CN102586274B

CN102586274B - Wild cabbage TT16 gene family and application thereof

Info

Publication number: CN102586274B
Application number: CN 201210018474
Authority: CN
Inventors: 柴友荣; 马丽娟; 闫楠; 雷波; 谌利; 吕俊; 李加纳; 马赑; 周清元
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2012-01-19
Filing date: 2012-01-19
Publication date: 2013-04-24
Anticipated expiration: 2032-01-19
Also published as: CN102586274A

Abstract

The invention discloses a wild cabbage TT16 gene family which comprises three members, namely a BoTT16-1 gene (the full-length cDNA (complementary deoxyribonucleic acid) sequence is as shown in SEQ ID No.8 (sequence identity number 8), a BoTT16-2 gene (the full-length cDNA sequence is as shown in SEQ ID No.10) and a BoTT16-3 gene (the full-length sequence is as shown in SEQ ID No.12), and the gene family can be applied to the molecular breeding of seed traits, such as the development of seed size, seed coat pigment accumulation and the like of Brassica crops.

Description

Wild cabbage TT16 gene family and application thereof

The application is that application number is 201010281905.2, and the applying date is 2010-09-15, and invention and created name is divided an application for " swede type rape and parent species Chinese cabbage thereof and wild cabbage TT16 gene family and application thereof ".

Technical field

The present invention relates to gene engineering technology field, be particularly related to swede type rape (Brassica napus) and parent species Chinese cabbage (Brassica rapa) thereof and wild cabbage (Brassica oleracea) TT16 (TRANSPARENT TESTA 16, transparent kind of skin 16; Claim again ABS, ARABIDOPSIS BSISTER; Or AGL32, AGAMOUS-LIKE 32) gene family and application thereof.

Background technology

The rape of Cruciferae (Brassicaceae) belongs to (Brassica) and comprises a lot of oil crops, vegetables and ornamental plant kind, for the mankind provide nutritive value abundant edible oil, vegetables and ornamental plant, and for livestock industry provides feed, has important economic worth.In the rape species, allotrtraploid species swede type rape is redoublingd after by species hybridization by 2 diploid species Chinese cabbages and wild cabbage and is formed.Swede type rape is second-biggest-in-the-world oil crops, extensively plants in the whole world, and cultivated area and output are only second to soybean.Chinese cabbage and wild cabbage also are important oil plant, vegetables and ornamental crops.To provide fundamental basis for the genetic evolution relation that discloses between them to the research of the comparative genomics of swede type rape and parent species Chinese cabbage and wild cabbage functional gene, and provide application foundation for the character improvement of Brassica Crops.

Seed coat color is one of important character of swede type rape.The cabbage type rape yellow seed strain has that kind of skin is thin, the cot rate is low, crude fiber content is low, oleaginousness is high, cake protein content advantages of higher, compares with black seed strain, and the quality of the economic worth of grouts and oil all increases.Although all have the stable natural yellow seed genotype of phenotype in Chinese cabbage and the wild cabbage, there is not natural For Yellow Seed Gene In Brassica Napus type in occurring in nature.Existing cabbage type rape yellow seed material is mainly created by modes such as distant hybirdization, exists yellow seed rate and yellow seed degree not high, and phenotype is unstable, easily affected by environment and make a variation, Breeding Efficiency is low, and breeding cycle is long, the shortcomings such as the negative correlation proterties is difficult to overcome can not satisfy production requirement far away.Therefore, the cabbage type rape yellow seed proterties of acquisition genetic stability becomes the important goal of swede type rape breeding.For a long time, the numerous investigators in the whole world have carried out broad research to this proterties, but up to the present still unclear for the molecule mechanism of yellow seed proterties formation, more do not create any report of yellow seed proterties by the transgenosis molecular breeding.

The flavonoid material is extensively to be present in botanic secondary metabolites, is the present-color material of the anthocyanin pigments such as redness, blueness and purple in the plant tissue.The main component of the Structure of Seed-coat pigments such as Arabidopis thaliana (Arabidopsis thaliana) is the polymkeric substance of pycnogenols (proanthocyanidin, PA) monomer, and it is synthetic through public phenylpropyl alcohol alkane approach-flavonoid path-pycnogenols approach.Present studies show that the biosynthetic regulation and control of flavonoid are to be finished by the synergy of transcription factor, and the space-time characterisation of these transcription factor expression is subject to precision control.Known WD40, MYB, bHLH, MADS-box, WRKY and bZIP transcription factor have all participated in the regulation and control of flavonoid path, and wherein MADS-box (TT16), WRKY (TTG2) and the effect of bZIP (TT1) transcription factor in this approach are also thoroughly not clear and definite.Rape belongs to and Arabidopis thaliana belongs to Cruciferae together, has nearer sibship.Arabidopis thaliana TT16 (AtTT16) genes encoding MADS-box transcription factor, research find that its expression and pycnogenols accumulation in inner seed coat for the BAN gene is essential.It is yellow seed proterties that Arabidopis thaliana tt16 mutant shows as transparent kind of skin, and the inner seed coat cell becomes irregular, infers that this gene may also participate in inner seed coat cytodifferentiation and growth, but concrete effect and mechanism it be unclear that.Therefore, in rape belongs to the TT16 gene being carried out homologous clone and Function Identification, is the important channel in screening cabbage type rape yellow seed site.

Rape belongs to and Arabidopis thaliana originates from same ancestors, before 1700～1,800 ten thousand, separated, the tripling of genomic level has occured in rape family plant, it is that rape belongs to elementary species: Chinese cabbage (AA group, 529Mbp), wild cabbage (CC group, 696Mbp) and black mustard (BB group, genome 632Mbp) etc. is equivalent to 3 times of arabidopsis gene group (157Mbp) approximately, and swede type rape (AACC group, genome 1132Mbp) is equivalent to wild cabbage and two genome sums of Chinese cabbage, be equivalent to approximately 6 times of arabidopsis gene group, that is to say, gene for single copy in Arabidopis thaliana may have respectively the copy of 3 correspondences in wild cabbage and Chinese cabbage, and 6 copies may be arranged in swede type rape.At present less to the research report of TT16 gene, and the tissue specificity of the number of members of TT16 gene in the rape species such as swede type rape, Chinese cabbage, wild cabbage, protein specificity, evolutionary relationship, expression and all have no report with the relation of yellow seed proterties etc.

Summary of the invention

In view of this, one of purpose of the present invention is to provide swede type rape and parent species Chinese cabbage and wild cabbage TT16 gene family.

For achieving the above object, the present invention adopts terminal rapid amplifying (RACE) technology of cDNA, clone respectively swede type rape and parent species Chinese cabbage thereof and wild cabbage TT16 gene family member's full-length cDNA and corresponding genome sequence, and carried out systems analysis.The result shows:

Described Chinese cabbage TT16 (BrTT16) gene family comprises following 3 members: BrTT16-1 gene, BrTT16-2 gene and BrTT16-3 gene; The full length cDNA sequence of described BrTT16-1 gene is shown in SEQ ID No.2, and the full length cDNA sequence of BrTT16-2 gene is shown in SEQ ID No.4, and the full length cDNA sequence of BrTT16-3 gene is shown in SEQ ID No.6;

Described wild cabbage TT16 (BoTT16) gene family comprises following 3 members: BoTT16-1 gene, BoTT16-2 gene and BoTT16-3 gene; The full length cDNA sequence of described BoTT16-1 gene is shown in SEQ ID No.8, and the full length cDNA sequence of BoTT16-2 gene is shown in SEQ ID No.10, and the full length cDNA sequence of BoTT16-3 gene is shown in SEQ IDNo.12;

Described swede type rape TT16 (BnTT16) gene family comprises following 6 members: BnTT16-1 gene, BnTT16-2 gene, BnTT16-3 gene, BnTT16-4 gene, BnTT16-5 gene and BnTT16-6 gene; The full length cDNA sequence of described BnTT16-1 gene is shown in SEQ ID No.14, the full length cDNA sequence of BnTT16-2 gene is shown in SEQ IDNo.16, the full length cDNA sequence of BnTT16-3 gene is shown in SEQ ID No.18, the full length cDNA sequence of BnTT16-4 gene is shown in SEQ ID No.20, the full length cDNA sequence of BnTT16-5 gene is shown in SEQ ID No.22, and the full length cDNA sequence of BnTT16-6 gene is shown in SEQ ID No.24.

Further, the genome sequence of described BrTT16-1 gene is shown in SEQ ID No.1, and the genome sequence of BrTT16-2 gene is shown in SEQ ID No.3, and the genome sequence of BrTT16-3 gene is shown in SEQ ID No.5;

The genome sequence of described BoTT16-1 gene is shown in SEQ ID No.7, and the genome sequence of BoTT16-2 gene is shown in SEQ ID No.9, and the genome sequence of BoTT16-3 gene is shown in SEQ ID No.11;

The genome sequence of described BnTT16-1 gene is shown in SEQ ID No.13, the genome sequence of BnTT16-2 gene is shown in SEQ ID No.15, the genome sequence of BnTT16-3 gene is shown in SEQ ID No.17, the genome sequence of BnTT16-4 gene is shown in SEQ ID No.19, the genome sequence of BnTT16-5 gene is shown in SEQ ID No.21, and the genome sequence of BnTT16-6 gene is shown in SEQ ID No.23.

12 TT16 genes and the AtTT16 gene of above-mentioned 3 species have higher homology, the genome sequence consistence is 67.1～70.3%, the coding region sequence consistence is 82.9～87.0%, the consistence of the aminoacid sequence of proteins encoded and similarity are respectively 73.0～78.2% and 78.2～85.7%, the aspects such as the sequence alignment of nucleic acid level and amino acid levels, system's generation cluster show that all they are Paralog genes of AtTT16 gene.Also has very high homology between 12 TT16 genes of these 3 species, the genome sequence consistence is 69.4～100.0%, the coding region sequence consistence is 85.2～100.0%, and the consistence of the aminoacid sequence of proteins encoded and similarity are respectively 75.1～100% and 80.4～100%; Wherein, the BnTT16-1 of swede type rape, BnTT16-4, BnTT16-6 gene derive from respectively BrTT16-1, BrTT16-2, the BrTT16-3 gene of Chinese cabbage, and the BnTT16-2 of swede type rape, BnTT16-3, BnTT16-5 gene derive from respectively BoTT16-1, BoTT16-2, the BoTT16-3 gene of wild cabbage.BnTT16, BrTT16, BoTT16 gene family have kept and the similar organ specificity of AtTT16 gene, mainly express in reproductive organ, and be the highest with flower and the expression of developmental seed, and descend gradually along with the developmental process of seed.In addition, the TT16 gene exists obvious difference at the black seed of swede type rape and Chinese cabbage and the expression in the yellow seed material, and in the black seed of wild cabbage and yellow seed material no significant difference, the yellow seed proterties that swede type rape and Chinese cabbage are described is relevant with the TT16 down regulation of gene expression, and the yellow seed proterties of wild cabbage and TT16 gene almost it doesn't matter.

Based on the above results, utilize any one or more gene or gene truncated segment in BnTT16 of the present invention, BrTT16, the BoTT16 gene family, can make up TT16 dna recombinant expression carrier and transformant, justice expression, Antisense Suppression, the RNA that is used for the TT16 gene disturbs etc.

Two of purpose of the present invention is to provide described swede type rape and parent species Chinese cabbage and the application of wild cabbage TT16 gene family in the molecular breeding of Brassica Crops seed properties.

Further, described swede type rape and parent species Chinese cabbage thereof and the application of wild cabbage TT16 gene family in the molecular breeding of cabbage type rape yellow seed proterties.

For achieving the above object, it is the RNA interference fragment that the present invention chooses the special conservative section BTT16I (nucleotide sequence is shown in the 353rd～966 bit base among the SEQ ID No.14) of swede type rape and parent species Chinese cabbage and wild cabbage TT16 gene family thereof, disturb carrier is carrier pFGC5941M as skeleton take the plant RNA of transforming based on pFGC5941, BTT16I inserted between the CaMV35S promotor of pFGC5941M and the OCS terminator simultaneously with antisense and just mode respectively form inverted repeats, make up rape and belonged to TT16 gene family rna interference vector pFGC5941M-BTT16I, and transformed in the swede type rape typical black seed kind two No. 10 by the Agrobacterium-mediated Transformation method, the proterties investigation of the positive transfer-gen plant of gained is found, after disturbing reticent BnTT16 gene family by RNA, the background proterties of transfer-gen plant is normal, transgenic seed obviously diminishes and plants skin pigment and obviously reduce, majority is tawny and yellowish brown, with the typical black seed formation sharp contrast of non-transgenic seed.Explanation is in the plants such as swede type rape, the TT16 gene is regulated and control the formation of the proterties such as seed size is grown, the accumulation of kind skin pigment simultaneously, can be applied to the molecular breeding of Brassica Crops seed properties, especially the molecular breeding of cabbage type rape yellow seed proterties, be beneficial to and create novel cabbage type rape yellow seed material, for increasing the size of seed, improve thousand grain weigth after also can overexpression.

Beneficial effect of the present invention is: the invention provides tissue specificity of the number of members of TT16 gene in swede type rape and parent species Chinese cabbage and wild cabbage, each member's full length cDNA sequence and genome sequence, proteins encoded feature, evolutionary relationship, expression etc., and confirmed that the TT16 gene family participates in the growth of seed size and the accumulation of kind skin pigment simultaneously, the invention provides thus the TT16 gene and improve application in the molecular breeding of cabbage type rape yellow seed proterties particularly at the Brassica Crops seed properties, application prospect is good.

Description of drawings

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is described in further detail below in conjunction with accompanying drawing, wherein:

Fig. 1 is the amplification of BnTT16 (A), BrTT16 (B), BoTT16 (C) gene family 5 ' cDNA end.

Fig. 2 is the amplification of BnTT16 (A), BrTT16 (B), BoTT16 (C) gene family 3 ' cDNA end.

Fig. 3 is the amplification of BnTT16 (A), BrTT16 (B), BoTT16 (C) gene family member full-length cDNA, wherein 1 adopt combination of primers FBT16-1+RBT16-2,2 adopt combination of primers FBT16-3+RBT16-4, and 3 adopt combination of primers FBT16-5+RBT16-6.

Fig. 4 is the amplification of BnTT16 (A), BrTT16 (B), BoTT16 (C) gene family member genomic dna, wherein 1 adopt combination of primers FBT16-1+RBT16-2,2 adopt combination of primers FBT16-3+RBT16-4, and 3 adopt combination of primers FBT16-5+RBT16-6.

Fig. 5 is the sequence alignment of BrTT16, BoTT16, BnTT16 gene family member and AtTT16 gene mRNA.

Fig. 6 is the aminoacid sequence comparison of BrTT16, BoTT16, BnTT16 family protein and AtTT16 albumen.

Fig. 7 is the cluster analysis of BrTT16, BoTT16, BnTT16 gene family member and AtTT16 gene mRNA.

Fig. 8 is the cluster analysis of BrTT16, BoTT16, BnTT16 family protein and AtTT16 albumen.

Fig. 9 is that BrTT16, BoTT16, BnTT16 gene family member's Southern hybridization identifies that wherein M is the molecular weight standard of digoxigenin labeled.

Figure 10 is the overall and member's of BrTT16, BoTT16, BnTT16 gene family organ specificity detection of expression.

Figure 11 is the expression of the overall and member of TT16 gene family in the black seed of Chinese cabbage, wild cabbage, swede type rape, the yellow seed material Main Reproductive Organs.

Figure 12 is the pcr amplification of RNA interference fragment BTT16I.

Figure 13 is the structure iron of rna interference vector pFGC5941M-BTT16I.

Figure 14 is that the enzyme during rna interference vector pFGC5941M-BTT16I makes up is cut the evaluation with PCR, and wherein A is Swa I+Aat II double digestion pMD19-T-BTT16I, and M is DNA marker, and CK represents enzyme and cuts front pMD19-T-BTT16I; B is Swa I+AatII double digestion pFGC5941M, and M is DNA marker, and CK represents enzyme and cuts front pFGC5941M; C is that clone's daughter bacteria liquid PCR of pFGC5941M-BTT16IA detects; D is BamH I+Xba I double digestion pMD19-T-BTT16I, and M is DNA marker, and CK represents enzyme and cuts front pMD19-T-BTT16I; E is BamH I+Xba I double digestion pFGC5941M-BTT16IA; M is DNA marker, and CK represents enzyme and cuts front pFGC5941M-BTT16IA; F is that clone's daughter bacteria liquid PCR of pFGC5941M-BTT16I detects, and M is DNA marker.

Figure 15 is that the Basta of regeneration plant rechecks evaluation, and wherein CK is the non-transgenic plant, and 1-3 is regeneration plant.

Figure 16 is the PCR detected result of regeneration plant, and wherein M is DNA marker, the positive contrast of CK, and 1 negative contrast, 2-15 is regeneration plant.

Figure 17 is the comparison of transgenic seed (left side) and non-transgenic seed (right side).

Embodiment

Hereinafter with reference to accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in the preferred embodiment, usually according to normal condition, the molecular cloning experiment guide (third edition for example, J. the work such as Pehanorm Brooker, Huang Peitang etc. translate, Science Press, 2002) described in condition, or the condition of advising according to manufacturer.

The vegetable material that preferred embodiment adopts: the Chinese cabbage material all comes from turnip type rape subspecies (B.rapa ssp.oleifera), comprises that black seed is that 09L597 and yellow seed are 09L600; The wild cabbage material all comes from kale mutation (B.oleracea var.acephala), comprises that black seed is that 09L598 and yellow seed are 09L599; Brassica napus comprises that black seed is 5B and seed look near isogenic line (black seed is that 09L588, yellow seed are 09L587), by Chongqing City's rape Engineering Technical Research Centre seed selection and land for growing field crops general planting, and has passed through the above simple inflorescence bagging selfing of 10 generations; In the black seed kind of swede type rape two No. 10 by the seed selection of cole crop institute of the Chinese Academy of Agricultural Sciences, seed is provided by Chongqing City's rape Engineering Technical Research Centre.

The reagent that preferred embodiment adopts: [5U/ μ l, attached 10 * PCR Buffer (contains Mg to the Easy-Taq archaeal dna polymerase ²⁺)] available from the Beijing Quanshijin Biotechnology Co., Ltd; [5U/ μ l, attached 10 * LA PCR Buffer II (contains Mg to LA Taq archaeal dna polymerase ²⁺)], λ-HindIII DNA marker etc. is available from precious biotechnology (Dalian) company limited; The DNA marker of restriction enzyme DraI, EcoRI, EcoRV, HindIII, nylon membrane, digoxigenin labeled etc. are available from Lithuania MBI Fermentas company; The pMD19-T carrier is available from precious biotechnology (Dalian) company limited, MS (Murashige and Skoog medium) substratum is available from Dutch Duchefa company, gelling gum is available from Zhejiang Zhongken Biotechnology Co., Ltd., and other molecule, biochemistry and plant tissue culture reagent are sowed rich garden supplies company limited available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd and Shanghai; The modified version plant RNA disturbs carrier is carrier pFGC5941M to improve on the basis of pFGC5941 to form, improvements are to adopt from the BnPAP2 gene intron 2 (BnPAP2I2) of swede type rape to replace the upper long PhChsA transcribed spacer of pFGC5941, and increase an AatII point of contact between transcribed spacer and promotor.

The test kit that preferred embodiment adopts: plant tissue RNA extraction agent box (W7021) is available from Shanghai China Shun biotechnology company limited in a small amount; glue recovery test kit and plasmid extraction test kit are available from Omego company in a small amount; the pMD19-T carrier connects test kit available from precious biotechnology (Dalian) company limited; GeneRacer Kit is available from American I nvitrogen company; PCR DIGProbe Synthesis Kit; DIG Easy Hyb; DIG Wash and Block Buffer Set; all available from German Roche company, RNA PCR Kit (AMV) Ver.3.0 is available from precious biotechnology (Dalian) company limited for DIG Nucleic AcidDetection Kit.

One, the clone of swede type rape and parent species Chinese cabbage thereof and wild cabbage TT16 gene family

1, the extraction of swede type rape, Chinese cabbage and wild cabbage genome DNA

Get the swede type rape 5B, the Chinese cabbage 09L597 that cultivate under the normal condition of land for growing field crops and the tender leaf of wild cabbage 09L598, adopt hexadecyl trimethyl ammonium bromide (CTAB) method to extract genome DNA, adopt electrophoretic method and spectrophotometry to estimate quality and the concentration of nucleic acid samples.1.0% agarose gel electrophoresis result shows, the genome DNA integrity of 3 species that extract is good, molecular-weight average is all greater than the 23kb band of λ-HindIII DNA Marker, RNA digestion is more complete, higher through spectrophotometry purity, can be directly used in pcr amplification and Southern hybridization.

2, the extraction of the total RNA of each organ of swede type rape, Chinese cabbage and wild cabbage

[swede type rape and wild cabbage take away the seed of spending rear 15 days (15D), 30 days (30D), 45 days (45D) and 55 days (55D) to get flower bud (Bu), flower (Fl) and the seed of 4 etap of the swede type rape 5B, the Chinese cabbage 09L597 that cultivate under the normal condition of land for growing field crops and wild cabbage 09L598; Chinese cabbage takes away the seed of spending rear 10 days (10D), 25 days (25D), 40 days (40D) and 45 days (45D)]; And root (Ro), hypocotyl (Hy), cotyledon (Co), stem (St), leaf (Le), flower bud, flower, pod skin (SP) and the seed of 4 etap of the swede type rape 09L587 that cultivates under the normal condition of land for growing field crops and 09L588, Chinese cabbage 09L597 and 09L600, wild cabbage 09L598 and 09L599 (swede type rape and wild cabbage are got the seed of 15D, 30D, 45D and 55D; Chinese cabbage is got the seed of 10D, 25D, 40D and 45D) totally 12 organs; Adopt the total RNA extraction agent of a small amount of plant box to extract the total RNA of each organ, adopt electrophoretic method and spectrophotometry to estimate quality and the concentration of nucleic acid samples.1.0% agarose gel electrophoresis result shows that total RNA feature band of acquisition is clear, and without obviously RNA degraded and DNA pollution, higher through spectrophotometry purity, can satisfy the basic demand of RACE operation.

3, the acquisition of swede type rape, Chinese cabbage and wild cabbage RACE the first chain cDNA

Get respectively swede type rape 5B, the flower bud of Chinese cabbage 09L597 and wild cabbage 09L598, total RNA of the seed of flower and 4 etap is mixed into the RNA sample that total amount is 5 μ g, adopt GeneRacer Kit to carry out a series of RACE operation by its specification sheets, final reverse transcription obtains to hold simultaneously grappling that the first chain cDNA of manual splice sequence is arranged at 3 ' end and 5 ', carrying out 1.0% agarose gel electrophoresis after PCR amplifies detects, the result shows, the first chain cDNA of three species presents size at the traction of 200bp～10kb, the center of gravity zone is at 500bp～4kb, most crucial district is about 1.5kb, illustrate that reverse transcription is more complete, obtain total cDNA of better quality, can be used for cloning swede type rape, the cDNA that Chinese cabbage and wild cabbage TT16 gene family are complete is terminal.

4, the clone of swede type rape, Chinese cabbage and wild cabbage TT16 gene family 5 ' cDNA end

According to the right result of AtTT16 gene order multiple ratio, reverse primer RTT16-50 corresponding to two points the most conservative (5 '-ggatcgttttgaagattaggctgagcaag-3 ') and RBT16RT (5 '-gctcgtgtggaggaatggagg-3 ') have been designed.Respectively take Chinese cabbage, wild cabbage, swede type rape the first chain cDNA as template, the primer 5 ' P that provides with GeneRacer Kit (5 '-cgactggagcacgaggacactga-3 ') and RTT16-50 pairing, the RACE one that carries out 5 ' cDNA end expands.50 μ l standard TaqPCR amplification systems are: 10 * PCR Buffer, 5.0 μ l, the MgCl of 25mmol/L ₂3.0 μ l, the dNTPs1.0 μ l of 10mmol/L, the forward primer 1.0 μ l of 10 μ mol/L, the reverse primer 1.0 μ l of 10 μ mol/L, the Taq enzyme 0.5 μ l of 5U/ μ l, template 0.5 μ l, adding distilled water to cumulative volume is 50 μ l.The pcr amplification loop parameter is: 94 ℃ of denaturations 2 minutes; 94 ℃ of sex change are 1 minute again, 52 ℃ of annealing 1 minute, and 72 ℃ were extended totally 30 circulations 1 minute; Last 72 ℃ were extended 10 minutes.

Expand production thing as template take one, primer 5 ' the NP that provides with GeneRacer Kit (5 '-ggacactgacatggactgaaggagta-3 ') and RBT16RT pairing, the RACE nest that carries out 5 ' cDNA end expands, pcr amplification system and one expands identical but template changes 0.1 μ l into, and the pcr amplification loop parameter is: 94 ℃ of denaturations 2 minutes; 94 ℃ of sex change are 1 minute again, 58 ℃ of annealing 1 minute, and 72 ℃ were extended totally 25 circulations 1 minute; Last 72 ℃ were extended 10 minutes.The PCR product carries out 1.0% agarose gel electrophoresis and detects (Fig. 1), adopting in a small amount, glue reclaims test kit recovery target fragment, be connected with the pMD19-T carrier, transform again the bacillus coli DH 5 alpha competent cell, with containing penbritin (Amp), it is clear that the LB flat board of IPTG and X-gal is cultured to blue hickie, picking hickie list bacterium colony, after increasing bacterium and cultivate with the LB liquid nutrient medium that contains Amp, get bacterium liquid and carry out the PCR detection, the positive colony sublist reveals obvious length polymorphism as a result, respectively selects 10 representative positive colony and entrusts the English Weihe River, Shanghai Jie Ji Bioisystech Co., Ltd to check order.Sequencing result shows: 5 ' cDNA end of BrTT16 gene family is between 429～681bp, and 5 ' cDNA end of BoTT16 gene family is between 448～658bp, and 5 ' cDNA end of BnTT16 gene family is between 526～649bp.NCBI BLASTn shows that these 5 ' cDNA are terminal to have very high consistence with AtTT16/ABS mRNA (AJ318098), shows that they belong to 5 ' the cDNA end of TT16 gene family really for rape.

5, the clone of swede type rape, Chinese cabbage and wild cabbage TT16 gene family 3 ' cDNA end

According to the right result of AtTT16 gene order multiple ratio, forward primer FTT16-30 (5 '-tgagctctct (g/a) ttctctg (c/t) gatgc-3 ') corresponding to two points the most conservative and FTT16-3N (5 '-ctcacatcggtctcatcgtcttctc-3 ') have been designed.Respectively take Chinese cabbage, wild cabbage, swede type rape the first chain cDNA as template, the primer 3 ' P that provides with GeneRacer Kit (5 '-gctgtcaacgatacgctacgtaacg-3 ') and FTT16-30 pairing, the RACE one that carries out 3 ' cDNA end expands.The pcr amplification system expands identical with the amplification cycles parameter with the RACE one of 5 ' cDNA end.

Expand production thing as template take one, primer 3 ' the N P that provides with GeneRacer Kit (5 '-cgctacgtaacggcatgacagtg-3 ') and FTT16-3N pairing, the RACE nest that carries out 3 ' cDNA end expands, and the pcr amplification system expands identical with the amplification cycles parameter with the RACE nest of 5 ' cDNA end.The PCR product carries out electrophoresis detection (Fig. 2), glue recovery, pMD19-T carrier cloning, conversion competent escherichia coli cell, the screening of positive colony, bacterium liquid PCR evaluation and order-checking as described in front method.Sequencing result shows: 3 ' cDNA end of BrTT16 gene family is between 778～853bp, 3 ' cDNA end of BoTT16 gene family is between 733～936bp, and 3 ' cDNA end of BnTT16 gene family [does not all comprise poly (A)] between 687～820bp.NCBI BLASTn shows that these 3 ' cDNA are terminal to have very high consistence with AtTT16/ABS mRNA gene, shows that they belong to 3 ' the cDNA end of TT16 gene family really for rape.

6, the clone of swede type rape, Chinese cabbage and wild cabbage TT16 gene family member full-length cDNA

Sequencing result according to BrTT16, BoTT16, BnTT16 gene family 5 ' and 3 ' cDNA end, design 3 forward primers and 3 reverse primers (table 1), obtained 3 couples of combination of primers: FBT16-1+RBT16-2, FBT16-3+RBT16-4, FBT16-5+RBT16-6; Respectively take Chinese cabbage, wild cabbage, swede type rape the first chain cDNA as template, adopt above-mentioned combination of primers and 50 μ l standard Taq pcr amplification systems, amplification BnTT16, BrTT16, each member's of BoTT16 gene family full-length cDNA, the pcr amplification loop parameter is: 94 ℃ of denaturations 2 minutes, 1 minute, 72 ℃ extensions of 1 minute, 54～57 ℃ annealing of 94 ℃ of sex change are 2 minutes again, totally 35 circulations, last 72 ℃ were extended 10 minutes; Front method is describedly carried out electrophoresis detection (Fig. 3), glue recovery, pMD19-T carrier cloning, is transformed competent escherichia coli cell for another example, the screening of positive colony, bacterium liquid PCR identifies and order-checking.

The amplimer of table 1 BrTT16, BoTT16, BnTT16 gene family member full-length cDNA

Result: take the total cDNA of Chinese cabbage the first chain as template, combination of primers is respectively FBNA10-6+RBRA10-10, FBT16-3+RBT16-4, FBT16-5+RBT16-6, each amplification obtains the full-length cDNA that 1 length is respectively 1060bp, 1242bp, 1123bp, respectively called after BrTT16-1mRNA, BrTT16-2mRNA and BrTT16-3mRNA.

Take the total cDNA of wild cabbage the first chain as template, combination of primers is respectively FBNA10-6+RBRA10-10, FBT16-3+RBT16-4, FBT16-5+RBT16-6, each amplification obtains the full-length cDNA that 1 length is respectively 1087bp, 1028bp, 1134bp, respectively called after BoTT16-1mRNA, BoTT16-2mRNA and BoTT16-3mRNA.

Take the total cDNA of swede type rape the first chain as template, combination of primers is FBT16-1+RBT16-2, and amplification obtains the full-length cDNA that 2 length are respectively 1060bp and 1084bp, respectively called after BnTT16-1mRNA and BnTT16-2mRNA; Combination of primers is FBT16-3+RBT16-4, and amplification obtains the full-length cDNA that 2 length are respectively 1214bp and 1243bp, respectively called after BnTT16-3mRNA and BnTT16-4mRNA; Combination of primers is FBT16-5+RBT16-6, and amplification obtains the full-length cDNA that 2 length are respectively 1128bp and 1123bp, respectively called after BnTT16-5mRNA and BnTT16-6mRNA.

Vector NTI Advance 9.0 multiple ratios are to showing, the full-length cDNA of the BnTT16 of gained, BrTT16, BoTT16 gene family all has the terminal clone of corresponding RACE subsequence corresponding with it, illustrates that they all are genes of transcribed expression.Multiple ratio is to also showing, terminal each the indicated separate gene of RACE has all obtained corresponding full-length cDNA in 3 species.

7, the clone of swede type rape, Chinese cabbage and wild cabbage TT16 gene family member genomic dna

According to the multiple comparison result of sequence of BrTT16, BoTT16, BnTT16 gene family member full-length cDNA, design detects each member's Specific primer pair: FBT16-1S+RBT16-12S, FBT16-2S+RBT16-12S, FBT16-34S+RBT16-3S, FBT16-34S+RBT16-4S, FBT16-56S+RBT16-5S, FBT16-56S+RBT16-6S (table 2); According to BrTT16, BoTT16, the clone result of BnTT16 gene family RACE end and full-length cDNA, respectively with Chinese cabbage 09L597, wild cabbage 09L598, the genome DNA of swede type rape 5B is template, adopt the amplimer combination of above-mentioned full-length cDNA to carry out identical PCR with 50 μ l standard Taq pcr amplification systems, amplification BnTT16, BrTT16, each member's of BoTT16 gene family genomic dna, the for another example described electrophoresis detection (Fig. 4) of carrying out of front method, glue reclaims, the pMD19-T carrier cloning, transform competent escherichia coli cell, the screening of positive colony, bacterium liquid PCR identifies and special primer is identified (carrying out grads PCR with above-mentioned Specific primer pair), at last order-checking.

Table 2 detects BrTT16, BoTT16, BnTT16 gene family member's special primer

Result: take the Chinese cabbage genome DNA as template, amplimer is combined as FBT16-1+RBT16-2, and Specific primer pair is FBT16-1S+RBT16-12S, the genomic dna that to obtain 1 length be 2615bp, its exon 1 and BrTT16-1cDNA are in full accord, called after BrTT16-1 gene; Amplimer is combined as FBT16-3+RBT16-4, and Specific primer pair is FBT16-34S+RBT16-4S, the genomic dna that to obtain 1 length be 2730bp, and its exon 1 and BrTT16-2cDNA are in full accord, called after BrTT16-2gene; Amplimer is combined as FBT16-5+RBT16-6, and Specific primer pair is FBT16-56S+RBT16-6S, the genomic dna that to obtain 1 length be 2674bp, and its exon 1 and BrTT16-3cDNA are in full accord, called after BrTT16-3gene.

Take the wild cabbage genome DNA as template, amplimer is combined as FBT16-1+RBT16-2, and Specific primer pair is FBT16-2S+RBT16-12S, the genomic dna that to obtain 1 length be 2625bp, its exon 1 and BoTT16-1cDNA are in full accord, called after BoTT16-1gene; Amplimer is combined as FBT16-3+RBT16-4, and Specific primer pair is FBT16-34S+RBT16-3S, the genomic dna that to obtain 1 length be 2632bp, and its exon 1 and BoTT16-2cDNA are in full accord, called after BoTT16-2gene; Amplimer is combined as FBT16-5+RBT16-6, and Specific primer pair is FBT16-56S+RBT16-5S, the genomic dna that to obtain 1 length be 2763bp, and its exon 1 and BoTT16-3cDNA are in full accord, called after BoTT16-3gene.

Take the swede type rape genome DNA as template, amplimer is combined as FBT16-1+RBT16-2, Specific primer pair is respectively FBT16-1S+RBT16-12S and FBT16-2S+RBT16-12S, obtain the genomic dna that 2 length are respectively 2616bp and 2622bp, its exon 1 is in full accord with BnTT16-1cDNA and BnTT16-2cDNA respectively, called after BnTT16-1gene and BnTT16-2gene; Amplimer is combined as FBT16-3+RBT16-4, Specific primer pair is respectively FBT16-34S+RBT16-3S and FBT16-34S+RBT16-4S, obtain the genomic dna that 2 length are respectively 2632bp and 2729bp, its exon 1 is in full accord with BnTT16-3cDNA and BnTT16-4cDNA respectively, called after BnTT16-3gene and BnTT16-4gene; Amplimer is combined as FBT16-5+RBT16-6, Specific primer pair is respectively FBT16-56S+RBT16-5S and FBT16-56S+RBT16-6S, obtain the genomic dna that 2 length are respectively 2707bp and 2678bp, its exon 1 is in full accord with BnTT16-5cDNA and BnTT16-6cDNA respectively, called after BnTT16-5gene and BnTT16-6gene.

Two, the analysis of swede type rape and parent species Chinese cabbage thereof and wild cabbage TT16 gene family

Carry out sequence alignment at Vector NTI Advance 9.0 softwares, open reading frame (ORF) is searched and is translated, carry out the CDD search of BLAST and protein sequence in http://www.ncbi.nlm.nih.gov website, provide the information biology on-line analysis software of link to carry out structural analysis of protein in websites such as http://bip.weizmann.ac.il/ and http://www.expasy.org, carry out in websites such as http://prodes.toulouse.inra.fr/multalin/multalin.html and http://www.ebi.ac.uk/clustalw/ gene and protein sequence multiple ratio to and cluster analysis.

1, the nucleic acid sequence analysis of swede type rape, Chinese cabbage and wild cabbage TT16 gene family

BrTT16, BoTT16, BnTT16 gene family member's genomic dna basic parameter is as shown in table 3, and the full-length cDNA basic parameter is as shown in table 4, and G+C content is as shown in table 5.

The basic parameter of table 3BnTT16, BrTT16, BoTT16 gene family member genomic dna

The basic parameter of table 4BnTT16, BrTT16, BoTT16 gene family member full-length cDNA

The G+C content (%) of table 5BnTT16, BrTT16, BoTT16 gene

By table 3～5 as can be known, BrTT16, BoTT16,12 members' of BnTT16 gene family genomic dna forms by 6 introns and 7 exons, the length of 6 introns is respectively between 99～298bp, 818～922bp, 70～118bp, 102～108bp, 83～103bp and 77～100bp, and all intron montage borders are standard compliant " GT...AG " rule all.12 member ORF are between 723～738bp, and G+C content is between 44.4～50.0%, apparently higher than non-coding region.12 members have abundant transcription initiation site polymorphism and the 1st intron mutability montage, cause each member's 5 ' UTR length and sequence difference larger, and length is between 121～321bp, and G+C content is between 36.4～44.8%.Because the polymorphism in tailing site, each member's 3 ' UTR length also exists difference, and between 189～206bp, G+C content is between 30.3～32.8%.Among 12 members, all there are typical tailing signal AAATAAA in BnTT16-3, BnTT16-4 and BrTT16-2 gene, BnTT16-1 and BoTT16-1 gene are not found typical tailing signal, and other member has then found a possible atypia tailing signal AATGAATGAATGAA.

NCBI BLASTn the analysis showed that, BnTT16, BrTT16, BoTT16 gene family and Arabidopis thaliana AtTT16/ABSmRNA (AJ318098) have highest homology, illustrate that 12 members of BnTT16, BrTT16, BoTT16 gene family that the clone obtains are the Paralog gene of AtTT16, the TT16 gene member in the same species consists of the Ortholog gene.

The nucleotide sequence of BnTT16, BrTT16, BoTT16 gene family member and AtTT16 gene in twos comparison result shown in table 6 and table 7, the multiple comparison result of mRNA as shown in Figure 5, the cluster analysis of mRNA is as shown in Figure 7.

The genome sequence comparison of table 6BnTT16, BrTT16, BoTT16 gene family member and AtTT16 gene

The coding region sequence comparison of table 7BnTT16, BrTT16, BoTT16 gene family member and AtTT16 gene

By table 6～7 as can be known, have higher homology between 12 TT16 genes of 3 species, the consistence of genome sequence is 69.4～100.0%, and the consistence of coding region sequence is 85.2～100.0%; They and AtTT16 gene also have very high homology, and the consistence of genome sequence is 67.1～70.3%, and the consistence of coding region sequence is 82.9～87.0%.BrTT16-1 and BnTT16-1 genomic level consistence are 99.8%, and the coding region consistence is 100.0%; BrTT16-2 and BnTT16-4 genomic level consistence are 99.7%, and the coding region consistence is 99.9%; BrTT16-3 and BnTT16-6 genomic level consistence are 99.9%, and the coding region consistence is 100.0%.BoTT16-1 and BnTT16-2 genomic level consistence are 99.7%, and the coding region consistence is 99.6%; BoTT16-2 and BnTT16-3 genomic level consistence are 100.0%, and the coding region consistence is 100%; BoTT16-3 and BnTT16-5 genomic level consistence are 97.4%, and the coding region consistence is 99.2%.BnTT16-1, the BnTT16-4 of swede type rape, BrTT16-1, BrTT16-2, the BrTT16-3 gene that the BnTT16-6 gene derives from respectively Chinese cabbage are described, and BnTT16-2, BnTT16-3, BnTT16-5 gene derive from respectively BoTT16-1, BoTT16-2, the BoTT16-3 gene of wild cabbage, swede type rape is the allotrtraploid species of Chinese cabbage and wild cabbage, has the summation of Chinese cabbage and wild cabbage TT16 gene.Characteristic variation base (Fig. 5) and phylogenetic relationship (Fig. 7) from gene order are also supported conclusions.

2, the proteins encoded analysis of swede type rape, Chinese cabbage and wild cabbage TT16 gene family

The basic parameter of BrTT16, BoTT16, BnTT16 family protein is as shown in table 8.

The basic parameter of table 8BrTT16, BoTT16, BnTT16 family protein

As shown in Table 8, BrTT16, BoTT16,12 albumen sizes of BnTT16 family are between 240～256aa, and molecular weight is between 28.11～30.41kDa, and iso-electric point is between 6.44～7.89; Major Members is similar to AtTT16, is slightly acidic albumen, but BnTT16-4, BrTT16-2 and BoTT16-2 are basic protein, and each protein sequence Semi-polarity amino acid proportion is the highest, secondly is hydrophobic amino acid.

The aminoacid sequence comparison result of BnTT16, BrTT16, BoTT16 family protein and AtTT16 albumen is shown in table 9 and table 10, and multiple comparison result as shown in Figure 6.

The consistence (%) of table 9BrTT16, BoTT16, BnTT16 family protein and AtTT16 protein sequence

The similarity (%) of table 10BrTT16, BoTT16, BnTT16 family protein and AtTT16 protein sequence

By table 9～10 and Fig. 6 as can be known, have very high homology between 12 TT16 albumen of 3 species, consistence is 75.1～100%, and similarity is 80.4～100%; They and AtTT16 also have very high homology, and consistence is 73.0～78.2%, and similarity is 78.2～85.7%.Simultaneously, still can clearly find out between 6 albumen of BnTT16 family and BoTT16, the BrTT16 family protein to have the evolution corresponding relation from the homology of protein level.

The cluster analysis of BrTT16, BoTT16, BnTT16 family protein and AtTT16 albumen as shown in Figure 8, BnTT16-1 and BrTT16-1 at first meet, BnTT16-2 and BoTT16-1 at first meet, then these 4 poly-be one group; BnTT16-3 and BrTT16-2 at first meet, and BnTT16-4 and BoTT16-2 at first meet, then these 4 poly-be one group; BnTT16-5 and BrTT16-3 at first meet, and BnTT16-6 and BoTT16-3 at first meet, then these 4 poly-be one group.Proving again rape belong to the evolutionary relationship of 3 species TT16 genes.

Software prediction BrTT16, BoTT16,12 albumen of BnTT16 family do not have signal peptide, N-glycosylation site and transbilayer helix.NetPhos 2.0 predictions show a plurality of phosphorylation sites of their ubiquities, BnTT16-1, BnTT16-2, BrTT16-1 and BrTT16-1 respectively have 11, BnTT16-5, BnTT16-6, BrTT16-3 and BoTT16-3 respectively have 9, BnTT16-4 and BrTT16-2 respectively have 6, BnTT16-3 and BoTT16-2 respectively have 5, and phosphorylation may participate in their protein-active and regulate.The WoLFPSORT prediction shows that these 12 albumen all are positioned can be combined with DNA in the nucleus.The PredictNLS prediction shows, BnTT16-1 and BrTT16-1 respectively have a nuclear localization signal RKVRERKKELLQQQL-GNLSRKKR, BnTT16-1 and BoTT16-1 respectively have a nuclear localization signal RKKELLQQQLGNLSRKRRM, BoTT16-2 has two nuclear localization signal KKKRR and SRKRRM, and other member respectively has a nuclear localization signal SRKRRM.SOPMA prediction shows, the secondary structure main body of 12 TT16 albumen is at random curling and α spiral, also has certain extended chain, does not contain β-bend, and large alpha-helix mainly concentrates on the middle part of albumen, and each member's secondary structure is similar.SWISS-MODEL and ESyPred3D fail to dope the tertiary structure of 12 TT16 albumen.

3, the number of members of swede type rape, Chinese cabbage and wild cabbage TT16 gene family detects

Adopting respectively restriction enzyme DraI, EcoRI, EcoRV and HindIII enzyme to cut the black seed of Chinese cabbage is that the black seed of 09L597, kale is that the black seed of 09L598, swede type rape is the genome DNA of 5B, then carry out 0.8% agarose gel electrophoresis, alkaline denaturation and neutralization, with capillary tube technique DNA is transferred on the positively charged nylon membrane.Adopt the 305bp fragment of combination of primers FTT16-32 and RTT16-50 amplification BoTT16-3mRNA coding region 3 ' end, and adopt PCR DIG ProbeSynthesis Kit with digoxin (digoxigenin, DIG)-dUTP on the target fragment mark as hybridization probe; The PCR loop parameter of probe mark is: 94 ℃ of denaturations 2 minutes, and 1 minute, 72 ℃ of 1 minute, 52 ℃ annealing of 94 ℃ of sex change were extended 1 minute again, totally 35 circulations, last 72 ℃ were extended 10 minutes.Adopt this probe 43 ℃ of Southern hybridization (DIG Easy Hyb) of respectively above-mentioned 3 species gene group DNA being carried out 16 hours, carry out immunodetection (DIG Washand Block Buffer Set and DIG Nucleic Acid Detection Kit) after the rigorous washing, and hybridization colour developing band is taken pictures.

The Southern results of hybridization as shown in Figure 9, the swede type rape genomic dna has not produced 4,5,6,6 hybridization bands through DraI, EcoRI, EcoRV, the cutting of HindIII enzyme, Chinese cabbage and wild cabbage genomic dna have produced 3,4,4,3 hybridization bands respectively through DraI, EcoRI, EcoRV, HindIII, basically identical with the gene number of members that the actual clone in front obtains, can determine substantially that swede type rape, Chinese cabbage, wild cabbage TT16 gene family number of members are respectively 6,3,3.

4, the tissue and organ specificity detection of expression of swede type rape, Chinese cabbage and wild cabbage TT16 gene family

Getting respectively the black seed of Chinese cabbage is that the black seed of 09L597, wild cabbage is total RNA that the black seed of 09L598, swede type rape is each 12 organ of 5B, adopt sxemiquantitative RT-PCR detect BrTT16, BoTT16, BnTT16 gene family in the different tissues organ overall expression and each member's tissue expression specificity.Adopt forward primer FBT16RT (5 '-gatgctcacatcggtctcatcg-3 ') and reverse primer RBT16RT (5 '-gctcgtgtggaggaatggagg-3 ') detection BrTT16, BoTT16, the overall expression of BnTT16 gene family in 12 organs.Adopt Specific primer pair FBT16-1S+RBT16-12S, FBT16-34S+RBT16-4S, FBT16-56S+RBT16-6S, FBT16-1S+RBT16-12S, FBT16-2S+RBT16-12S, FBT16-34S+RBT16-3S, FBT16-56S+RBT16-5S, FBT16-2S+RBT16-12S, FBT16-34S+RBT16-3S, FBT16-34S+RBT16-4S, FBT16-56S+RBT16-5S, FBT16-56S+RBT16-6S detects respectively BrTT16-1, BrTT16-2, BrTT16-3, BoTT16-1, BoTT16-2, BoTT16-3, BnTT16-1, BnTT16-2, BnTT16-3, BnTT16-4, BnTT16-5, the expression of BnTT16-6 gene in 12 organs.Adopt 50 μ l standard Taq pcr amplification systems, the amplification cycles parameter is: 94 ℃ of denaturations 2 minutes, 1 minute, 72 ℃ extensions of 1 minute, 62～68 ℃ (selecting according to primer) annealing of 94 ℃ of sex change are 1 minute again, totally 35 circulations, and last 72 ℃ were extended 10 minutes.

Detected result as shown in figure 10, internal standard gene 26S rRNA through the PCR of 20 circulations in 12 organs of Chinese cabbage, wild cabbage, the black seed material of swede type rape, all amplified brightness more consistent band, initial total RNA amount, reverse transcription efficient, PCR efficient that each organ is described etc. is more consistent, and the tissue and organ specificity expression result who carries out on this basis is believable.The overall expression of BrTT16 gene family is the strongest in 10 days seeds at flower with after spending, and secondly is to spend rear 25 days, 40 days seeds and flower bud; Each member's expression characteristic is to totally similar, expresses at flower and after spending the byest force in 10 days in the seed, reduces gradually along with the seed development process, do not express in root, hypocotyl, cotyledon, stem, leaf and pod skin or only has faint expression; In 3 members, the gene expression abundance of BrTT16-1 gene is the highest, at flower bud, flower, spend after predominant expression in 10 days, 25 days seeds, in hypocotyl, cotyledon, stem, leaf and pod skin, weak expression is arranged also, in root without expressing; The organ specificity of BrTT16-3 gene is similar with BrTT16-1, and just gene expression abundance is lower slightly; The gene expression abundance of BrTT16-2 gene in reproductive organ is similar with BrTT16-3 to BrTT16-1, but the expression in other organ is very weak or nothing.The BoTT16 gene family totally has expression in the main histoorgan of wild cabbage, but abundance is different, spend rear 15 days seeds the highest, flower, spend rear 30 days, 45 days seeds, pod skin, flower buds, spend rear 55 days seeds to take second place, also have weak in leaf, hypocotyl, cotyledon, stem, the root or trace is expressed; Each member's expression characteristic is to totally similar, but the organ specificity of BoTT16-1 gene is the strongest, only expresses in reproductive organ, especially predominant expression in 15 days seeds after spending; BoTT16-2 and BoTT16-3 gene are also mainly expressed in reproductive organ, but the expression of weak or trace is also arranged in vegetative organ.The overall expression of BnTT16 gene family is the strongest in 15 days seeds at flower with after spending, secondly be spend rear 30 days, 45 days, spend rear 55 days seeds, flower bud, in hypocotyl, cotyledon, stem, leaf, pod skin, root, also have weak or utmost point weak expression; Each member's organ specificity is namely mainly expressed in reproductive organ to totally similar, expresses the highlyest in developmental seed, and reduces gradually along with the seed development process; But also variant between each member, on expression level, BnTT16-2 and BnTT16-4 gene are the highest, then are followed successively by BnTT16-5, BnTT16-6, BnTT16-3, BnTT16-1 gene; On organ specificity, BnTT16-5 is the poorest, except predominant expression in the seed of growing, certain expression is arranged also in other major organs; BnTT16-1 is the most special, only in 15 days, 30 days, 45 days the seed expression is arranged after spending and spending; All the other members are placed in the middle, and except the expression characteristic with BnTT16-1 gene, majority is also flower bud, have certain expression in 55 days seeds even the pod skin after spending.To sum up, rape belongs to the TT16 gene family and has kept and the similar organ specificity of Arabidopis thaliana TT16 gene, namely mainly in reproductive organ, express, the highest to express in flower and the developmental seed, and the gradually decline along with the developmental process of seed, the function that inner seed coat cell development and the accumulation of kind skin pigment are regulated in this and their participations of prediction is consistent.But, no matter be rape belong to and Arabidopis thaliana between, rape plant in belonging between or plant in the Ortholog gene between, all there is certain disproportionation in the organ specificity of TT16.

5, swede type rape, Chinese cabbage and wild cabbage TT16 gene family detect at the different expression of black seed, yellow seed storeroom

Getting respectively the black seed of Chinese cabbage is that 09L597 and yellow seed are that the black seed of 09L600, wild cabbage is that 09L598 and yellow seed are that the black seed of 09L599, swede type rape seed look near isogenic line is that 09L588 and yellow seed are total RNA of 09L587 Main Reproductive Organs, and employing sxemiquantitative RT-PCR detects BrTT16, BoTT16, the BnTT16 gene family is overall and the expression of each member in the different tissues organ.Primer and amplification cycles parameter are the same.

Detected result as shown in figure 11, the organ specificity of BrTT16 gene family in the yellow seed material of Chinese cabbage is similar to black seed material, but BrTT16-1 and BrTT16-3 gene are obviously reduced than black seed material on the expression level, and the BrTT16-2 gene is slightly higher than black seed material.The organ specificity of BoTT16 gene family in the yellow seed material of wild cabbage is similar to black seed material, and expression level does not have notable difference yet.The organ specificity of BnTT16 gene family in the cabbage type rape yellow seed material is similar to black seed material, but BnTT16-1, BnTT16-2, BnTT16-5 and BnTT16-6 gene are obviously than black seed material downward modulation, BnTT16-3 and BnTT16-4 gene and black seed material no significant difference on the expression level.The above results explanation, the yellow seed proterties of swede type rape and Chinese cabbage should be relevant with the TT16 down regulation of gene expression, and the yellow seed proterties of wild cabbage and TT16 almost it doesn't matter; In addition, the different TT16 gene family members degree that participates in yellow seed proterties is also different.

Three, the application of swede type rape and parent species Chinese cabbage thereof and wild cabbage TT16 gene family

1, rape belongs to the structure of TT16 gene family rna interference vector

BrTT16, BoTT16, BnTT16 gene family and each member's of Arabidopis thaliana MADS gene superfamily mRNA is carried out multiple ratio pair, and selecting BrTT16, BoTT16, the special conservative section of BnTT16 gene family is that RNA disturbs target.Take swede type rape 5B the first chain cDNA as template, adopt combination of primers FBTT16I+RBTT16I cloning RNA interference fragment BTT16I (nucleotide sequence is shown in the 353rd～966 bit base among the SEQ ID No.14), and add that at its 5 ' end BamHI and AatII restriction enzyme site, 3 ' end add XbaI and SwaI restriction enzyme site; Employing standard 50 μ l Taq pcr amplification system (template 0.5 μ l, Taq archaeal dna polymerase 1.5U), the amplification cycles parameter is: 94 ℃ of denaturations 2 minutes, and 1 minute, 72 ℃ of 1 minute, 58 ℃ annealing of 94 ℃ of sex change were extended 1 minute again, totally 30 circulations, last 72 ℃ were extended 10 minutes; The PCR product carries out electrophoresis detection (Figure 12), glue recovery, pMD19-T carrier cloning, conversion competent escherichia coli cell, the screening of positive colony, bacterium liquid PCR evaluation and order-checking as described in front method, get recombinant vectors pMD19-T-BTT16I.

Double digestion from pMD19-T-BTT16I goes out antisense fragment BTT16IA with SwaI and AatII, pFGC5941M carrier with open loop behind same double digestion is connected (being about to the antisense fragment is inserted between the CaMV35S promotor and transcribed spacer of pFGC5941M) again, connect product and transform DH5 α competent cell, LB plate screening with the kantlex that contains 100mg/L (Kan) is cultivated, picking list bacterium colony, after increasing bacterium and cultivate with the LB liquid nutrient medium that contains Kan, getting bacterium liquid carries out composite PCR and detects that (combination of primers is F35S3N+FBTT16I and RBTT16I+RBnPAP2I2, sequence sees Table 11, detected result is seen Figure 14), clone's of choosing the full positive extracts plasmid, gets intermediate carrier pFGC5941M-BTT16IA.

Table 11TT16 gene family rna interference vector makes up and transgenosis detects the primer

Double digestion from pMD19-T-BTT16I goes out just fragment BTT16IS with BamHI and XbaI, be connected with pFGC5941M-BTT16IA through open loop behind the same double digestion again (be about to transcribed spacer and OCS terminator that just fragment is inserted into intermediate carrier between), connect product and transform DH5 α competent cell, cultivate with the LB plate screening that contains 100mg/L Kan, picking list bacterium colony, after increasing the bacterium cultivation, getting bacterium liquid carries out composite PCR and detects that (combination of primers is F35S3N+RBnPAP2I2, FBnPAP2I2+ROCST5N, sequence sees Table 11, detected result is seen Figure 14), clone's of choosing the full positive extracts plasmid, gets rna interference vector pFGC5941M-BTT16I (structure is seen Figure 13).

2, rna interference vector transforms agrobacterium tumefaciens

Adopt liquid nitrogen cold shock method to transform the agrobacterium tumefaciens lba4404 competent cell pFGC5941M-BTT16I, coat on the YEB flat board that contains 75mg/L Kan, 40mg/L Rifampin (Rif) and 20mg/L Streptomycin sulphate (Str), be inverted for 28 ℃ and cultivated 2 days, picking resistance bacterium colony, be inoculated in to contain in the aforementioned identical antibiotic YEB liquid nutrient medium and cultivate, get bacterium liquid and carry out the composite PCR detection, the bacterium liquid that detected result is correct uses glycerine in-80 ℃ of preservations, namely gets the Agrobacterium engineering strain.

3, rna interference vector transforms black seed swede type rape

With frozen Agrobacterium engineering strain thaw the activation after be cultured to logarithmic phase, 5000rpm collected thalline in centrifugal 10 minutes, [MS+1.0mg/L 2 with the MSm liquid nutrient medium, 4-dichlorophenoxyacetic acid (2,4-D)+and 1.0mg/L 6-benzylaminopurine (6-BA)+100 μ M Syringylethanone (AS), pH5.8] regulate bacterial concentration to OD ₆₀₀About 0.3, for contaminating.Choose in the black seed kind of full swede type rape two No. 10 seed, soaked 1～2 hour with clear water, use again 95% alcohol immersion 1 minute, aseptic water washing 3 times, soaked 15 minutes with 0.1% mercuric chloride solution, aseptic water washing is clean, is inoculated in the MS solid medium again, 25 ℃ of illumination cultivation 7 days cut the hypocotyl of aseptic seedling as genetically modified explant; With hypocotyl segment, preculture is 3 days in the access pre-training substratum (MS+1.0mg/L 6-BA+1.0mg/L 2,4-D, pH5.8); Hypocotyl after the preculture is immersed in the aforementioned Agrobacterium engineering bacteria liquid of getting ready and contaminated 10 minutes, sucks unnecessary bacterium liquid with aseptic thieving paper, and 23 ℃ of dark cultivations 2 days in the substratum (MS+1.0mg/L 6-BA+50 μ M AS, pH5.8) are trained in access altogether again; Hypocotyl after cultivating was altogether immersed the middle vibration of MSk liquid nutrient medium [MS+1.0mg/L 2,4-D+1.0mg/L 6-BA+500mg/L cephamycin (Cef)] washing sterilization 30 minutes, repeated secondary, blotted surface-moisture with aseptic thieving paper; Access again the middle illumination cultivation of callus induction substratum [MS+1.0mg/L 2,4-D+1.0mg/L 6-BA+500mg/L Cef+10mg/L grass fourth phosphine (Basta), pH5.8] more than 14 days, to growing macroscopic kanamycin-resistant callus tissue; Access again division culture medium [MS+4.0mg/L 6-BA+2.0mg/L zeatin (ZT)+5.0mg/LAgNO ₃+ 500mg/L Cef+10mg/L Basta, pH5.8] middle continuation cultivation, the callus induction differentiation; Access again the middle illumination cultivation of stem division culture medium (MS+3.0mg/L 6-BA+2.0mg/L ZT+500mg/L Cef+10mg/L Basta, pH5.8) to growing little stem; Access again the middle illumination cultivation of long shoot substratum (MS+0.005mg/L 6-BA+500mg/L Cef+10mg/L Basta, pH5.8) to growing stem and blade; Access again the middle illumination cultivation of root media (MS+0.5mg/L indolylacetic acid+500mg/L Cef, pH5.8) to growing flourishing root system; Seedling after taking root is transplanted in the basin alms bowl that contains sterilization perlite-vermiculite-turfy soil (mass ratio is 1: 1: 1) mixture after domestication, manages by greenhouse pot culture, finally obtains 14 strain regeneration plants.

4, the evaluation of transfer-gen plant

(1) Basta of transfer-gen plant rechecks and identifies

Leaflet tablet at regrowth drips the 1 Basta solution that concentration is 50mg/L, and observing blade after 3 days has unchanged.The result reacts not obvious or only has the plant of faint variation may positive plant as shown in figure 15, may negative plant and drip that the zone that Basta solution is arranged becomes withered and yellow even downright bad plant occurs.

(2) PCR of transfer-gen plant identifies

Adopt the CTAB method to extract the genome DNA of regeneration plant, again take this total DNA as template, adopt respectively combination of primers F35S3N+FBTT16I and FPbarT+RPbarT to carry out pcr amplification, whether detect pFGC5941M-BTT16I successfully is incorporated in the genome of deceiving the seed swede type rape, to contain the positive contrast of Agrobacterium engineering bacteria liquid of pFGC5941M-BTT16I, with the negative contrast of non-transgenic plant.The result amplifies the band consistent with positive control as shown in figure 16 in the partial regeneration plant, all positive plant may be transfer-gen plant in two kinds of combination of primers detect.Recheck the result relatively with Basta, show as the plant of resistance in Basta rechecks, its PCR detects great majority and also presents the positive.The result of comprehensive several respects has at least 7 strains can be defined as positive transfer-gen plant in the 14 strain regeneration plants.

5, the investigation of transfer-gen plant proterties

In the process that transfer-gen plant grows, the morphological specificitys such as the growing way of transfer-gen plant and non-transgenic plant, plant type, flower, flower bud, leaf, pod are compared observation, found that, both and no significant difference, growing way is basically identical, plant type does not have considerable change yet, pattern is aureus, blade is blunt sharp type, it is also normal to bear pods, after explanation is disturbed the BnTT16 gene family silence that makes in the black seed swede type rape by RNA, comprise that the background proterties of substantially growing does not obviously change.Simultaneously, transgenic seed and non-transgenic seed are compared.The result as shown in figure 17, transgenic seed is compared with the non-transgenic seed 2 obvious proterties variations has been occured: the one, and the kind skin pigment of transgenic seed obviously reduces, the seed outward appearance is tawny and yellowish brown, has formed sharp contrast with the typical black kind skin of non-transgenic seed; The 2nd, transgenic seed obviously diminishes, and the thousand seed weight of non-transgenic seed is 4.64g, and transgenosis suppresses that the thousand grain weigth of the most fierce plant is 2.23g, has descended 51.94%.The above results shows that the brassica plant TT16 gene families such as swede type rape participate in the accumulation of kind of skin pigment and the growth of seed size simultaneously.Simultaneously explanation, disturb the BnTT16 gene family silence make in the black seed swede type rape by RNA after, can reach and suppress kind of the effect of skin pigment accumulation, be conducive to create novel cabbage type rape yellow seed material.

Need to prove, swede type rape of the present invention and parent species Chinese cabbage thereof and wild cabbage TT16 gene family, except above-mentioned employing RNA perturbation technique is applied to the molecular breeding of swede type rape seed properties, also can adopt other technology such as Antisense Suppression to mediate the down-regulated expression of endogenous TT16 gene or gene family to reduce kind of a skin pigment accumulation, also can carry out accumulation even the increase seed size of overexpression to increase pycnogenols by just transgenosis, also can be applied to the molecular breeding of other Brassica Crops seed properties except swede type rape.Even adopt the RNA perturbation technique, in preferred embodiment, the used pFGC5941M carrier, also can adopt other carrier to make up rna interference vector; The gained rna interference vector also can adopt other method to carry out Plant Transformation except the improvement Ye Panfa that adopts the agrobacterium tumefaciens lba4404 mediation transforms.And, in preferred embodiment 12 members of disclosed swede type rape and parent species Chinese cabbage thereof (coming from the turnip type rape subspecies) and wild cabbage (coming from the kale mutation) TT16 gene family, the research method and the result of study that provide according to preferred embodiment, come from swede type rape, other TT16 allelotrope sequence of Chinese cabbage and wild cabbage, other subspecies that perhaps come from these 3 species, the TT16 gene order of the ecotype or kind, perhaps the gene order with above-mentioned 6 members is having at least 98% conforming any nucleotide sequence more than the 80bp continuously, can be applied to realize purposes of the present invention or effect in the molecular breeding of Brassica Crops seed properties.

Explanation is at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although by invention has been described with reference to the preferred embodiments of the present invention, but those of ordinary skill in the art is to be understood that, can make various changes to it in the form and details, and not depart from the spirit and scope of the present invention that appended claims limits.

Claims

1. wild cabbage TT16 gene family is characterized in that: comprise following 3 members: BoTT16-1 gene, BoTT16-2 gene and BoTT16-3 gene; The full length cDNA sequence of described BoTT16-1 gene is shown in SEQ ID No.8, and the full length cDNA sequence of BoTT16-2 gene is shown in SEQ ID No.10, and the full length cDNA sequence of BoTT16-3 gene is shown in SEQ ID No.12.

2. wild cabbage TT16 gene family according to claim 1, it is characterized in that: the genome sequence of described BoTT16-1 gene is shown in SEQ ID No.7, the genome sequence of BoTT16-2 gene is shown in SEQ ID No.9, and the genome sequence of BoTT16-3 gene is shown in SEQ ID No.11.