtgtc ctgaaacagt 13080

 

tgattcatag ttgcttgact gatgtgcgta aatctaaaaa tatatatgct gattccattt 13140

 

tgcaacgttt gtcttggtgg ctctgcaggt atgattatgt cttgtaacat attcatttta 13200

 

ttcttgactc ataaaatacc aatctataat ttcatattgt agcccttcct cgaagctcca 13260

 

tgatccagct cttcatctca tgcttcacaa ggtagatcat atttaatgct ttttgctctt 13320

 

tttttatcag gtctaaatga gtaagtgtgg ataaatcagc aaactttata ttaattcctc 13380

 

cttgcttttc tgtctgaatc tttcccaatc aattcattta tgggatctgt agcttacaga 13440

 

ttatatctcc ttgccggatt atacctgtca taatatatat aattcccaag gcaccttcag 13500

 

atcttgagat ttgccagtta cactcagttt ttatttgcct taagaattct aaaccaattt 13560

 

gacttcctac atttttcgtc tatttgcagg tcatgcaaaa ggtgtttgca ctgctattga 13620

 

ctgatttgcg gagattaggt gctataataa tatatgcaga cttctcaaag gtcattatcg 13680

 

acacagtgaa atttgatcta tctgcagcta aggcttattg tgaaagcttg ctatcaacgg 13740

 

tgcggaatag gttggtttct tccttgctcg atatacatgt ctatgacctt aagtctcatg 13800

 

gactgcactc attttcctgc catgattttt gtgcttcgcc tctgacagtg atatctttga 13860

 

gtggatattg ctcgagccgg ttcactactg gcactcatta cttttcatgg accaggttgg 13920

 

aattcttttt tcttatactt tccttttgta tttttaatct ctgattgtta aacctggcaa 13980

 

aacttgttgt aaattgtaaa attccaactc ctcaacgaga gagaaaatga aaaagtgaca 14040

 

atgcatgcta ttccagtaaa accagaatgg gccaaggaat tataactgat gagtagaacc 14100

 

ctttcacctt gatggttaat aaaattatag aaccaattta ccatcattag tttttgttta 14160

 

atcaggtttc ttgatcttgc cttttctcat atttttcctt cccattgagg tatctctttt 14220

 

cttctacatt ttagtataac tacgctggta tccgagccga tgatgaaatt tcacttgacg 14280

 

aagtgactat tgaaccaaag tggagtgttg cacgacattt acctgagtat attgaggttt 14340

 

ggagacagaa ccctcactca aaccaatgtt catttagtga aaacaaaaaa catgtggcaa 14400

 

cacatggacc atttttcttg gacactgacg catttcatct atcttggcag agggatttca 14460

 

ttataatcat tgccaagttt atatttgacc cctggaaatt tgcgatagaa aataaaaagg 14520

 

gcagttcaga gagtttagag gcacagatga tagaatatct aagagaacag gtagcatatt 14580

 

aaagccaatt cgcttgatct acttttcatc aatcaaaaga ttgtctcctc atttcttttt 14640

 

tgttttcttt tatgcagatt ggatcaacct tcataaatat gcttgtcaaa aaggttgatg 14700

 

atatcatgtc acacatgaag gagataaatg tttcagatgc atctcgggtt tctggtcaag 14760

 

ctcccaaagg agattactcg ttggaattta tacaggttat ctctgctgtt ctggctcttg 14820

 

accaaaacgt ccagcaagat gttctggtaa ttactcccat tctttttcat tttaacatat 14880

 

ggcgtattca caagtcttct gaatagatta atcttgaata tgaaaagtcc actgaagcgt 14940

 

tgtttatctt tacagttaaa aaccctcttt ataactgatt tcattcacaa atccaatctt 15000

 

atctctggaa atgtggttct tgaaaccatg tttaccaata gagctggatt tttttttgaa 15060

 

tgggttttaa cgatagtctt ctcaattttt gtcctctaga cctacaatct tgctgattta 15120

 

atctcttttc tataggtaat gagaaagagt ctgttgaagt acattaaagt aaaagagtgt 15180

 

gctgctgaag ccgaatttct tgatcctggg ccatccttca tcttacctaa tgtagcttgc 15240

 

aggtattatt tgaccaaaac aaatgctcgt gatttaaata agcattcttg tgattggcta 15300

 

cgtctagtaa tagagaaatt gttttccgaa atcgagttgt cttttggcca caaacataaa 15360

 

tccctgcgaa tcccctcctt tagagacatc ctggttcacc cttgtggtga tcatcacatc 15420

 

aaatctacac agcaatatca aacttgcaac actaaagaaa agaaaactta gacccctaaa 15480

 

tcctagacaa taaatacttg acttttttgc agtagtaagc agtaagacag ctggaaatgt 15540

 

atacactgag ttttgattgt actgtttttc ttgttgcagc aactgtgatg cctacagaga 15600

 

cctagatata tgcagagacc cagcactgtt aacagagaaa gaatggtcat gtgcggatac 15660

 

acaatgtggg aagatatatg acagagagca gatggagagt agccttctag agatggtgag 15720

 

acagagagaa agaatgtacc atatgcagga tgtagtgtgc attaggtgca atcaggtgaa 15780

 

agcggctcat ttgacagagc agtgtgagtg ttctggttcc tttagatgca aggagagtgg 15840

 

gtcagagttc agcaagagga tggagatatt catggatata gcaaagcgcc agaagtttag 15900

 

actgttagaa gaatacatct cttggatcat atatggccct agctattaa 15949

 

 

<210> 2

<211> 6486

<212> DNA

<213> archaeal dna polymerase epsilon(AT1G08260) cDNA sequence 6486bp

 

<400> 2

atgagcggag ataatcgaag acgggatcga aaggatacgc ggtggtcgaa gaagcctaag 60

 

gtagtgaaca ctgcggaaga tgaactcgaa tctaaactcg gatttgggct tttctctgaa 120

 

ggtgaaaccc gacttgggtg gttactcaca ttctcatcgt catcttggga agatcgcgat 180

 

actgggaaag tctatagttg tgtggacctt tactttgtta ctcaggacgg cttttctttc 240

 

aagacaaagt acaagttccg tccttacttt tatgcagcca cgaaagacaa aatggaattg 300

 

gaactcgaag cctacttgag acgccgatat gaaagacaag ttgctgatat tgagattgtt 360

 

gaaaaagaag atcttgatct taaaaatcat ctatctggcc tgcaaaaaaa atacctaaaa 420

 

atatcatttg atactgtgca acaattgatg gaagtcaagc gggatttgtt acatattgtc 480

 

gaacgaaacc aggcaaaatt tgatgcactt gaagcatacg aatccatatt ggctggaaaa 540

 

cgagaacaac gtcctcaaga ttgcttagac tctatcgtgg acctccgcga gtacgatgta 600

 

ccttatcatg ttcgatttgc aattgataat gatgtgcgga gtgggcaatg gtacaatgtc 660

 

agtatttcca gcaccgatgt catattggag aagaggacag atcttcttca acgcgcagaa 720

 

gtccgtgttt gtgccttcga tatagagaca acaaagcttc cgttgaagtt tcctgatgct 780

 

gaatatgatc aaattatgat gatatcctac atggtcgatg ggcaaggttt tctaattatt 840

 

aacagagagt gtgttggcga agatgtagaa gatctggaat atacaccaaa accagaattt 900

 

gaagggtatt tcaaagttac taatgttaaa aatgaggttg aactcctcca aagatggttt 960

 

taccatatgc aagagttgaa acccggtatt tatgttacat acaacggtga cttttttgac 1020

 

tggccattta tagaacggag agcatctcat catgggataa aaatgaatga ggaattggga 1080

 

ttccgttgtg atcagaatca aggtgaatgt cgagctaaat tcgcttgtca tttggattgt 1140

 

tttgcttggg ttaaacgtga tagctatctt cctcagggaa gccatggtct gaaggctgtt 1200

 

acaaaggcaa aactaggtta tgatccactg gaagtgaacc cagaggacat ggttcggttt 1260

 

gcgatggaaa agccccagac aatggcctcc tactctgtct ctgatgctgt tgcaacttat 1320

 

tacctataca tgacatatgt caatccattc attttttctc tcgcaactat catcccaatg 1380

 

gtccctgatg aggttttacg taaagggagt ggcacccttt gtgaaatgct tctgatggtt 1440

 

gaggcttaca aggcaaatgt tgtatgtccc aacaaaaatc aagctgaccc tgagaaattc 1500

 

taccaaaatc aacttcttga aagtgagaca tatattggtg gtcatgtaga gtgccttgaa 1560

 

agtggtgttt tcagatctga cattccaacc agttttaaac tcgattcatc ggcataccag 1620

 

caactgatcg ataaccttgg tcgtgatctg gaatatgcta tcactgtaga aggaaaaatg 1680

 

agaatggatt caatttccaa ttatgatgaa gtgaaggacg aaatcaagga gaagcttgag 1740

 

aagttacgag atgaccctat acgggaagag gggcctctta tttatcacct tgatgtcgca 1800

 

gccatgtatc caaatatcat cttaacaaac aggcttcagc caccatcaat agttacggat 1860

 

gagatttgca cagcatgtga tttcaaccgc ccgggaaaga cctgtcttag aaaacttgaa 1920

 

tgggtttggc gtggtgtgac attcatggga aagaaaagtg attattacca tttgaagaag 1980

 

cagattgagt ctgaatttgt tgatgctggt gcaaatataa tgtcttcaaa atcgttcctt 2040

 

gatctgccaa aggtggatca acaatctaag ctaaaagaac gactaaaaaa atattgtcaa 2100

 

aaggcttata aacgagtact tgacaagcca attactgaag ttcgtgaagc tgggatatgc 2160

 

atgagagaaa accctttcta tgtggatact gttcggagct ttcgagatag gaggtatgaa 2220

 

tacaaaacac ttaacaaggt ctggaaggga aaattgtcag aggccaaggc aagtggtaat 2280

 

tcaatcaaga tccaggaagc acaggacatg gtagtggttt acgattcctt gcagctagct 2340

 

cataagtgta tacttaattc cttctatgga tatgtcatgc gaaagggtgc aagatggtat 2400

 

tccatggaaa tggctggtgt agttacctat actggagcca aaatcataca gaatgcccgt 2460

 

ttgcttattg agcgaattgg gaaaccactt gagttggata cagatggtat atggtgttgt 2520

 

ctccctggat cttttcctga gaactttact tttaaaacaa tagacatgaa gaaactcacc 2580

 

atctcgtatc cttgtgttat gcttaatgtt gatgtggcaa agaataatac aaatgatcaa 2640

 

tatcagactc ttgtagatcc tgtacgcaag acatataaat cacatagtga atgctcaatt 2700

 

gaatttgaag tagatggacc gtacaaggca atgattattc ctgcatcgaa agaagaagga 2760

 

atcttaatca agaagcgtta tgctgttttc aatcatgatg gaaccttagc agaactcaag 2820

 

gggtttgaga tcaagcgtag aggggagctg aaactcatta aagttttcca ggccgagctt 2880

 

tttgacaaat ttctccatgg atcaactctt gaagagtgtt attctgctgt tgcagcggtt 2940

 

gctgatcgat ggcttgacct actcgataat caaggaaaag atattgccga tagtgaactg 3000

 

ctagattata tatcagaatc aagcacaatg agcaaatcgt tagcagatta tggtgagcaa 3060

 

aagtcatgtg cagtgaccac agcaaaacgc ctggctgaat ttcttggtgt tacaatggtc 3120

 

aaagataaag gactacgttg ccagtacatt gttgcttgtg aaccaaaggg tacaccggta 3180

 

agcgaacgtg ctgttcctgt tgccatattt acaacaaatc cggaagttat gaagtttcat 3240

 

ttgcgaaaat ggtgcaagac atcatcagat gtcggaattc gtctgattat tgactggtcg 3300

 

tattacaagc agcgccttag ttcagctatc caaaaagtta ttaccattcc tgctgcaatg 3360

 

cagaaggtgg caaatcccgt gcctagggtg cttcatcctg actggctcca taaaaaggtc 3420

 

cgtgagaaag acgacaaatt tcgccagaga aaactagttg atatgttcag ctcagcaaat 3480

 

aaagatgttg tgttggacac agatcttccg gtgactaaag ataatgtgga agatattgag 3540

 

gatttttgca aagaaaatag gccttccgtc aaagggccaa agccaattgc gcgttcctat 3600

 

gaagtcaata aaaaacagtc tgaatgtgag cagcaagaga gctgggatac agagttccat 3660

 

gatatctcat ttcagaacat tgacaagagc gtaaactacc aaggatggct tgaactgaaa 3720

 

aagagaaaat ggaaagtaac tctagagaag aagaaaaaac gaaggttggg tgatctaaga 3780

 

tcttcaaacc aggttgatac tcacgagata aatcaaaaag tcggtcaggg aagaggaggt 3840

 

gtgggttcat actttaggag gcctgaagaa gctttgacta gttcacattg gcagataata 3900

 

cagctggtcc caagtccaca gagcggacaa ttttttgctt gggtggttgt ggaagggttg 3960

 

atgcttaaga tcccattgtc gatcccaagg gtgttttaca ttaattccaa agttcctata 4020

 

gatgaatatt tccaaggaaa gtgtgtcaac aagattcttc ctcatggaag gccttgttat 4080

 

agtttgactg aggtcaagat tcaagaagat caatttaaaa aagaaagtaa aaagcgtgca 4140

 

gctcttcttg cagaccctgg agttgagggc atatatgaaa ctaaggtgcc tctggagttt 4200

 

agtgctatat gtcagattgg atgtgtatgc aaaattgata acaaagccaa gcaccgtaac 4260

 

acccaagatg gatgggaagt tggtgaactt cacatgaaaa ctacaactga gtgtcactac 4320

 

ttgaagcgtt caattccact tgtttacttg tataacagca cctcaacagg gcgtgctata 4380

 

tatgttttat attgtcatgt atcgaagctt atgtctgccg ttgtagttga tccttttaac 4440

 

ggcaatgaat tattaccatc tgccttagag agacagttta gagatagttg cctagaatta 4500

 

tcacttgatt cattgtcttg ggatggtatc cgtttccagg tgcactacgt tgaccatccc 4560

 

gaagctgcca agaagataat acaaagagca ataagtgaat acagagaaga aaattgtgga 4620

 

ccaactgttg ctgtcattga atgccctgac tttaccttca tgaaggaagg tattaaggcc 4680

 

ctagatgatt tcccatgtgt gaggattccc ttcaacgacg atgacaatag ttatcagcct 4740

 

gtctcctggc aacgtccagc tgcaaaaata gcaatgttcc gttgtgctgc agcatttcag 4800

 

tggttggatc ggaggattac ccaatcaaga tacgcccatg tgcctctggg gaattttgga 4860

 

cttgattggc taacattcac aatagatatc ttcctatcca gggcactccg tgaccagcaa 4920

 

caggtccttt gggtgtcaga taatggtgtt ccagaccttg gaggtataaa caatgaagag 4980

 

gcattctttg ccgatgaggt gcaacagaca agtttagtat tccctggagc gtatagaaaa 5040

 

gtgtctgtgg agttaaagat ccataacttg gctgttaatg cccttctgaa aagcaacctg 5100

 

gtgaatgaaa tggaaggagg cggttttatg ggatttgaac aggatgtgaa tcctagagga 5160

 

attaattcaa acgataacac cagttttgat gagaccactg gatgtgctca agcatttcgt 5220

 

gtcctgaaac agttgattca tagttgcttg actgatgtgc gtaaatctaa aaatatatat 5280

 

gctgattcca ttttgcaacg tttgtcttgg tggctctgca gcccttcctc gaagctccat 5340

 

gatccagctc ttcatctcat gcttcacaag gtcatgcaaa aggtgtttgc actgctattg 5400

 

actgatttgc ggagattagg tgctataata atatatgcag acttctcaaa ggtcattatc 5460

 

gacacagtga aatttgatct atctgcagct aaggcttatt gtgaaagctt gctatcaacg 5520

 

gtgcggaata gtgatatctt tgagtggata ttgctcgagc cggttcacta ctggcactca 5580

 

ttacttttca tggaccagta taactacgct ggtatccgag ccgatgatga aatttcactt 5640

 

gacgaagtga ctattgaacc aaagtggagt gttgcacgac atttacctga gtatattgag 5700

 

agggatttca ttataatcat tgccaagttt atatttgacc cctggaaatt tgcgatagaa 5760

 

aataaaaagg gcagttcaga gagtttagag gcacagatga tagaatatct aagagaacag 5820

 

attggatcaa ccttcataaa tatgcttgtc aaaaaggttg atgatatcat gtcacacatg 5880

 

aaggagataa atgtttcaga tgcatctcgg gtttctggtc aagctcccaa aggagattac 5940

 

tcgttggaat ttatacaggt tatctctgct gttctggctc ttgaccaaaa cgtccagcaa 6000

 

gatgttctgg taatgagaaa gagtctgttg aagtacatta aagtaaaaga gtgtgctgct 6060

 

gaagccgaat ttcttgatcc tgggccatcc ttcatcttac ctaatgtagc ttgcagcaac 6120

 

tgtgatgcct acagagacct agatatatgc agagacccag cactgttaac agagaaagaa 6180

 

tggtcatgtg cggatacaca atgtgggaag atatatgaca gagagcagat ggagagtagc 6240

 

cttctagaga tggtgagaca gagagaaaga atgtaccata tgcaggatgt agtgtgcatt 6300

 

aggtgcaatc aggtgaaagc ggctcatttg acagagcagt gtgagtgttc tggttccttt 6360

 

agatgcaagg agagtgggtc agagttcagc aagaggatgg agatattcat ggatatagca 6420

 

aagcgccaga agtttagact gttagaagaa tacatctctt ggatcatata tggccctagc 6480

 

tattaa 6486

 

 

<210> 3

<211> 2000

<212> PRT

The aminoacid sequence 2161aa of <213> archaeal dna polymerase epsilon

 

<400> 3

 

Met Ser Gly Asp Asn Arg Arg Arg Asp Arg Lys Asp Thr Arg Trp Ser

1 5 10 15

Lys Lys Pro Lys Val Val Asn Thr Ala Glu Asp Glu Leu Glu Ser Lys

20 25 30

Leu Gly Phe Gly Leu Phe Ser Glu Gly Glu Thr Arg Leu Gly Trp Leu

35 40 45

Leu Thr Phe Ser Ser Ser Ser Trp Glu Asp Arg Asp Thr Gly Lys Val

50 55 60

Tyr Ser Cys Val Asp Leu Tyr Phe Val Thr Gln Asp Gly Phe Ser Phe

65 70 75 80

Lys Thr Lys Tyr Lys Phe Arg Pro Tyr Phe Tyr Ala Ala Thr Lys Asp

85 90 95

Lys Met Glu Leu Glu Leu Glu Ala Tyr Leu Arg Arg Arg Tyr Glu Arg

100 105 110

Gln Val Ala Asp Ile Glu Ile Val Glu Lys Glu Asp Leu Asp Leu Lys

115 120 125

Asn His Leu Ser Gly Leu Gln Lys Lys Tyr Leu Lys Ile Ser Phe Asp

130 135 140

Thr Val Gln Gln Leu Met Glu Val Lys Arg Asp Leu Leu His Ile Val

145 150 155 160

Glu Arg Asn Gln Ala Lys Phe Asp Ala Leu Glu Ala Tyr Glu Ser Ile

165 170 175

Leu Ala Gly Lys Arg Glu Gln Arg Pro Gln Asp Cys Leu Asp Ser Ile

180 185 190

Val Asp Leu Arg Glu Tyr Asp Val Pro Tyr His Val Arg Phe Ala Ile

195 200 205

Asp Asn Asp Val Arg Ser Gly Gln Trp Tyr Asn Val Ser Ile Ser Ser

210 215 220

Thr Asp Val Ile Leu Glu Lys Arg Thr Asp Leu Leu Gln Arg Ala Glu

225 230 235 240

Val Arg Val Cys Ala Phe Asp Ile Glu Thr Thr Lys Leu Pro Leu Lys

245 250 255

Phe Pro Asp Ala Glu Tyr Asp Gln Ile Met Met Ile Ser Tyr Met Val

260 265 270

Asp Gly Gln Gly Phe Leu Ile Ile Asn Arg Glu Cys Val Gly Glu Asp

275 280 285

Val Glu Asp Leu Glu Tyr Thr Pro Lys Pro Glu Phe Glu Gly Tyr Phe

290 295 300

Lys Val Thr Asn Val Lys Asn Glu Val Glu Leu Leu Gln Arg Trp Phe

305 310 315 320

Tyr His Met Gln Glu Leu Lys Pro Gly Ile Tyr Val Thr Tyr Asn Gly

325 330 335

Asp Phe Phe Asp Trp Pro Phe Ile Glu Arg Arg Ala Ser His His Gly

340 345 350

Ile Lys Met Asn Glu Glu Leu Gly Phe Arg Cys Asp Gln Asn Gln Gly

355 360 365

Glu Cys Arg Ala Lys Phe Ala Cys His Leu Asp Cys Phe Ala Trp Val

370 375 380

Lys Arg Asp Ser Tyr Leu Pro Gln Gly Ser His Gly Leu Lys Ala Val

385 390 395 400

Thr Lys Ala Lys Leu Gly Tyr Asp Pro Leu Glu Val Asn Pro Glu Asp

405 410 415

Met Val Arg Phe Ala Met Glu Lys Pro Gln Thr Met Ala Ser Tyr Ser

420 425 430

Val Ser Asp Ala Val Ala Thr Tyr Tyr Leu Tyr Met Thr Tyr Val Asn

435 440 445

Pro Phe Ile Phe Ser Leu Ala Thr Ile Ile Pro Met Val Pro Asp Glu

450 455 460

Val Leu Arg Lys Gly Ser Gly Thr Leu Cys Glu Met Leu Leu Met Val

465 470 475 480

Glu Ala Tyr Lys Ala Asn Val Val Cys Pro Asn Lys Asn Gln Ala Asp

485 490 495

Pro Glu Lys Phe Tyr Gln Asn Gln Leu Leu Glu Ser Glu Thr Tyr Ile

500 505 510

Gly Gly His Val Glu Cys Leu Glu Ser Gly Val Phe Arg Ser Asp Ile

515 520 525

Pro Thr Ser Phe Lys Leu Asp Ser Ser Ala Tyr Gln Gln Leu Ile Asp

530 535 540

Asn Leu Gly Arg Asp Leu Glu Tyr Ala Ile Thr Val Glu Gly Lys Met

545 550 555 560

Arg Met Asp Ser Ile Ser Asn Tyr Asp Glu Val Lys Asp Glu Ile Lys

565 570 575

Glu Lys Leu Glu Lys Leu Arg Asp Asp Pro Ile Arg Glu Glu Gly Pro

580 585 590

Leu Ile Tyr His Leu Asp Val Ala Ala Met Tyr Pro Asn Ile Ile Leu

595 600 605

Thr Asn Arg Leu Gln Pro Pro Ser Ile Val Thr Asp Glu Ile Cys Thr

610 615 620

Ala Cys Asp Phe Asn Arg Pro Gly Lys Thr Cys Leu Arg Lys Leu Glu

625 630 635 640

Trp Val Trp Arg Gly Val Thr Phe Met Gly Lys Lys Ser Asp Tyr Tyr

645 650 655

His Leu Lys Lys Gln Ile Glu Ser Glu Phe Val Asp Ala Gly Ala Asn

660 665 670

Ile Met Ser Ser Lys Ser Phe Leu Asp Leu Pro Lys Val Asp Gln Gln

675 680 685

Ser Lys Leu Lys Glu Arg Leu Lys Lys Tyr Cys Gln Lys Ala Tyr Lys

690 695 700

Arg Val Leu Asp Lys Pro Ile Thr Glu Val Arg Glu Ala Gly Ile Cys

705 710 715 720

Met Arg Glu Asn Pro Phe Tyr Val Asp Thr Val Arg Ser Phe Arg Asp

725 730 735

Arg Arg Tyr Glu Tyr Lys Thr Leu Asn Lys Val Trp Lys Gly Lys Leu

740 745 750

Ser Glu Ala Lys Ala Ser Gly Asn Ser Ile Lys Ile Gln Glu Ala Gln

755 760 765

Asp Met Val Val Val Tyr Asp Ser Leu Gln Leu Ala His Lys Cys Ile

770 775 780

Leu Asn Ser Phe Tyr Gly Tyr Val Met Arg Lys Gly Ala Arg Trp Tyr

785 790 795 800

Ser Met Glu Met Ala Gly Val Val Thr Tyr Thr Gly Ala Lys Ile Ile

805 810 815

Gln Asn Ala Arg Leu Leu Ile Glu Arg Ile Gly Lys Pro Leu Glu Leu

820 825 830

Asp Thr Asp Gly Ile Trp Cys Cys Leu Pro Gly Ser Phe Pro Glu Asn

835 840 845

Phe Thr Phe Lys Thr Ile Asp Met Lys Lys Leu Thr Ile Ser Tyr Pro

850 855 860

Cys Val Met Leu Asn Val Asp Val Ala Lys Asn Asn Thr Asn Asp Gln

865 870 875 880

Tyr Gln Thr Leu Val Asp Pro Val Arg Lys Thr Tyr Lys Ser His Ser

885 890 895

Glu Cys Ser Ile Glu Phe Glu Val Asp Gly Pro Tyr Lys Ala Met Ile

900 905 910

Ile Pro Ala Ser Lys Glu Glu Gly Ile Leu Ile Lys Lys Arg Tyr Ala

915 920 925

Val Phe Asn His Asp Gly Thr Leu Ala Glu Leu Lys Gly Phe Glu Ile

930 935 940

Lys Arg Arg Gly Glu Leu Lys Leu Ile Lys Val Phe Gln Ala Glu Leu

945 950 955 960

Phe Asp Lys Phe Leu His Gly Ser Thr Leu Glu Glu Cys Tyr Ser Ala

965 970 975

Val Ala Ala Val Ala Asp Arg Trp Leu Asp Leu Leu Asp Asn Gln Gly

980 985 990

Lys Asp Ile Ala Asp Ser Glu Leu Leu Asp Tyr Ile Ser Glu Ser Ser

995 1000 1005

Thr Met Ser Lys Ser Leu Ala Asp Tyr Gly Glu Gln Lys Ser Cys

1010 1015 1020

Ala Val Thr Thr Ala Lys Arg Leu Ala Glu Phe Leu Gly Val Thr

1025 1030 1035

Met Val Lys Asp Lys Gly Leu Arg Cys Gln Tyr Ile Val Ala Cys

1040 1045 1050

Glu Pro Lys Gly Thr Pro Val Ser Glu Arg Ala Val Pro Val Ala

1055 1060 1065

Ile Phe Thr Thr Asn Pro Glu Val Met Lys Phe His Leu Arg Lys

1070 1075 1080

Trp Cys Lys Thr Ser Ser Asp Val Gly Ile Arg Leu Ile Ile Asp

1085 1090 1095

Trp Ser Tyr Tyr Lys Gln Arg Leu Ser Ser Ala Ile Gln Lys Val

1100 1105 1110

Ile Thr Ile Pro Ala Ala Met Gln Lys Val Ala Asn Pro Val Pro

1115 1120 1125

Arg Val Leu His Pro Asp Trp Leu His Lys Lys Val Arg Glu Lys

1130 1135 1140

Asp Asp Lys Phe Arg Gln Arg Lys Leu Val Asp Met Phe Ser Ser

1145 1150 1155

Ala Asn Lys Asp Val Val Leu Asp Thr Asp Leu Pro Val Thr Lys

1160 1165 1170

Asp Asn Val Glu Asp Ile Glu Asp Phe Cys Lys Glu Asn Arg Pro

1175 1180 1185

Ser Val Lys Gly Pro Lys Pro Ile Ala Arg Ser Tyr Glu Val Asn

1190 1195 1200

Lys Lys Gln Ser Glu Cys Glu Gln Gln Glu Ser Trp Asp Thr Glu

1205 1210 1215

Phe His Asp Ile Ser Phe Gln Asn Ile Asp Lys Ser Val Asn Tyr

1220 1225 1230

Gln Gly Trp Leu Glu Leu Lys Lys Arg Lys Trp Lys Val Thr Leu

1235 1240 1245

Glu Lys Lys Lys Lys Arg Arg Leu Gly Asp Leu Arg Ser Ser Asn

1250 1255 1260

Gln Val Asp Thr His Glu Ile Asn Gln Lys Val Gly Gln Gly Arg

1265 1270 1275

Gly Gly Val Gly Ser Tyr Phe Arg Arg Pro Glu Glu Ala Leu Thr

1280 1285 1290

Ser Ser His Trp Gln Ile Ile Gln Leu Val Pro Ser Pro Gln Ser

1295 1300 1305

Gly Gln Phe Phe Ala Trp Val Val Val Glu Gly Leu Met Leu Lys

1310 1315 1320

Ile Pro Leu Ser Ile Pro Arg Val Phe Tyr Ile Asn Ser Lys Val

1325 1330 1335

Pro Ile Asp Glu Tyr Phe Gln Gly Lys Cys Val Asn Lys Ile Leu

1340 1345 1350

Pro His Gly Arg Pro Cys Tyr Ser Leu Thr Glu Val Lys Ile Gln

1355 1360 1365

Glu Asp Gln Phe Lys Lys Glu Ser Lys Lys Arg Ala Ala Leu Leu

1370 1375 1380

Ala Asp Pro Gly Val Glu Gly Ile Tyr Glu Thr Lys Val Pro Leu

1385 1390 1395

Glu Phe Ser Ala Ile Cys Gln Ile Gly Cys Val Cys Lys Ile Asp

1400 1405 1410

Asn Lys Ala Lys His Arg Asn Thr Gln Asp Gly Trp Glu Val Gly

1415 1420 1425

Glu Leu His Met Lys Thr Thr Thr Glu Cys His Tyr Leu Lys Arg

1430 1435 1440

Ser Ile Pro Leu Val Tyr Leu Tyr Asn Ser Thr Ser Thr Gly Arg

1445 1450 1455

Ala Ile Tyr Val Leu Tyr Cys His Val Ser Lys Leu Met Ser Ala

1460 1465 1470

Val Val Val Asp Pro Phe Asn Gly Asn Glu Leu Leu Pro Ser Ala

1475 1480 1485

Leu Glu Arg Gln Phe Arg Asp Ser Cys Leu Glu Leu Ser Leu Asp

1490 1495 1500

Ser Leu Ser Trp Asp Gly Ile Arg Phe Gln Val His Tyr Val Asp

1505 1510 1515

His Pro Glu Ala Ala Lys Lys Ile Ile Gln Arg Ala Ile Ser Glu

1520 1525 1530

Tyr Arg Glu Glu Asn Cys Gly Pro Thr Val Ala Val Ile Glu Cys

1535 1540 1545

Pro Asp Phe Thr Phe Met Lys Glu Gly Ile Lys Ala Leu Asp Asp

1550 1555 1560

Phe Pro Cys Val Arg Ile Pro Phe Asn Asp Asp Asp Asn Ser Tyr

1565 1570 1575

Gln Pro Val Ser Trp Gln Arg Pro Ala Ala Lys Ile Ala Met Phe

1580 1585 1590

Arg Cys Ala Ala Ala Phe Gln Trp Leu Asp Arg Arg Ile Thr Gln

1595 1600 1605

Ser Arg Tyr Ala His Val Pro Leu Gly Asn Phe Gly Leu Asp Trp

1610 1615 1620

Leu Thr Phe Thr Ile Asp Ile Phe Leu Ser Arg Ala Leu Arg Asp

1625 1630 1635

Gln Gln Gln Val Leu Trp Val Ser Asp Asn Gly Val Pro Asp Leu

1640 1645 1650

Gly Gly Ile Asn Asn Glu Glu Ala Phe Phe Ala Asp Glu Val Gln

1655 1660 1665

Gln Thr Ser Leu Val Phe Pro Gly Ala Tyr Arg Lys Val Ser Val

1670 1675 1680

Glu Leu Lys Ile His Asn Leu Ala Val Asn Ala Leu Leu Lys Ser

1685 1690 1695

Asn Leu Val Asn Glu Met Glu Gly Gly Gly Phe Met Gly Phe Glu

1700 1705 1710

Gln Asp Val Asn Pro Arg Gly Ile Asn Ser Asn Asp Asn Thr Ser

1715 1720 1725

Phe Asp Glu Thr Thr Gly Cys Ala Gln Ala Phe Arg Val Leu Lys

1730 1735 1740

Gln Leu Ile His Ser Cys Leu Thr Asp Val Arg Lys Ser Lys Asn

1745 1750 1755

Ile Tyr Ala Asp Ser Ile Leu Gln Arg Leu Ser Trp Trp Leu Cys

1760 1765 1770

Ser Pro Ser Ser Lys Leu His Asp Pro Ala Leu His Leu Met Leu

1775 1780 1785

His Lys Val Met Gln Lys Val Phe Ala Leu Leu Leu Thr Asp Leu

1790 1795 1800

Arg Arg Leu Gly Ala Ile Ile Ile Tyr Ala Asp Phe Ser Lys Val

1805 1810 1815

Ile Ile Asp Thr Val Lys Phe Asp Leu Ser Ala Ala Lys Ala Tyr

1820 1825 1830

Cys Glu Ser Leu Leu Ser Thr Val Arg Asn Ser Asp Ile Phe Glu

1835 1840 1845

Trp Ile Leu Leu Glu Pro Val His Tyr Trp His Ser Leu Leu Phe

1850 1855 1860

Met Asp Gln Tyr Asn Tyr Ala Gly Ile Arg Ala Asp Asp Glu Ile

1865 1870 1875

Ser Leu Asp Glu Val Thr Ile Glu Pro Lys Trp Ser Val Ala Arg

1880 1885 1890

His Leu Pro Glu Tyr Ile Glu Arg Asp Phe Ile Ile Ile Ile Ala

1895 1900 1905

Lys Phe Ile Phe Asp Pro Trp Lys Phe Ala Ile Glu Asn Lys Lys

1910 1915 1920

Gly Ser Ser Glu Ser Leu Glu Ala Gln Met Ile Glu Tyr Leu Arg

1925 1930 1935

Glu Gln Ile Gly Ser Thr Phe Ile Asn Met Leu Val Lys Lys Val

1940 1945 1950

Asp Asp Ile Met Ser His Met Lys Glu Ile Asn Val Ser Asp Ala

1955 1960 1965

Ser Arg Val Ser Gly Gln Ala Pro Lys Gly Asp Tyr Ser Leu Glu

1970 1975 1980

Phe Ile Gln Val Ile Ser Ala Val Leu Ala Leu Asp Gln Asn Val

1985 1990 1995

Gln Gln

2000

 

 

<210> 4

<211> 26

<212> DNA

<213> mutant pol2a-1 genotype identification primer (genome band)

 

<400> 4

aaggtgaatg tcgagctaaa ttcgct 26

 

 

<210> 5

<211> 33

<212> DNA

<213> mutant pol2a-1 genotype identification primer (genome band)

 

<400> 5

accaatatat gtctcacttt caagaagttg att 33

 

 

<210> 6

<211> 21

<212> DNA

<213> mutant pol2a-1 genotype identification primer (T-DNA inserts band)

 

<400> 6

ctatggctct ttatgggttg c 21

 

 

<210> 7

<211> 19

<212> DNA

<213> mutant pol2a-1 genotype identification primer (T-DNA inserts band)

 

<400> 7

attttgccga tttcggaac 19

 

 

<210> 8

<211> 21

<212> DNA

<213> mutant pol2a-2 point mutation sequencing primer

 

<400> 8

tgcgatggaa aagccccagg t 21

 

 

<210> 9

<211> 21

<212> DNA

<213> mutant pol2a-2 point mutation sequencing primer

 

<400> 9

tgccttgtaa gcctgtgctg a 21

 

 

<210> 10

<211> 21

<212> DNA

<213> mutant pol2a-3 genotype identification primer (genome band)

 

<400> 10

aaacggatac catcccaaga c 21

 

 

<210> 11

<211> 21

<212> DNA

<213> mutant pol2a-3 genotype identification primer (genome band)

 

<400> 11

aaagaaattg ccatcacatg c 21

 

 

<210> 12

<211> 19

<212> DNA

<213> mutant pol2a-3 genotype identification primer (T-DNA inserts band)

 

<400> 12

attttgccga tttcggaac 19

 

 

<210> 13

<211> 21

<212> DNA

<213> mutant pol2a-3 genotype identification primer (T-DNA inserts band)

 

<400> 13

aaagaaattg ccatcacatg c 21

 

 

<210> 14

<211> 19

<212> DNA

<213> Real Time PCR internal reference primer

 

<400> 14

ggtggtattg acaagcgtg 19

 

 

<210> 15

<211> 19

<212> DNA

<213> Real Time PCR internal reference primer

 

<400> 15

gatttcatcg tacctagcc 19

 

 

<210> 16

<211> 25

<212> DNA

<213> Real Time PCR detects the PCR primer of POL2A genetic expression in reduction division process in pol2a-1 and pol2a-2

 

<400> 16

tgtcatttgg attgttttgc ttggg 25

 

 

<210> 17

<211> 20

<212> DNA

<213>

 

<400> 17

accgaaccat gtcctctggg 20

 

 

<210> 18

<211> 20

<212> DNA

<213>

 

<400> 18

tggcctccta ctctgtctct 20

 

 

<210> 19

<211> 21

<212> DNA

<213>

 

<400> 19

acctcatcag ggaccattgg g 21

 

 

<210> 20

<211> 22

<212> DNA

<213>

 

<400> 20

gcaccctttg tgaaatgctt ct 22

 

 

<210> 21

<211> 33

<212> DNA

<213>

 

<400> 21

accaatatat gtctcacttt caagaagttg att 33

 

 

<210> 22

<211> 21

<212> DNA

<213>

 

<400> 22

tggtggtcat gtagagtgcc t 21

 

 

<210> 23

<211> 23

<212> DNA

<213>

 

<400> 23

tggtatgccg atgaatcgag ttt 23

 

 

<210> 24

<211> 25

<212> DNA

<213>

 

<400> 24

gcacagcatg tgatttcaac cgccc 25

 

 

<210> 25

<211> 24

<212> DNA

<213>

 

<400> 25

tcccatgaat gtcacaccac gcca 24

 

 

<210> 26

<211> 25

<212> DNA

<213>

 

<400> 26

aagcagcgcc ttagttcagc tatcc 25

 

 

<210> 27

<211> 25

<212> DNA

<213>

 

<400> 27

atggagccag tcaggatgaa gcacc 25

 

 

<210> 28

<211> 37

<212> DNA

<213> builds the primer of the carrier of the RNAi interference POL2 expression containing DMC1 promotor

 

<400> 28

cattctagac catggggtcc caagtccaca gagcgga 37

 

 

<210> 29

<211> 34

<212> DNA

<213> builds the primer of the carrier of the RNAi interference POL2 expression containing DMC1 promotor

 

<400> 29

gatgtcgacg ggcccgcacg ccctgttgag gtgc 34

 

 

Claims (13)

1. the POL2A protein as shown in SEQ ID NO.1, comprises different functional domains: hold C to hold from N and be respectively: nitrogen terminal domains, 3 '-5 ' 5 prime excision enzyme activity structural domain, 5 '-3 ' polymerase activity structural domain, central catalytic active domain, PCNA binding domains and Zinc finger domain.

2. qualification Arabidopis thaliana as described in SEQ ID NO.1 pOL2Athree mutant materials of gene: the genotypic primer of SALK_096341C, CS6947 and SALK_124217, these three mutant materials called after successively pol2a-1, pol2a-2with pol2a-3; It is characterized in that primer nucleotide sequences is for shown in SEQ ID NO.4-SEQ ID NO.13, wherein, mutant pol2a-1genotype identification primer (genome band): sEQ ID NO.4 and SEQ ID NO.5,mutant pol2a-1genotype identification primer (T-DNA inserts band): sEQ ID NO.6 and SEQ ID NO.7,mutant pol2a-2point mutation sequencing primer: sEQ ID NO.8 and SEQ ID NO.9,mutant pol2a-3genotype identification primer (genome band): sEQ ID NO.10 and SEQ ID NO.11,mutant pol2a-3genotype identification primer (T-DNA inserts band): sEQ ID NO.12 and SEQ ID NO.13.

3. determine a method for POL2A concrete function in meiotic recombination, it is characterized in that concrete steps are as follows:

(1) corresponding weak mutant is obtained; Or build a RNAi carrier by the promoters driven of reduction division specifically expressing, and obtain positive transgenic plant;

(2) use regular growth biology techniques, analyze the activity of pollen granule, the dyeing of quadrantal magenta and exhibition sheet method and observe mutant pollen mother cells process, determine the maiotic concrete link of this effect gene;

(3) chromosome fluorescence in-situ hybridization is passed through, analyze POL2A gene affect the interaction event of homologous chromosomes, immunofluorescence experiment analyzes location and the change of the various albumen in the reduction division process that may participate in mutant, determines the function of this gene in reduction division further;

(4) by the hybridization between multimutation body, analyze this gene and do mutually from intergenic heredity in different genetic recombination passage, as sPO11, RAD51, MUS81deng, after two material that isozygotys compares with the reduction division phenotype of respective single mutant, determine the position of this gene in path;

(5) utilize fluorescence labels site system, the pollen granule number combined by the fluorescence that statistics offspring tetrad is different, is calculated the recombination frequency between several fluorescent mark, thus analyzes this effect gene meiotic recombination frequency or distribution situation;

(6) according to existing result of study, in conjunction with the concrete biochemical function of this known gene, the concrete function of this gene in reduction division is comprehensively drawn.

4. one kind for vitro detection pol2a-1with pol2a-2in in reduction division process pOL2Athe method of genetic expression, it is characterized in that concrete steps are: get the inflorescence that mutant is not bloomed, with TRIzol method extracting paddy rice RNA, and use DNaseI DNA digestion, then reverse transcription forms cDNA, and carry out quantitative fluorescent PCR with it for template and analyze, fluorescence quantification PCR primer nucleotide sequence is as shown in SEQ ID NO.14 ~ SEQ ID NO.27, wherein, Real Time PCR internal reference primer: sEQ ID NO.14 and SEQ ID NO.15,real Time PCR detects pol2a-1with pol2a-2in in reduction division process pOL2Athe PCR primer of genetic expression: sEQ ID NO.16 ~ SEQ ID NO.27.

5. one kind dMC1the special interference of Meiosis of promoters driven pOL2 ^rNAicarrier construction and transgenic line, be specially: select one section of nearly N end duplicate about 400bp base sequence in two genes to be building up to as target sequence and contain dMC1in the RNAi carrier of promotor, carry out Agrobacterium-mediated Transformation, thus obtain the special POL2 afunction mutant material of Meiosis, amplimer nucleotides sequence is classified as sEQ ID NO.28 and SEQ ID NO.29.

6. Arabidopis thaliana as described in claim 2 pOL2Athe weak mutant of gene pol2a-1with pol2a-2plant phenotype and male meiosis abnormal, specifically by observation and the cytologic experiment of plant, determine pol2a-1with pol2a-2abnormal Circumstances In Meiosis phenotype.

7. utilize an experiment for fluorescence in situ hybridization, determine pol2a-1the method of interaction between chromosomes situation in mutant, is specially: utilize digoxigenin labeled to be positioned at arabidopsis gene group chromosome long arm near-end grain end BAC F1N21, by observing pol2a-1in mutant male meiosis process, the difference of this signal and wild-type, determines pol2a-1the interaction of nonhomologous chromosome is there is in mutant.

8. one kind utilizes immunofluorescence experiment to observe mutant pol2a-1the method that in mutant, can synaptonemal complex correctly be formed, is specially: that is located in mutant and wild-type by synaptonemal complex formation associated protein ASY1 and ZYP1 is relatively analyzed.

9. one kind utilizes immunofluorescence experiment to observe mutant pol2a-1the location of γ-H2AX and RAD51 and change in mutant, thus determine whether POL2A sudden change have impact on the method for meiotic recombination process double center chain fracture restoration.

10. one kind utilizes the method for immunofluorescence to observe MLH1 albumen at diakinesis stage wild-type and mutant pol2a-1middle location, and then determine pOL2Athe method of responsive type regrouping process is interfered in impact.

11. one kind utilizes hereditary path analysis pOL2Athe method of mechanism of action in Arabidopis thaliana reduction division process, it is characterized in that: by the hybridization by some key gene mutant of upstream and downstream in reduction division unknown mutation body and known reduction division process, analyze the reduction division phenotype of two material that isozygotys and the similarities and differences of the parental phenotypes that isozygotys in F2 colony.

12. 1 kinds are existed by hybridization pol2a-1fluorescence labels site system is introduced, the pollen granule number combined by the fluorescence that statistics offspring tetrad is different, research in mutant pOL2Aaffect the method for meiotic recombination frequency or distribution.

13. 1 kinds utilize the function of Arabidopis thaliana POL2A in meiotic recombination, in the recombination frequency of directed change crop, carry out the application in Crop Genetic Breeding.