CN101255429A - Cabbage pollen wall growth related gene PGBc2 and separation method thereof - Google Patents

Abstract

The invention discloses a growth of Chinese cabbage pollen wall related gene PGBc2 and separation thereof. The upgrowth of Chinese cabbage pollen wall related gene includes nucleotide sequence indicated in SEQ ID No. 2. Separation clone and functional verification of the gene is helpful to illustrate molecular mechanism in growth of pollen and male sterility of plants, and has practical meaning for artificially controlling fertility of plant, cultivating superior varieties.

Description

Chinese cabbage pollen wall development related gene PGBc2 and separation method thereof

Technical field

The present invention relates to the genetically engineered field, be specifically related to a kind of Chinese cabbage pollen wall development related gene PGBc2 and separation method thereof.

Background technology

Pollen development and plants male sterility are closely related, are the ideal model of processes such as the growth of research cell, division, cytodifferentiation and the transmission of iuntercellular information simultaneously.Therefore, pollen development research is the focus in the life science field always.Pollen development process complexity relates to numerous expression of gene regulation and control.Big characteristics of discovering pollen gene expression are exactly to express the gene synthetic with cell walls in a large number, that regulation and control are relevant.The most characteristic part of pollen granule is pollen wall (Blackmore and Barnes, 1990; Owen andMakaroff, 1995).By extine with intine is two-layer forms, on pollen is brought into normal play function, play a part very important, especially in pollen germination and pollen tube growth process, pollen wall be to cause pollen can not finish the one of the main reasons of its biological function unusually.

Few about the gene or the protein research at present that act on the pollen wall growth, in the model animals Arabidopis thaliana, only found the unusual mutant that several extines are grown, for example ms2, flp1, nef1 and dex1, and 2 extines and simultaneously unusual mutant ms33 and the ms1 of inwall.The protein function of the coded by said gene of these mutant correspondences all fully not clear and definite (Aarts et al, 1997; Paxson-Sowders et al, 1997,2001; Shukla et al., 1998; Fei and Sawhney, 2001; Ariizumi et al, 2003,2005).In Chinese cabbage, do not have as yet and act on gene or the separated report of mutant that pollen wall is grown.

Hydration pollen during pollen germination can discharge the protein that much is present in the pollen wall, and perhaps from some protein of the inner secretion of pollen, these protein are considered to pollen wall or the pollen tube wall is grown necessary (Cosgrove et al, 1997,2000; Allen et al, 1993), polygalacturonase (PG) is exactly one of them.It is a kind of cell walls hydrolysis and loose enzyme, participates in the degraded of pectin, and cell wall structure is disintegrated.Although present research infers the PG in the pollen tube and may allow pollen tube to pass through style by degraded style cell walls, perhaps may act on the cell walls of itself and quicken self growth (Brown et al, 1990; Allen et al, 1993; Hadfield et al, 1998; Honys et al, 2003).But conclusive evidence is few.Therefore, the clone and the Function Identification of the gene that the pollen wall growth is relevant remain an important job.

Summary of the invention

The objective of the invention is at the deficiencies in the prior art, provide a kind of Chinese cabbage pollen wall to grow relevant gene PGBc2 and separation method thereof.

The objective of the invention is to be achieved through the following technical solutions:

A kind of Chinese cabbage pollen wall development related gene PGBc2, it comprises the nucleotide sequence shown in the SEQ ID No.2.

Further, also be included in mutant, allelotrope and the derivative that add, replace, insert or delete one or more Nucleotide generations in the nucleotide sequence shown in the SEQ ID No.2.

The separation method of the described Chinese cabbage pollen wall of a kind of claim 1 development related gene PGBc2 may further comprise the steps:

(1), be that bud gene expression difference carry out cDNA-AFLP analyze to the sterile strain system of ' short pin Huang ' Chinese cabbage genic male sterility dual purpose with educating strain with the A8/T18 primer, acquisition can educated the gene fragment Bc-A8T18 that strain is a specifically expressing in the bud, and this fragment comprises the described nucleotide sequence of SEQ ID No.1 and add, replace, insert or delete the derivative that one or more Nucleotide generate in the nucleotide sequence shown in the SEQID No.1;

(2), utilize 3 ' the terminal and 5 ' end of the cDNA of the relevant gene of RACE technology amplification Bc-A8T18 fragment, wherein, forward special primer S1 sequence is 5 '-CTCCTCTCCAGGTTCTGACATTACT-3 '; Oppositely special primer A1 sequence is 5 '-CTGCTCGGTGTTGGAGATTGG-3 '; Respectively with 3 ' RACE and 5 ' RACE anchor primer amplification, 3 ' terminal and 5 ' end;

(3), with the cDNA sequence and the dna sequence dna thereof of conventional pcr amplification Bc-A8T18 fragment genes involved, this dna sequence dna is the nucleotide sequence shown in the SEQ ID No.2.According to the cDNA full length sequence design special primer T1 and the T2 of two ends splicing, the cDNA sequence of amplification Bc-A8T18 fragment genes involved from bud cDNA, its dna sequence dna of amplification from genomic dna.The sequence of T1 and T2 is respectively: T1,5 '-TGGGATTTTCAGCCAATG-3 '; T2,5 '-AGAGCATACATCATAGAC-3 '.

The invention has the beneficial effects as follows: the present invention adopts the cDNA-AFLP technology the sterile strain of ' short pin Huang ' Chinese cabbage kernel sterility dual serial is and can educates strain to be that the bud gene expression difference is analyzed, cooperate the RACE technical point from obtaining and pollen wall development related gene PGBc2 simultaneously, for understanding fully that further sterile strain is the reason of pollen abortion, and illustrate the molecular mechanism that the plant pollen wall is grown and male sterile takes place and provide research basic, and then for the manual creation sterile material, cultivating improved seeds provides material conditions.

Description of drawings

Fig. 1 is the amplification figure of Chinese cabbage PGBc2 cDNA and DNA full length sequence;

The Chinese cabbage PGBc2 aminoacid sequence figure of Fig. 2 for inferring;

Fig. 3 is the spatial and temporal expression characteristics synoptic diagram of Chinese cabbage PGBc2;

The in situ hybridization analysis chart that Fig. 4 expresses for Chinese cabbage PGBc2;

Fig. 5 is an antisense PGBc2 expression vector synoptic diagram;

Fig. 6 detects the expression synoptic diagram of PGBc2 in the transgenosis cabbage heart for Northern hybridization;

The in-vitro pollen germination of Fig. 7 antisense PGBc2 gene cabbage heart is observed figure;

Fig. 8 antisense PGBc2 gene cabbage heart pollen scanning electron microscopic observation figure;

Fig. 9 antisense PGBc2 gene cabbage heart pollen development transmission electron microscope observing figure.

Embodiment

The present invention is to separate the gene PGBc2 that obtains a fertile flower flower bud specifically expressing the bud by the gene expression difference analysis from the educated strain of Chinese cabbage ajhGMS ' Bcajh97-01A/B ', sequential analysis shows that this gene has 4 conserved domains of plant PG gene family, and has higher similarity with known PG gene, but being different from known PG gene family member, is a new PG gene therefore.RT-PCR, Northern hybridization and in situ hybridization shows, the transcript of PGBc2 occurs in the tapetum in cabbage rolls medicine tetrad period and microspore tetrad, and the expression amount in tapetum is than the expression amount height in microspore tetrad.Keep very strong expression signal subsequently always in tapetum, degenerate up to the degraded of tapetum, the expression level in sporule begins to strengthen from mononuclear microspore period, lasts till the pollen maturation phase always.By Antisense RNA Technique,, verified the function of this gene with the Chinese cabbage cell that the inverted defined gene importing of PGBc2 normally can be educated.Find that PGBc2 expression by inhibitation system or reticent transfer-gen plant pollen cause pollen normally not sprout because of being obstructed of pollen wall growth, the plant male fertile reduces, and shows that PGBc2 is an important pollen wall development related gene.The separating clone of this gene helps to illustrate pollen development and plants male sterility molecule mechanism, has important practice significance to producing artificial controlling plant fertility, cultivating improved seeds.

Realize that concrete technological step of the present invention is as follows:

1. the separation of Chinese cabbage PGBc2 gene and sequential analysis

Utilizing the sterile strain system of cDNA-AFLP technical Analysis Chinese cabbage ajhGMS ' Bcajh97-01A/B ' and can educating strain is the bud gene expression difference, what screen that the A8/T18 primer amplification obtains can educate the gene fragment Bc-A8T18 that strain is a specifically expressing in the bud, long 250bp, concrete sequence is seen SEQ ID No.1.Obtain the 3 ' end of the cDNA of this fragment genes involved by RACE technology amplification, 2 amplifications show that all the purpose clip size is about 1400bp, shown in Figure 1A swimming lane 1,2; 3 ' RACE product cloning transforms DH 5 α competent cells to the pGEM-TEasy carrier, and blue hickie screening positive clone, alkaline lysis extracting plasmid, enzyme are cut the order-checking of evaluation (Figure 1B) back.Similarly, obtain 5 ' terminal (Fig. 1 C swimming lane 1,2) of the cDNA of this fragment genes involved by RACE technology amplification, enzyme is cut evaluation (Fig. 1 C swimming lane 3,4) and is checked order after cloning.

CDNA full length sequence design primer according to the two ends splicing, the cDNA sequence (Fig. 1 D swimming lane 1) and the dna sequence dna (Fig. 1 D swimming lane 2) thereof of conventional PCR method amplification Bc-A8T18 fragment genes involved, enzyme is cut evaluation (Fig. 1 D swimming lane 1 is cut evaluation for the monoclonal enzyme of cDNA, and Fig. 1 D swimming lane 2 is cut evaluations for dna single clone's enzyme) and is checked order after cloning.

Dna sequence dna total length 1594bp, concrete sequence is seen SEQ ID No.2.Utilize the information biology related software to analyze this gene order, find that it contains 3 exons and 2 introns, long 1 188 bp of maximum open reading frame, 395 amino acid of encoding.Has one section hydrophobic signal peptide at the N of the protein sequence of supposing end.In database, carry out homology search and find that the polygalacturonase PGA3 sequence of the aminoacid sequence inferred and the supposition of Arabidopis thaliana pollen expression has 86% consistence and 94% similarity.Has a typical PG avtive spot in the aminoacid sequence that search is found to infer in the protein structure database.Compare with the aminoacid sequence of other plant PG that deliver, comprise peculiar 4 conserved domains of plant PG (Fig. 2 underscore partly is plant PG functions peculiar structural domain) in its aminoacid sequence.These presentation of results, this gene are typical PG genes, called after PGBc2.

2.PGBc2 the spatial and temporal expression characteristic analysis

(1) RT-PCR and the Northern hybridization analysis PGBc2 expression in the buds of 5 grades of different sizes of Chinese cabbage ajhGMS ' Bcajh97-01A/B ' fertile plant (size in the vertical footpath of bud is respectively to the V level from the I level: less than 1mm, 1.2 ~ 1.6mm, 1.8 ~ 2.2mm, 2.4 ~ 2.8mm, 3.0 ~ 3.4mm, period before these buds that vary in size correspond respectively to microsporocyte and form and form, tetrad period, monokaryon pollen granule period and mature pollen period) and open flower, tender angle fruit, scape and scape blade.Found that, the expression pattern that RT-PCR analyzes (as shown in Figure 3A) and Northern hybridization analysis (shown in Fig. 3 B) PGBc2 all shows identical result, its transcript in III level bud just tetrad begin to occur period, and expression amount is higher, subsequently continuous expression in the monokaryon pollen granule bud in period, the V level bud that comprises the mature pollen grain, the flower of opening and tender angle fruit.(among Fig. 3, B1～B5 represents the I～V level bud of ' Bcajh97-01B ' and all do not detect the expression of PGBc2 in the II before reduction division, I level bud and scape and the scape blade; F, Si, S, L represent the open flower of Bcajh97-01B ', tender angle fruit, scape and blade successively; The Actin-1 that omnipresence is expressed in contrast.)。

(2) utilize hybridization in situ technique further to analyze the expression of PGBc2 in the bud of different developmental phases.Find that hybridization signal at first occurs, and the signal in tapetum is than the signal in microspore tetrad strong (Fig. 4 B) in the tapetum in tetrad period and microspore tetrad; In the mononuclear microspore later stage, tapetum begins degraded, but still can see very strong hybridization signal, and in the mononuclear microspore at this moment, hybridization signal is better than tetrad period (Fig. 4 C); To the mature pollen phase, tapetum is degraded fully, and at this moment, expression signal only detects in pollen granule, and the signal power is similar to mononuclear microspore period (Fig. 4 D); And in the sepal of the flower pesticide (Fig. 4 A) of tetrad before period, all detected buds, petal, center pillar, gynoecium, and (Fig. 4 E, F) all do not detect the expression signal of PGBc2 in the contrast of hybridizing with just probe.

3. utilize the function of Antisense RNA Technique checking PGBc2

Make up the antisense PGBc2 expression vector (Fig. 5) that contains CaMV35S constitutive promoter and BcA9 organizing specific type promotor respectively, change cabbage heart (mutation of Chinese cabbage) plant over to by agrobacterium-mediated transformation, induce Kan by the cultivation of cabbage heart aseptic seedling, the pre-cultivation of cabbage heart cotyledon-cotyledon petiole explant, common cultivation, differentiation culture ^RThe generation of seedling, numerous Kan is expanded in growth in aseptic bottle ^RA series of processes such as seedling, root culture, transplanting transformed plant obtain transfer-gen plant 35S-pgbc2 (transformed plant that contains the CaM35S constitutive promoter) and the A9-pgbc2 (transformed plant that contains BcA9 tapetum specifically expressing type promotor) of PGBc2 respectively.Choose transgenosis until carrying out Northern hybridization, with Chinese cabbage ' Bcajh97-01A/B ' plant bud (B among Fig. 6), flower (F among Fig. 6) and non-transgenic cabbage heart plant (non-tra among Fig. 6) bud and flower is contrast, and the cDNA that uses PGBc2 is as the template label probe.The result shows, the bud of a wherein strain transfer-gen plant 35S-pgbc2 who chooses and the expression signal that Hua Zhongjun does not detect PGBc2 have detected very weak hybridization signal at the bud of another strain with in spending; Select good strains in the field for seed in the A9-pgbc2 plant of getting 4, have in the spending of the bud of 1 strain and 1 strain to still have more weak expression signal, do not detect tangible hybridization signal at the bud and the Hua Zhongjun of other 2 strains.All detect very high PGBc2 expression level at the bud that can educate strain ' Bcajh97-01B ' (the B strain among Fig. 6 is ' Bcajh97-01B ', and A strain is ' Bcajh97-01A ') and non-transgenic cabbage heart plant with in spending on the contrary.Being expressed in of these presentation of results PGBc2 is subjected in the transfer-gen plant than obvious suppression, and through the Northern blot hybridization, the expression that obtains PGBc2 is subjected to the obviously transfer-gen plant of inhibition.

35S-pgbc2 and A9-pgbc2 transfer-gen plant are carried out the morphology character observation, find that 35S-pgbc2 and A9-pgbc2 nourish and grow normally, and can normally bloom, the growth of floral organ is not distinguished with the obvious of non-transgenic plant yet.Through the bud pollination selfing, transfer-gen plant can be born the fruit pod, but compares with the fruit pod that not genetically modified wild-type plant selfing produces, and its fruit pod is not full, and each fruit pod only produces 1 ~ 5 seed.And normal non-transgenic plant fruit pod is full, can produce about 15 seeds.The normal pollen of non-transgenic plant is awarded respectively on the column cap of transfer-gen plant 35S-pgbc2 and A9-pgbc2, and fruit pod size of bearing and product seed number and wild-type plant do not have difference.The planting seed that the transfer-gen plant selfing produces can be grown by normal growth, obtains T ₁For each 50 strain of plant.These explanation transfer-gen plant female fertilities and ovule are grown normal, and its fertility reduces be attributed to the unusual of pollen function.Late detection is found the not obviously difference of proterties that 35S-pgbc2 and A9-pgbc2 plant are presented, and therefore is collectively referred to as pgbc2.The stripped normal germination rate of pgbc2 plant pollen obviously reduces (Fig. 7 I), nuclear and inclusion normal (Fig. 7 B～D) compare that moves in pollen tube when in-vitro pollen germination (Fig. 7 A) is with pollen germination normally with non-transgenic plant (WT among Fig. 7), pollen tube explosion (Fig. 7 E) during the pollen germination of pgbc2 plant about 81%, simultaneously, nuclear energy enters pollen tube (Fig. 7 F～H) of explosion.Scanning electron microscopic observation shows, compares with the normal pollen (Fig. 8 E, F) of non-transgenic adjoining tree (WT), the lopsided pollen that the transfer-gen plant 35S-pgbc2 of PGBc2 expression by inhibitation system and A9-pgbc2 generation outer wall reticulate pattern are shallow (Fig. 8 A ~ D).Transmission electron microscope observing shows that transfer-gen plant pgbc2 begins to come off at the pollen germination ditch place outer wall of three nuclear pollen, and interior change in wall thickens (Fig. 9 F, H), and scope is wider than the germinal furrow of non-transgenic plant (Fig. 9 E, G), and thickened degree is big; The mature pollen phase is because outer wall destroys pgbc2 extine tryphine excessive (Fig. 9 J, L).Hurdle, Fig. 9 left side is the pollen development process of non-transgenic adjoining tree, and right hurdle is the pollen development process of transfer-gen plant pgbc2, and Ba represents [; Exine represents extine; Fm represents Isolated microspore; Gf represents germinal furrow; In represents inwall; Ne represents nexine; Np represents monokaryon pollen; Tc represents tectum; Tp represents three nuclear pollen.These presentation of results PGBc2 gene has participated in the growth of pollen wall.

In conjunction with specific embodiments, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the invention.

The separation and the clone of embodiment 1 PGBc2 gene

(1) the segmental separation of Bc-A8T18 is that bud gene expression difference carry out cDNA-AFLP analyze to the sterile strain system of Chinese cabbage ajhGMS ' Bcajh97-01A/B ' with educating strain with the A8/T18 primer, detect and to educate the gene band Bc-A8T18 that strain is a specifically expressing in the bud, cut off differential fragment on the polyacrylamide gel with knife blade, be dissolved in 100 μ L TE (10mmolL ^-1Tris, pH 7.5,1mmolL ^-1EDTA, pH 8.0), and carry out pcr amplification as template, condition is identical with the pre-amplification among the cDNA-AFLP.PCR product 1.5% agarose gel electrophoresis reclaims the test kit purifying through gel.Reclaim product and be connected to pGEM-T Easy Vector (Promega) and be transformed into E.coli DH 5 α competent cells, through blue hickie screening recombinant plasmid, order-checking, last RT-PCR checking obtains the nucleotide sequence of this band.

(2) 3 ' of the cDNA of the gene that the Bc-A8T18 fragment is relevant terminal and 5 ' the terminal amplification according to double-stranded cDNA synthetic agent box (SMART ^TMPCR cDNA Synthesis Kit) characteristic Design of center tap and primer 3 ' RACE and 5 ' RACE anchor primer P3 and P5; According to differential fragment Bc-A8T18 design forward special primer S1, with anchor primer P3 amplification 3 ' end, reverse primer A1 and anchor primer P5 amplification 5 ' end.The sequence of anchor primer P5 is: 5 '-AAGCAGTGGTATCAACGCAGAGT-3 '; The sequence of anchor primer P3 is: 5 '-GAGGCGGCCGACATGTTTTT-3 '; Forward special primer S1 sequence is 5 '-CTCCTCTCCAGGTTCTGACATTACT-3 '; Oppositely special primer A1 sequence is 5 '-CTGCTCGGTGTTGGAGATTGG-3 '.PCR product D NA gel reclaims test kit and reclaims, insert pGEM-T Easy carrier, transform DH 5 α competent cells, blue hickie screening positive clone, alkaline lysis extracting plasmid, after enzyme was cut evaluation, recombinant plasmid was served the order-checking of Hai Boya Bioisystech Co., Ltd, obtained 3 ' the terminal and 5 ' end of the cDNA of the relevant gene of Bc-A8T18 fragment.

(3) full-length clone of PGBc2 gene according to the cDNA full length sequence design special primer T1 of two ends splicings (sequence is: 5 '-TGGGATTTTCAGCCAATG-3 ') and T2 (sequence is: 5 '-AGAGCATACATCATAGAC-3 ').Conventional PCR method is the cDNA sequence of amplification Bc-A8T18 fragment genes involved from bud cDNA once more; With same special primer amplification in genomic dna is obtained its dna sequence dna.

Embodiment 2 utilizes the function of Antisense RNA Technique checking PGBc2

(1) structure of antisense expression vector designs primer according to PGBc2 cDNA full length sequence between 546 bases of inner the 85th base to the of PGBc2 sequence, and amplification 462bp sequence is seen SEQ ID No:3 as the inverted defined gene fragment.And according to the polyclone restriction enzyme site of plant binary carrier pBI121, design contains two pcr amplification primers of BamH I and Xba I restriction endonuclease sites and three protection bases respectively, and upstream primer is 5 '-ACA GGATCCGAGCTTTTACCGTTTTTA-3 ', downstream primer are 5 '-GCC TCTAGATGGCATCTATTGAAGATA-3 ' (the line part is a restriction enzyme site).With ' Bcajh97-01B ' bud cDNA is that template is carried out pcr amplification.Product cloning is to pGEM-T Easy carrier, positive colony plasmid called after pTPGBc2A.

(2) structure of CaMV35S constitutive promoter expression vector and BcA9 organizing specific type promoter expression vector is connected antisense PGBc2 fragment with mixing through the binary vector pBI121 of BamH I and Xba I double digestion, product transforms DH 5 α competent cells, is containing Kan 50mgL ^-1Solid LB culture medium flat plate on overnight incubation.Picking list bacterium colony is containing Kan 50mgL ^-1Liquid LB substratum in shaking culture spend the night, its plasmid DNA of alkaline lysis extracting adopts methods such as PCR and digestion with restriction enzyme to identify positive colony.Positive colony plasmid called after pBI35S-PGBc2A.The BcA9 promotor from Chinese cabbage, separate obtain, the organizing specific type promotor of specifically expressing in anther tissue.Mix with antisense PGBc2 fragment, BcA9 promotor with through the pBI121 of Hind III and BamH I double digestion and to be connected, as above method transforms, identifies acquisition positive colony plasmid, called after pBIBcA9-PGBc2A.

(3) antisense expression vector changes above-mentioned plant expression vector pBI35S-PGBc2A that builds and pBIBcA9-PGBc2A plasmid over to Agrobacterium LBA4404 to the genetic transformation of cabbage heart, and the genetic transformation of cabbage heart is realized by the tissue culture approach.The composition of each substratum is as follows:

Cultivate and be total to culture medium in advance: 1/2 times of concentration NH ₄ ⁺The salt+2% sucrose+0.8% agar+3mgL of MS substratum ^-1BAP+1mgL ^-1NAA+7.5mgL ^-1AgNO ₃(pH value 5.8).

Division culture medium: 1/2 times of concentration NH ₄ ⁺The salt+2% sucrose+0.8% agar+3mgL of MS substratum ^-1BAP+1mgL ^-1NAA+7.5mgL ^-1AgNO ₃+ 7.5mgL ^-1Kan+300mgL ^-1Amp (pH value 5.8).

Root media: the salt of 1/2 MS substratum and VITAMIN+0.8% agar+5mgL ^-1Kan (pH value 5.8).

Plant-growth regulator adds before autoclaving, AgNO ₃Add behind autoclaving with microbiotic, autoclaved condition is 121 ℃ and keeps 20min, 50 ℃ of insulations.

This experiment is an explant with cotyledon-cotyledon petiole, and it is dark that cotyledon petiole is inserted in the pre-culture medium 2 ~ 3mm, cultivates 3d, keeps flat after Agrobacterium is infected altogether on the culture medium, cultivates 2d.Cabbage heart cotyledon-cotyledon petiole explant of cultivating altogether behind the 2d is changed on the division culture medium, and approximately about 20d, resistant buds length to be differentiated arrives a certain size, carefully from base portion its cutting-out is transferred to continued growth on the new division culture medium.The resistant buds that obtains is subculture and screening on division culture medium, and every 20d left and right sides rolling bottle is once removed false positive plant wherein.When treating resistant plant length to 2 ~ 3cm, it is downcut from callus, forward on the root media.That treats that resistant plant grows a large amount of whites on root media must shape during root, opens bottleneck hardening 2 ~ 3d, carefully removes the substratum on the root, moves into water content and is in 60% the compost, preserves moisture and cultivates after 5 ~ 7 days, forwards outdoor cultivation again to.

Transfer-gen plant detects through PCR detection, Southern and Northern blot hybridization, and screening obtains PGBc2 genetic expression reticent and downtrod transfer-gen plant 35S-pgbc2 and A9-pgbc2.

(4) the outer and interior pollen germination situation of body of the form of transfer-gen plant and cytological observation detection bodies, the semisolid medium that is used for in-vitro pollen germination consists of: 15% sucrose+0.4mmolL-1 boric acid+0.4mmolL-1 CaNO3+0.1% agar.The time of gathering flower is about 9:00 in the morning, the flower that 5 flower pesticide have just split is adopted in every strain, adopting the back shakes off pollen to template earlier, abundant mixing, its point is coated in the substratum on the slide glass, does not add cover glass, slide glass is placed the culture dish that is covered with moistening filter paper, place dark, 20 ~ 25 ℃ of constant temperatures to cultivate down, examine under a microscope germination rate behind 4 ~ 6h.The scanning electron microscopic observation POLLEN MORPHOLOGY, adopt the flower of firm opening, pollen is coated in lightly on the metal Stage microscope that posts double faced adhesive tape with tweezers clamping flower pesticide, under Philips XL-40 type scanning electron microscope, observes pollen shape, full degree and surface texture behind the metal spraying, take pictures.Transmission electron microscope observing pollen development process is got the flower pesticide in the different big small buds, and the preparation ultrathin section(ing) is observed pollen anther development process.

At last, note also that what more than enumerate only is a specific embodiment of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.

Sequence table

＜110〉Zhejiang University

＜120〉Chinese cabbage pollen wall development related gene PGBc2 and separation method thereof

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Claims (3)

1, a kind of Chinese cabbage pollen wall development related gene PGBc2, it is characterized in that: it comprises the nucleotide sequence shown in the SEQ ID No.2.

2, Chinese cabbage pollen wall development related gene PGBc2 according to claim 1 is characterized in that: also be included in and add, replace, insert or delete mutant, allelotrope and the derivative that one or more Nucleotide generate in the nucleotide sequence shown in the SEQ ID No.2.

3, the separation method of the described Chinese cabbage pollen wall of a kind of claim 1 development related gene PGBc2 is characterized in that, may further comprise the steps:

(1) the segmental separation of Bc-A8T18: is that bud gene expression difference carry out cDNA-AFLP analyze to the sterile strain system of Chinese cabbage with educating strain with the A8/T18 primer, detect and to educate the gene band Bc-A8T18 that strain is a specifically expressing in the bud, cut off differential fragment on the polyacrylamide gel with knife blade, be dissolved in 100 μ L TE, and carry out pcr amplification as template, condition is identical with the pre-amplification among the cDNA-AFLP, PCR product 1.5% agarose gel electrophoresis, reclaim the test kit purifying through gel, reclaim product and be connected to pGEM-T Easy Vector and be transformed into E.coli DH 5 α competent cells.

Through blue hickie screening recombinant plasmid, order-checking, last RT-PCR checking obtains the nucleotide sequence of this band.

(2) 3 ' of the cDNA of the gene that the Bc-A8T18 fragment is relevant terminal and 5 ' terminal amplification: according to the characteristic Design 3 ' RACE and 5 ' the RACE anchor primer P3 and the P5 of double-stranded cDNA synthetic agent box center tap and primer; According to differential fragment Bc-A8T18 design forward special primer S1 and reverse special primer A1, forward special primer S1 and anchor primer P3 amplification 3 ' end, oppositely special primer A1 and anchor primer P5 amplification 5 ' end.The sequence of anchor primer P5 is: 5 '-AAGCAGTGGTATCAACGCAGAGT-3 '; The sequence of anchor primer P3 is: 5 '-GAGGCGGCCGACATGTTTTT-3 '; Forward special primer S1 sequence is 5 '-CTCCTCTCCAGGTTCTGACATTACT-3 '; Oppositely special primer A1 sequence is 5 '-CTGCTCGGTGTTGGAGATTGG-3 '; The PCR product reclaims test kit through dna gel and reclaims, and inserts pGEM-T Easy carrier, transforms DH 5 α competent cells, blue hickie screening positive clone, alkaline lysis extracting plasmid is after enzyme is cut evaluation, check order, obtain 3 ' the terminal and 5 ' end of the cDNA of the relevant gene of Bc-A8T18 fragment.

(3) full-length clone of PGBc2 gene: according to the cDNA full length sequence design special primer T1 and the T2 of two ends splicing, the sequence of T1 is 5 '-TGGGATTTTCAGCCAATG-3 ', and the sequence of T2 is 5 '-AGAGCATACATCATAGAC-3 '; Conventional PCR method is the cDNA sequence of amplification Bc-A8T18 fragment genes involved from bud cDNA once more; With same special primer amplification in genomic dna is obtained its dna sequence dna SEQ ID No.2.

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