CN102559701A

CN102559701A - Chinese cabbage TT8 gene family and application thereof

Info

Publication number: CN102559701A
Application number: CN2012100185047A
Authority: CN
Inventors: 柴友荣; 殷家明; 赵文军; 谢伶俐; 肖霞; 李加纳; 林呐; 张瑞熙; 许本波
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2012-01-19
Filing date: 2012-01-19
Publication date: 2012-07-11

Abstract

The invention discloses a Chinese cabbage TT8 gene family comprising two members, namely a BrTT8-1 gene with a full-length cDNA sequence shown as SEQ ID No.2 and a BrTT8-2 gene with a full-length cDNA sequence shown as SEQ ID No.4. The gene family can be applied to the molecular breeding based on seed traits such as seed coat color, seed size and the like of a brassica plant.

Description

Chinese cabbage TT8 gene family and application thereof

The application is that application number is 201010281920.7, and the applying date is 2010-09-15, and invention and created name is divided an application for " swede type rape and parent's species Chinese cabbage thereof and wild cabbage TT8 gene family and application thereof ".

Technical field

The present invention relates to gene engineering technology field, particularly swede type rape (Brassica napus) and parent's species Chinese cabbage (Brassica rapa) thereof and wild cabbage (Brassica oleracea) TT8 (TRANSPARENT TESTA 8, transparent kind of skin 8; Claim bHLH42 again, BASIC HELIX-LOOP-HELIX 42, alkaline helix-loop-helix 42) 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, has important economic value for livestock industry provides feed.In the rape species, allotrtraploid species swede type rape is redoublingd after through 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's species Chinese cabbage and wild cabbage functional gene, and application foundation will be provided for the character improvement of rape genus crop.

The seed 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 economic worth of grouts all increases with the quality of oil.Though all have the stable natural yellow seed genotype of phenotype in Chinese cabbage and the wild cabbage, there is not natural cabbage type rape yellow seed genotype in occurring in nature.Existing cabbage type rape yellow seed material is mainly created through modes such as distant hybirdization, exists yellow seed rate and yellow seed degree not high, and phenotype is unstable; Be prone to affected by environment and make a variation, seed selection efficient is low, and breeding cycle is long; 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 through the transgenic molecular breeding.

Flavonoid is the important secondary metabolites of plant, has the various biological function, wherein Kuromanine (anthocyanin, AN), (proanthocyanidin PA) with flavonol is the important component of plant organ surface colour such as Cruciferae to pycnogenols.Rape belongs to and Arabidopis thaliana (Arabidopsis thaliana) belongs to Cruciferae together, has nearer sibship.In Arabidopis thaliana kind skin, pycnogenols mainly is accumulated in inner seed coat and coloured chalaza zone, is the nucleus of kind of skin pigment.Arabidopis thaliana TT8 (AtTT8) positive regulative transcription factor of bHLH type of genes encoding (AtbHLH42); Have in the cell of Kuromanine and pycnogenols by abduction delivering in accumulation; TT2, TT8 and TTG1 form a transcription factor complex; " late period " structure gene of regulation and control flavones route of synthesis such as the expression of DFR, BAN etc., thus regulate and control pycnogenols, Kuromanine, the mucous accumulation of kind skin.The seed of Arabidopis thaliana tt8 two mutants kind of the skin that shows transparency is yellow seed proterties.Therefore, in rape belongs to, the TT8 gene being carried out homologous clone and Function Identification, is the important channel in screening cabbage type rape yellow seed site, also can be used in the rape species molecular breeding with pycnogenols, Kuromanine, kind skin mucus correlated character.

Rape belongs to and Arabidopis thaliana originates from same ancestors, before 1700～1,800 ten thousand, separates, and the tripling of genomic level has taken place rape family plant; It is that rape belongs to elementary species: Chinese cabbage (AA group; 529Mbp), wild cabbage (CC group, 696Mbp) with black mustard (the BB group, 632Mbp) genome of grade 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, is equivalent to 6 times of arabidopsis gene group approximately, that is to say; Gene for single copy in Arabidopis thaliana possibly have the copy of 3 correspondences respectively in wild cabbage and Chinese cabbage, and in swede type rape, has 6 copies.At present, the relation of proterties such as the number of members of TT8 gene in rape species such as swede type rape, Chinese cabbage, wild cabbage, protein specificity, evolutionary relationship, the tissue specificity of expression, yellow seed and transgenic molecular breeding etc. all do not appear in the newspapers.

Summary of the invention

In view of this, one of the object of the invention is to provide swede type rape and parent's species Chinese cabbage and wild cabbage TT8 gene family.

For achieving the above object; The present invention adopts terminal rapid amplifying (RACE) technology of cDNA; Cloned swede type rape and parent's species Chinese cabbage thereof and wild cabbage TT8 gene family member's full-length cDNA and corresponding genome sequence respectively, and it has been carried out systems analysis.The result shows:

Said Chinese cabbage TT8 (BrTT8) gene family comprises following 2 members: BrTT8-1 gene and BrTT8-2 gene; The full length cDNA sequence of said BrTT8-1 gene is shown in SEQ ID No.2, and the full length cDNA sequence of BrTT8-2 gene is shown in SEQ ID No.4;

Said wild cabbage TT8 (BoTT8) gene family comprises following 2 members: BoTT8-1 gene and BoTT8-2 gene; The full length cDNA sequence of said BoTT8-1 gene is shown in SEQ ID No.6, and the full length cDNA sequence of BoTT8-2 gene is shown in SEQ ID No.8;

Said swede type rape TT8 (BnTT8) gene family comprises following 2 members: BnTT8-1 gene and BnTT8-2 gene; The full length cDNA sequence of said BnTT8-1 gene is shown in SEQ ID No.10, and the full length cDNA sequence of BnTT8-2 gene is shown in SEQ ID No.12.

Further, the genome sequence of said BrTT8-1 gene is shown in SEQ ID No.1, and the genome sequence of BrTT8-2 gene is shown in SEQ ID No.3; The genome sequence of said BoTT8-1 gene is shown in SEQ ID No.5, and the genome sequence of BoTT8-2 gene is shown in SEQ ID No.7; The genome sequence of said BnTT8-1 gene is shown in SEQ ID No.9, and the genome sequence of BnTT8-2 gene is shown in SEQ IDNo.11.

Has very high homology between 6 TT8 genes of above-mentioned 3 species; The consistence of genome sequence is 69.7～99.9%; The mRNA consistence is 85.8%～99.8%; The consistence of coding region is 93.2%～99.9%, and wherein the consistence of BnTT8-1 gene and BoTT8-1 gene is up to 99.9%, and the consistence of BnTT8-2 gene and BrTT8-1 gene is up to 99.1%.They and AtTT8 gene also have very high homology, and the consistence of genome sequence is 44.2～52.4%, and the consistence of mRNA is 78.0%～82.1%, and the consistence of coding region is 80.1%～83.6%.Has very high homology between the albumen of 6 TT8 genes encodings; Consistence is 89.6%～100%; Similarity is 90.6%～100%; Wherein proteic similarity of BnTT8-1 albumen and BoTT8-1 and consistence are 99.6%, and proteic similarity of BnTT8-2 albumen and BrTT8-1 and consistence are 100%.They and AtTT8 albumen also have very high homology, and consistence is 71.5%～77.2%, and similarity is 77.4%～84.1%.Phylogenetic analysis shows that AtTT8 gets together with BrTT8-2 earlier, and BnTT8-2 gets together with BrTT8-1 earlier, and these two groups are got together then; BnTT8-1 and BoTT8-1 get together, and be though BoTT8-2 is independent, also very near with BnTT8-1 and BoTT8-1.All show from aspects such as gene structure, protein structure, nucleic acid sequence homology, amino acid sequence homology, phyletic evolutions; The BnTT8-1 gene originates from the BoTT8-1 gene; The BnTT8-2 gene originates from the BrTT8-1 gene, and these 6 TT8 genes vertical homologous gene that all is the AtTT8 gene.The TT8 gene family is mainly expressed in the reproductive organ of Chinese cabbage, wild cabbage, swede type rape, and is the highest with developmental seed expression, and descends gradually along with the developmental process of seed.In addition; The TT8 gene family exists than evident difference in the overall expression black, yellow seed storeroom of Chinese cabbage and swede type rape; And the different gene membership table reveals different black, yellow seed differential expression pattern; And the TT8 gene family is overall and the member is black at wild cabbage, the expression no significant difference in the yellow seed material, explain that TT8 gene family down-regulated expression is relevant with the yellow seed proterties of Chinese cabbage and swede type rape, and the TT8 gene family is not the major reason of wild cabbage Huang seed proterties.

Based on The above results; Utilize any one or more gene or gene truncated segment in BrTT8 of the present invention, BoTT8, the BnTT8 gene family; Can make up TT8 dna recombinant expression carrier and transformant, the justice expression, Antisense Suppression, RNA that is used for the TT8 gene disturbed etc.

Two of the object of the invention is to provide the application in the molecular breeding of rape genus crop seed proterties of said swede type rape and parent's species Chinese cabbage and wild cabbage TT8 gene family thereof.

Further, said swede type rape and parent's species Chinese cabbage thereof and the application of wild cabbage TT8 gene family in the molecular breeding of cabbage type rape yellow seed proterties.

For achieving the above object; The present invention inserts BrTT8, BoTT8, BnTT8 gene family member's full length cDNA sequence respectively in the pCAMBIA2301G carrier and (inserts fragment by the CaMV35S promoters driven; Stop transcribing by the Nos terminator); Just plant expression vector pBrTT8-1, pBrTT8-2, pBoTT8-2 and pBnTT8-1 have been made up; With their arabidopsis thaliana transformation tt8 two mutants (seed is a transparent kind of skin), obtained the BnTT8-1 transfer-gen plant through kantlex (Kan) resistance screening, this transfer-gen plant T with Flower Dip method ₂Recovered the Vandyke brown of wild-type for the kernel seed coat colour of seed, proved that the TT8 gene that rape such as swede type rape belongs to has the function of answering with the AtTT8 gene pairs.Therefore, TT8 gene family of the present invention has using value in the molecular breeding of seed properties.Certainly; The genome sequence or the full length cDNA sequence justice of any gene in swede type rape and parent's species Chinese cabbage and the wild cabbage TT8 gene family are inserted between the CaMV35S promotor and Nos terminator of pCAMBIA2301G carrier, can be made up just plant expression vector.

Simultaneously; The present invention also chooses the part encoding sequence (shown in the 837th～1653 bit base among the SEQ ID No.10) of BnTT8-1 gene; Its antisense fragment is inserted between the CaMV35S promotor and Nos terminator of pCAMBIA2301G carrier; Made up antisense plant expression vector pBnTT8A, and changed No. 15, swede type rape typical black seed kind Hunan oil over to, obtained suppressing the transgenic line of endogenous BnTT8 genetic expression through agriculture bacillus mediated hypocotyl infestation method.Discover that the color of the antisense transgene plant results seed of GUS dyeing tests positive has been compared obviously with contrast and shoaled, though do not reach the golden yellow proterties of standard, relatively near yellow seed.Subject form characteristic of antisense BnTT8 transgenic progeny plant and not genetically modified adjoining tree no significant difference, but the general plumpness of transgenic seed descends, particle shape departs from sphere, and thousand seed weight drops to 2.54g by the 3.15g of contrast.The BnTT8 gene is described except participation regulation and control kind of skin pigment is synthetic, also some other seed properties such as seed sizes have been regulated and control in participation, can be used for the molecular breeding of seed properties such as rape genus crop kernel seed coat colour, seed size.Certainly; The all or part of encoding sequence antisense of any gene in swede type rape and parent's species Chinese cabbage and the wild cabbage TT8 gene family is inserted between the CaMV35S promotor and Nos terminator of pCAMBIA2301G carrier, can make up the antisense plant expression vector.

Beneficial effect of the present invention is: the invention provides the number of members of TT8 gene in swede type rape and parent's species Chinese cabbage and wild cabbage, each member's full length cDNA sequence and genome sequence, proteins encoded characteristic, evolutionary relationship, tissue specificity of expression etc.; And confirmed that TT8 gene family down-regulated expression is relevant with the yellow seed proterties of Chinese cabbage and swede type rape; The invention provides the TT8 gene thus and belong to the particularly application in the molecular breeding of cabbage type rape yellow seed proterties of crop seed character improvement rape, application prospect is good.

Description of drawings

In order to make the object of the invention, technical scheme and advantage clearer, will combine accompanying drawing that the present invention is made further detailed description below, wherein:

Fig. 1 is the agarose gel electrophoresis of BrTT8, BoTT8, the terminal amplification of BnTT8 gene family 5 ' cDNA.

Fig. 2 is the agarose gel electrophoresis of BrTT8, BoTT8, the terminal amplification of BnTT8 gene family 3 ' cDNA.

Fig. 3 is the amplification of BrTT8, BoTT8 and BnTT8 gene family member full-length cDNA, and wherein A and B are respectively FBnTT816+RBrTT819, FBnTT816+RBrTT821 amplification BrTT8 gene family full-length cDNA; C and D are respectively FBoTT8+RBoTT8-1, FBoTT8+RBoTT8-2 amplification BoTT8 gene family full-length cDNA; E and F are respectively FBoTT8+RBoTT8-1, FBrTT8+RBrTT8-1 amplification BnTT8 gene family full-length cDNA.

Fig. 4 is the nucleotide sequence comparison of BnTT8, BrTT8, BoTT8 gene family member and AtTT8 gene mRNA.

Fig. 5 is the cluster analysis of BnTT8, BrTT8, BoTT8 gene family member and AtTT8 gene.

Fig. 6 is that BnTT8, BrTT8 and BoTT8 gene family member's Southern hybridization is identified.

Fig. 7 is the proteic aminoacid sequence comparison of BnTT8, BrTT8 and BoTT8 gene family albumen and AtTT8.

Fig. 8 is BnTT8, BrTT8 and BoTT8 gene family proteins encoded and the proteic cluster analysis of other plant TT8.

Fig. 9 detects the overall and expression of each member in the different tissues organ of BnTT8, BrTT8 and BoTT8 gene family for RT-PCR.

Figure 10 detects the expression of overall and each member of BnTT8, BrTT8 and BoTT8 gene family in black, yellow seed near isogenic line reproductive organ for RT-PCR.

Figure 11 is the Expression element in the T-DNA zone in the pCAMBIA2301G justice plant expression vector.

Figure 12 is pBnTT8-1 carrier arabidopsis thaliana transformation tt8 two mutants T ₁For the screening of transformant, wherein A is T ₁For the Kan resistance screening of plant, B is the T of Kan resistance ₁For plant seedling stage, C is the T of Kan resistance ₁For the plant initial bloom stage.

Figure 13 is the Arabidopis thaliana T of Kan resistance ₁GUS dyeing for plant is identified.

Figure 14 is the T behind Arabidopis thaliana wild type seeds (A), the BnTT8-1 arabidopsis thaliana transformation tt8 two mutants ₂For seed (B), Arabidopis thaliana tt8 two mutants seed (C).

Figure 15 is the electrophoresis detection in the antisense plant expression vector pBnTT8A building process, and wherein A cuts BnTT8A for going up enzyme from pMD18-T, and B is the gus gene that downcuts 1.9kb from pCAMBIA2301G, and C is the detection of pBnTT8A.

Figure 16 is the T-DNA section figure of antisense plant expression vector pBnTT8A.

Figure 17 transforms the anti-Kan plant of partial regeneration behind the Hunan oil 15 for pBnTT8A.

Figure 18 transforms Hunan oil 15 backs (right side) and the blade GUS dyeing that contrasts (left side) for pBnTT8A.

Figure 19 transforms Hunan oil 15 each T of back for pBnTT8A ₃The seed of strain system (CK is the non-transgenic contrast).

Embodiment

Below will carry out detailed description to the preferred embodiments of the present invention with reference to accompanying drawing.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. work such as Sa nurse 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 the typical black seed is that 06K130 and yellow seed are 06K124; The wild cabbage material all comes from kale mutation (B.oleracea var.acephala), comprises that the typical black seed is that 06K158 and yellow seed are 06K165; Brassica napus comprises that the typical black seed is that 5B, No. 15, black seed kind Hunan oil, the black seed of seed look near isogenic line are that L1 and yellow seed are L2, provides the land for growing field crops general planting by Chongqing City's rape Engineering Technical Research Centre.

Main agents and test kit that preferred embodiment adopts: Taq archaeal dna polymerase (5U/ μ l) is available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd; Dna molecular amount standard λ-HindIII digest DNA Marker and DL-2000plus Marker, LATaq archaeal dna polymerase (5U/ μ l), pMD18-T support agent box, RNA PCR Kit (AMV) Ver.3.0 are available from precious biotechnology (Dalian) ltd; Restriction enzyme EcoRI, EcoRV etc. are available from U.S. New England Biolabs company; Restriction enzyme SacI etc., T ₄Dna ligase (10U/ μ l) is available from Lithuania MBI Fermentas company; MS minimum medium (Murashige&Skoog medium, including vitamins) is available from Dutch Duchefa Biochemie company; Pillar plant tissue RNA extraction agent box, a small amount of glue in a small amount reclaims test kit, a small amount of plasmid extraction test kit available from Shanghai China Shun biotechnology ltd; GeneRacer Kit is available from American I nvitrogen company, and Southern hybridization and the reagent (box) that detects, nylon membrane are available from German Roche company.

The key instrument that preferred embodiment adopts: PTC-200 Programmable Thermal Controller PCR appearance is available from U.S. MJ Research company; UVP HL-2000 hybridization ultraviolet interlinkage appearance is available from U.S. UVP company, and other is molecular biosciences and learns the genetically engineered common instrument.

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

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

Get the tender leaf of the swede type rape 5B, Chinese cabbage 06K130 and the wild cabbage 06K158 that cultivate under the normal condition of land for growing field crops; Adopt CTAB (CTAB) method to extract genome DNA, adopt electrophoretic method and spectrophotometry to estimate the quality and the concentration of nucleic acid samples.1.0% agarose gel electrophoresis result shows; The genome DNA good in integrity of 3 species that extract; Molecular-weight average is all greater than the 23kb band of λ-HindIII DNA Marker; RNA digestion is more complete, and it is higher to detect purity through spectrophotometry, can directly be used for pcr amplification and Southern hybridization.

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

Get root (Ro), hypocotyl (Hy), cotyledon (Co), stem (St), leaf (Le), flower bud (Bu), flower (Fl), the back 10 days seed (10D) of blooming, the back 20 days seed (20D) of blooming of the Chinese cabbage 06K130 that cultivates under the normal condition of land for growing field crops, wild cabbage 06K158, swede type rape 5B, back 30 days seed (30D) and pod skin (SP) totally 11 organs of blooming; And the Chinese cabbage 06K124 that cultivates under the normal condition of land for growing field crops, wild cabbage 06K165, (swede type rape is got the seed of 10D, 20D and 30D to the seed of the flower bud of swede type rape L1 and L2, flower and development in different stages; Chinese cabbage and wild cabbage are got the seed of 10D and 30D) etc. Main Reproductive Organs; Adopt the pillar total RNA of the total RNA extraction agent of plant box each organ of extraction in a small amount, adopt electrophoretic method and spectrophotometry to estimate the quality and the concentration of nucleic acid samples.1.0% agarose gel electrophoresis result shows that total RNA characteristic band of 3 species of extraction is clear, and does not have obvious RNA degraded and DNA pollution, and it is higher to detect purity through spectrophotometry, can satisfy the basic demand of RACE operation.

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

Get flower bud, flower, 10D, the 20D of Chinese cabbage 06K130, wild cabbage 06K158, swede type rape 5B respectively, total RNA of 30D seed mixes; Adopt GeneRacer Kit to carry out a series of RACE operation by its specification sheets; Obtain Chinese cabbage, wild cabbage, the swede type rape first chain cDNA (3 ' end and 5 ' end grappling simultaneously have the manual splice sequence) respectively, carry out 1.0% agarose gel electrophoresis after PCR amplifies and detect, the result shows; Total cDNA of 3 species demonstrates the traction of size at 200bp～10kb; The center of gravity zone is between 500bp～4kb, and most crucial district explains that reverse transcription is complete about 1.5kb; Obtained the total cDNA of high-quality RACE, it is terminal to can be used for cloning the complete cDNA of Chinese cabbage, wild cabbage and swede type rape TT8 gene family.

4, the terminal clone of Chinese cabbage, wild cabbage and swede type rape TT8 gene family 5 ' cDNA

Adopt 9.0 couples of Vector NTI Suite from Arabidopis thaliana (NM_1170502; AJ277509), morning glory (Ipomoeatricolor) (AB154370), lily (Lilium) (AB222076), ipomoea digitata purpurea (Ipomoea purpurea) (AB154369), that the nucleotide sequence of petunia (Ipomoea nil) TT8 or coding bHLH transcription factor (AB232775) carries out multiple ratio is right, has designed 2 forward primers (FTT8-3 and FTT8-3N) and the special primer of 2 reverse primers (RTT8-5 and RTT8-5N) (table 1) as the Chinese cabbage of increasing, wild cabbage and swede type rape TT8 gene family member cDNA end to 5 ' and 3 ' terminal conservative region.

Be template with Chinese cabbage, wild cabbage and the swede type rape first chain cDNA respectively, carry out the terminal RACE of 5 ' cDNA one with combination of primers 5 ' P+RTT8-5 and expand.50 μ l standard Taq pcr amplification systems are: 10 * PCR Buffer, 5 μ l, the MgCl of 25mmol/L ₂3 μ l, the dNTPs 1 μ l of 10mmol/L, the forward primer 1 μ l of 10 μ mol/L, the reverse primer 1 μ l of 10 μ mol/L, the Taq enzyme 0.5 μ l of 5U/ μ l, template 2 μ l, adding distilled water to TV is 50 μ l.The pcr amplification program is: 94 ℃ of preparatory sex change 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.Be template with the thing of expanding production again, carry out the terminal RACE nest of 5 ' cDNA with combination of primers 5 ' NP+RTT8-5N and expand that the pcr amplification system is identical with an expansion but template changes 0.1 μ l into, the pcr amplification program is: 94 ℃ of preparatory sex change 2 minutes; 94 ℃ of sex change are 1 minute again, 56 ℃ of annealing 1 minute, and 72 ℃ were extended totally 28 circulations 1 minute; Last 72 ℃ were extended 10 minutes.The PCR product carries out 1.0% agarose gel electrophoresis and detects, and BrTT8 has obtained the broadband about a 600bp, BoTT8 has obtained the band of a 550bp, and BnTT8 has obtained the very bright broadband (Fig. 1) about a 350-550.Adopting in a small amount, glue reclaims test kit recovery target fragment; Be connected with the pMD18-T carrier; Transformed into escherichia coli DH5 α competent cell again, it is clear to be cultured to blue hickie with the LB flat board that contains penbritin (Amp), IPTG and X-gal, picking hickie list bacterium colony; After using the LB liquid nutrient medium that contains Amp to increase the bacterium cultivation; Get bacterium liquid and carry out PCR and identify that the positive colony sublist reveals tangible length polymorphism as a result, each species is selected a plurality of representative mono-clonals and is entrusted Shanghai English to complete biotechnology ltd to check order.

Table 15 ' and 3 ' RACE in gene specific primer (GSP) and GeneRacer test kit primer

Sequencing result shows: it is 633bp 5 ' the cDNA end of [not comprising poly (A), down together] that the BrTT8 gene family obtains 1 length; The BoTT8 gene family obtains that 2 length are respectively 455, the 5 ' cDNA of 532bp is terminal, all has very high homology with AtTT8 mRNA; The BnTT8 gene family obtains the terminal clone of 20 5 ' cDNA subsequence, all have very high homology with AtTT8 mRNA, but actual capabilities is only represented 2 separate gene, and the difference of fragment length is caused by the mutability transcription site.NCBI BLASTn analysis revealed they and AtTT8 mRNA (NM 117050.2) have very high homology, and really to belong to 5 ' cDNA of TT8 gene family terminal for rape.

5, the terminal clone of Chinese cabbage, wild cabbage and swede type rape TT8 gene family 3 ' cDNA

Be template with Chinese cabbage, wild cabbage, the swede type rape first chain cDNA respectively, use combination of primers FTT8-3+3 ' P to carry out the terminal RACE of 3 ' cDNA one and expand.The pcr amplification system expands identical with program with the terminal RACE one of 5 ' cDNA.Be template with the thing of expanding production again, use combination of primers FTT8-3N+3 ' NP to carry out the terminal RACE nest of 3 ' cDNA and expand that the pcr amplification system is identical with the RACE nest expansion of 5 ' cDNA end with program.The PCR product carries out 1.0% agarose gel electrophoresis and detects, and BrTT8 has obtained the broadband about a 500～550bp, BoTT8 has obtained the band of a 500bp, and BnTT8 has obtained the very bright broadband (Fig. 2) about a 450-550.Glue recovery, T carrier cloning, transformed into escherichia coli competent cell, Amp resistance and the screening of blue hickie, hickie mono-clonal bacterium liquid PCR identify and order-checking.

Sequencing result shows: the BrTT8 gene family obtain 10 length be respectively 545,489,488,430 (4), 428,392 and the 3 ' cDNA of 382bp terminal; Actual capabilities are represented 2 separate gene, and fragment length difference is caused by mutability poly (A) tailing site; The BoTT8 gene family obtain 8 length be respectively 455,450,428 (3), 406,377 and the 3 ' cDNA of 320bp terminal, actual capabilities are represented 2 separate gene, fragment length difference is caused by mutability poly (A) tailing site; It is terminal that the BnTT8 gene family obtains 15 3 ' cDNA, and actual capabilities are represented 2 separate gene, and fragment length difference is caused by mutability poly (A) tailing site.These 3 ' cDNA of NCBI BLASTn analysis revealed are terminal all to have very high homology with AtTT8 mRNA, really belongs to 3 ' the cDNA end of TT8 gene family for rape.

6, Chinese cabbage, wild cabbage and swede type rape TT8 gene family member Full Length cDNA Cloning

According to the BrTT8 that is obtained, BoTT8, BnTT8 gene family 5 ' and 3 ' cDNA end sequence, designed forward primer FBrTT8 respectively with 2 reverse primers (RBrTT8-1 and RBrTT8-2) combinations, the BrTT8 gene family member's that is used to increase full-length cDNA; Designed forward primer FBoTT8 respectively with 2 reverse primers (RBoTT8-1, RBoTT8-2) combinations, the BoTT8 gene family member's that is used to increase full-length cDNA; These 4 combination of primers of front be used to jointly to increase full-length cDNA (table 2) of BnTT8 gene family member.With Chinese cabbage, wild cabbage and the swede type rape first chain cDNA is template, adopts above-mentioned combination of primers and 50 μ l standard Taq pcr amplification systems, amplification Chinese cabbage, wild cabbage and each member's of swede type rape TT8 gene family full-length cDNA; The pcr amplification program is: 94 ℃ of preparatory sex change 2 minutes, and 1 minute, 60 ℃ annealing of 94 ℃ of sex change were extended 3 minutes for 1 minute, 72 ℃ again, totally 35 circulations, last 72 ℃ were extended 10 minutes.The PCR product carries out 1.0% agarose gel electrophoresis and detects, and the combination of primers FBrTT8+RBrTT8-1 of BrTT8, FBrTT8+RBrTT8-2 have all obtained the band (Fig. 3 A, B) of treaty 1.9kb; The combination of primers FBoTT8+RBoTT8-1 of BoTT8, FBoTT8+RBoTT8-2 have also all obtained the band (Fig. 3 C, D) of treaty 1.9kb; The combination of primers FBoTT8+RBoTT8-1 of BnTT8, FBrTT8+RBrTT8-1 have all obtained the band (Fig. 3 E, F) of a treaty 1.9kb, but combination of primers FBrTT8+RBrTT8-2, FBoTT8+RBoTT8-2 do not obtain enough band clearly.Glue recovery, T carrier cloning, transformed into escherichia coli competent cell, Amp resistance and the screening of blue hickie, hickie mono-clonal bacterium liquid PCR identify and order-checking.

Result: adopt combination of primers FBrTT8+RBrTT8-1, FBrTT8+RBrTT8-2 to obtain 2 full length cDNA sequences of BrTT8 gene family respectively; Length is respectively 1932,1861bp [does not comprise poly (A); Down together]; Multiple ratio has very high homology to analysis revealed they and AtTT8mRNA, and has 5 ' and 3 ' the RACE end corresponding with them, and it is distinguished called after BrTT8-1 mRNA and BrTT8-2 mRNA; Adopt combination of primers FBoTT8+RBoTT8-1, FBoTT8+RBoTT8-2 to obtain 2 full length cDNA sequences of BoTT8 gene family respectively; Length is respectively 1795,1798bp; Multiple ratio has very high homology to analysis revealed they and AtTT8 mRNA; And exist 5 ' and the 3 ' RACE corresponding terminal, with its difference called after BoTT8-1 mRNA and BoTT8-2mRNA with them; Adopt combination of primers FBoTT8+RBoTT8-1, FBrTT8+RBrTT8-1 to obtain 2 full length cDNA sequences of BnTT8 gene family respectively; Length is respectively 1793,1932bp; Multiple ratio has very high homology to analysis revealed they and AtTT8 mRNA; And exist 5 ' and the 3 ' RACE corresponding terminal, with its difference called after BnTT8-1 mRNA and BnTT8-2 mRNA with them.

Table 2BrTT8, BoTT8, BnTT8 gene family member's full-length cDNA and genome sequence amplimer

7, the clone of Chinese cabbage, wild cabbage and swede type rape TT8 gene family member genome sequence

With Chinese cabbage, wild cabbage and swede type rape genome DNA is template; Employing can amplify the combination of primers of BrTT8, BoTT8, each member's full-length cDNA of BnTT8 gene family; Increase respectively BrTT8, BoTT8, each member's of BnTT8 gene family corresponding genome sequence, amplification program is identical with the amplification of full-length cDNA.In addition; Consider in BrTT8, BoTT8, each member's of BnTT8 gene family the genome sequence and possibly have some particular structural; The member who has can not directly expand and the genome total length; Need to splice after the segmentation amplification, this research has designed special primer (FBrTT8S, RBrTT8S1, RBrTT8S2, FBoTT8S, FBoTT8S1, FBoTT8S) and the public primers F TT8I5 of forward (table 2) according to BrTT8, BoTT8, each member's of BnTT8 gene family full length cDNA sequence again; It is matched with the forward and reverse primer that can amplify full-length cDNA respectively; Increase respectively BrTT8, BoTT8, each member's of BnTT8 gene family 5 ' and 3 ' terminal sequence fragment is utilized two segmental consensus sequences to splice again, thereby is obtained complete genome sequence.

Result: adopt combination of primers FBrTT8+RBrTT8S1, FBrTT8+RBrTT8S2 to amplify the upper reaches fragment of BrTT8-1 and BrTT8-2 respectively; FTT8I5+RBrTT8-1, FTT8I5+RBrTT8-2 amplify the downstream fragment of BrTT8-1 and BrTT8-2; Again the upstream and downstream fragment sequence is spliced on software VectorNTI 9.0, obtain that length is respectively 3903, BrTT8-1 and the BrTT8-2 genome sequence of 3838bp.Adopt combination of primers FBoTT8+RBoTT8-1, FBoTT8+RBoTT8-2 to increase respectively, obtain that length is respectively 2974, BoTT8-1 and the BoTT8-2 genome sequence of 2972bp.Adopt combination of primers FBoTT8+RBoTT8-1 to increase, obtaining length is the BnTT8-1 genome sequence of 2972bp; Adopt the upstream and downstream fragment of combination of primers FBrTT8+RBrTT8S1 and FTT8I5+RBrTT8-1 amplification BnTT8-2 respectively; The upstream and downstream fragment sequence is spliced on software VectorNTI 9.0, obtaining length is the BnTT8-2 genome sequence of 3914bp again.

Two, the analysis of swede type rape and parent's species Chinese cabbage thereof and wild cabbage TT8 gene family

Sequence alignment, ORFs (ORF) are searched and are translated on Vector NTI Advance 9.0 softwares and carry out.The BLAST of nucleic acid and protein sequence analyzes, the conserved structure domain search of protein sequence carries out on NCBI (http://www.ncbi.nlm.nih.gov) website, and structural analysis of protein provides in http://bip.weizmann.ac.il/ and http://www.expasy.org website on the information biology on-line analysis software of link and carries out.

1, the nucleic acid sequence analysis of Chinese cabbage, wild cabbage and swede type rape TT8 gene family

Relatively find through sequence; The structural similitude of BrTT8, BoTT8,6 members of BnTT8 gene family and AtTT8 gene; All form by 6 introns and 7 exons; Its intron shearing site all has conserved sequence AG|GT ... The characteristic of AG|GT, these members' nucleotide sequence structure characteristic are seen table 3-1,3-2 and 3-3.

Utilize the Nucleotide consistence of 9.0 couples of BrTT8 of Vector NTI, BoTT8, BnTT8 gene family member and AtTT8 gene to carry out comparing in twos (table 4, Fig. 4).The result shows to have higher homology between 6 members of BrTT8, BoTT8, BnTT8 gene family, and the consistence of genome sequence is 69.7～99.9%, and the mRNA consistence is 85.8%～99.8%, and the consistence of coding region is 93.2%～99.9%; They and AtTT8 gene also have very high homology, and the consistence of genome sequence is 44.2～52.4%, and the consistence of mRNA is 78.0%～82.1%, and the consistence of coding region is 80.1%～83.6%; Simultaneously, the consistence of BnTT8-1 and BoTT8-1 is up to 99.9%, and the consistence of the mRNA sequence of BnTT8-2 and BrTT8-1 is up to 99.1%.Therefore BrTT8, BoTT8,6 members of BnTT8 gene family all are vertical homologous genes of AtTT8, and produce through doubling separate afterwards by a common original ancestry gene with Arabidopis thaliana.

Phyletic evolution graph of a relation (Fig. 5) shows that AtTT8 gets together with BrTT8-2 earlier, and BnTT8-2 gets together with BrTT8-1 earlier, and these two groups are got together again and formed one big type then; BnTT8-1 and BoTT8-1 get together and form one type, and BoTT8-2 becomes one type separately, but evolutionary relationship and BnTT8-1/BoTT8-1 are very near.BrTT8, BoTT8,6 members' of BnTT8 gene family nucleotide sequence comparison and Evolution analysis show that the BnTT8-1 gene originates from the BoTT8-1 gene, and the BnTT8-2 gene originates from the BrTT8-1 gene.

The substruction of table 3-1BnTT8, BrTT8, BoTT8 gene family member nucleotide sequence

The substruction of table 3-2BnTT8, BrTT8, BoTT8 gene family member nucleotide sequence

The substruction of table 3-3BnTT8, BrTT8, BoTT8 gene family member nucleotide sequence

Table 4 rape belongs to genome sequence consistence (on arrange roman), the mRNA consistence (row's roman down) of 6 TT8 genes and AtTT8 gene and coding region consistence (italic) (%)

2, the number of members of Chinese cabbage, wild cabbage and swede type rape TT8 gene family detects

Multiple ratio according to the BrTT8 that clones, BoTT8, BnTT8 gene family member sequence is right; Design forward primer FTT8A (5 '-gagctcagaagaagaaatgacaatgtcaga-3 ') and reverse primer RTT8A (5 '-tctagatttcttccctctcactttcgccct-3 '); With the BnTT8-1 full-length cDNA is template; The 947bp fragment of amplification BnTT8-1 middle part is as Southern hybridization probe sequence, and with PCR method (PCR DIG Probe Synthesis Kit) probe carried out digoxin-dUTP (digoxigenin (DIG)-dUTP) mark; The PCR program of probe mark is: 94 ℃ of preparatory sex change 2 minutes, and 1 minute, 55 ℃ annealing of 94 ℃ of sex change were extended 1 minute for 1 minute, 72 ℃ again, totally 40 circulations, last 72 ℃ were extended 10 minutes.Choose the restriction enzyme EcoRI that do not have recognition site in the probe sequence, EcoRV, HindIII, XbaI respectively enzyme cut Chinese cabbage and wild cabbage genome DNA; Choose EcoRI, EcoRV, SacI, XbaI respectively enzyme cut the swede type rape genome DNA; Carry out 0.8% agarose gel electrophoresis, alkaline denaturation and neutralization then, DNA is transferred on the positively charged nylon membrane with capillary tube technique.Mark is good probe and nylon membrane are hybridized (DIG Easy Hyb) at 40.9 ℃ of Southern that carried out 16 hours; Carry out immunodetection (DIG Wash and Block Buffer Set and DIG Nucleic Acid Detection Kit) after the medium rigorous washing, and hybridization colour developing band is taken pictures.

The Southern results of hybridization is as shown in Figure 6, and the Chinese cabbage genome DNA all produces 2 dense bands and 1 blanking bar through EcoRI, EcoRV, XbaI enzyme cutting, and the HindIII enzyme is cut and produced 1 blanking bar; The wild cabbage genome DNA produces 2 dense bands and 1 blanking bar through EcoRI, XbaI enzyme cutting, and the EcoRV enzyme is cut and produced 2 dense bands, and the HindIII enzyme is cut and produced 1 dense band and 2 blanking bars; The swede type rape genome DNA is cut through EcoRI, EcoRV enzyme and is produced 2 dense bands and 1 blanking bar, and the SacI enzyme is cut and do not produced obvious band, and XbaI enzyme cutting produces 1 dense band.The Southern results of hybridization shows that BrTT8 and BoTT8 gene family member possibly be 2, and is consistent with this research clone result; Though the Southern results of hybridization shows BnTT8 gene family member and possibly be 2-3; But this research has been carried out clone repeatedly to the genome sequence and the cDNA of swede type rape; These two genes of BnTT8-1 and BnTT8-2 have finally only been obtained; And do not have the corresponding gene of acquisition and BrTT8-2 and BoTT8-2, and there be the 3rd BnTT8 gene even infer swede type rape BnTT8 family thus, also possibly not express or take place some structure disappearances.

3, Chinese cabbage, wild cabbage and swede type rape TT8 gene family proteins encoded are analyzed

Relatively find the basic parameter and the physico-chemical property similar (table 5) of BrTT8, BoTT8, BnTT8 gene family member proteins encoded through sequence.

Adopt 9.0 couples of BnTT8 of Vector NTI Suite, BrTT8, BoTT8 gene family member's proteins encoded and AtTT8 albumen to carry out comparison (Fig. 7) in twos; The result shows; Has very high homology between 6 TT8 albumen of BrTT8, BoTT8, BnTT8 gene family coding; Consistence is 89.6%～100%, and similarity is 90.6%～100%; They and AtTT8 albumen also have very high homology, and consistence is 71.5%～77.2%, and similarity is 77.4%～84.1%; Simultaneously, the similarity of BnTT8-1 and BoTT8-1 and consistence are all up to 99.6%, and the similarity of BnTT8-2 and BrTT8-1 and consistence are all up to 100% (table 6).Show that BnTT8,6 members of BrTT8 and BoTT8 gene family all are vertical homologous genes of AtTT8.

The essential property of table 5BnTT8, BrTT8, BoTT8 gene family member proteins encoded

Table 6 rape belongs to 6 TT8 albumen and the proteic consistence of AtTT8 (left side) and similarity (right side) (%)

The proteic cluster analysis result of BnTT8, BrTT8 and BoTT8 gene family member's proteins encoded and other plant TT8 shows (Fig. 8); BnTT8-1 and BoTT8-1 get together and form one type; BoTT8-2 is one type separately; It is to get together with BrTT8-2 after one type that BnTT8-2 and BrTT8-1 gather again, and last and AtTT8 albumen is got together and formed one big type.The proteic homology of TT8 of the radish of proteic aminoacid sequence of BnTT8, BrTT8 and BoTT8 and Cruciferae, Arabidopis thaliana is the highest; Have than higher homology with the bHLH transcription factor of other plant such as morning glory, lily, ipomoea digitata purpurea, petunia, also the proteic aminoacid sequence of other bHLH with the many plants that comprise Arabidopis thaliana has quite high homology on the conservative territory of bHLH.Proteic cluster analysis has proved that further rape belongs to the evolutionary relationship of 3 species TT8 genes, and promptly the BnTT8-1 gene originates from the BoTT8-1 gene, and the BnTT8-2 gene originates from the BrTT8-1 gene, and Chinese cabbage and wild cabbage are parent's species of swede type rape.

All there are more potential phosphorylation site in NetPhos 2.0 prediction BnTT8,6 albumen of BrTT8 and BoTT8 family; Wherein BrTT8-1 and BrTT8-2 respectively have 33; BoTT8-1 and BoTT8-2 have 34 and 33 respectively, and BnTT8-1 and BnTT8-1 have 34 and 33 respectively.All exist the N-glycosylation site on proteic the 282nd the N residue of NetNGlyc1.0 these 6 TT8 of prediction.All do not predict signal peptide and membrane spaning domain in these 6 proteic aminoacid sequences of TT8.But they all are positioned the WoLFPSORT prediction in the nucleus.The SOPMA prediction shows; These 6 proteic secondary structures of TT8 are closely similar, and alpha-helix accounts for 45.87%～52.21%, especially at proteic nearly C-end a major spiral arranged; Curl at random and account for 30.90%～38.58%; Extended chain accounts for 11.52%～13.63% and mainly be distributed in proteic N-end territory, and β-corner accounts for 3.75%～4.41%, and alpha-helix mainly links to each other with a spot of β-corner by curling at random.

4, the tissue and organ specificity detection of expression of Chinese cabbage, wild cabbage and swede type rape TT8 gene family

Adopt sxemiquantitative RT-PCR to detect Chinese cabbage, wild cabbage and swede type rape TT8 gene family overall expression and each member's the specifically expressing in the different tissues organ.Get respectively in Chinese cabbage 06K130, wild cabbage 06K158, the swede type rape oil 821 seed total RNA of totally 11 organs of root, hypocotyl, cotyledon, stem, leaf, flower bud, flower, pod skin and 10D, 20D, 30D; Adopting RNA PCR Kit (AMV) Ver.3.0 is total cDNA with total RNA reverse transcription of each organ, as the template of pcr amplification.According to the sequence of Arabidopis thaliana 26S rRNA, design primers F 26S and R26S (table 7) are with the conservative fragments of the house-keeping gene Bn26S gene 534bp in amplification Chinese cabbage, wild cabbage and the swede type rape, the cDNA concentration that is used to detect and regulate reverse transcription; The pcr amplification program is: 94 ℃ of preparatory sex change 2 minutes, and 1 minute, 60 ℃ annealing of 94 ℃ of sex change were extended 1 minute for 1 minute, 72 ℃ again, totally 21 circulations, last 72 ℃ were extended 10 minutes.Through multiple compare of analysis to Chinese cabbage, wild cabbage and the swede type rape TT8 gene family member sequence of having cloned; Design the overall expression level that following combination of primers: FTT8I1+RTT8-5 is used to detect the BrTT8 gene family, FBrTT8S+RBrTT8S1, FBrTT8S+RBrTT8S2 are respectively applied for the specifically expressing that detects BrTT8-1, BrTT8-2; FTT8I5+RTT8A is used to detect the overall expression level of BoTT8 gene family, and FBoTT8S+RBoTT8S2, FBoTT8S+RBoTT8S1 are respectively applied for the specifically expressing that detects BoTT8-1, BoTT8-2; FTT8I5+RTT8A is used to detect the overall expression level of BnTT8 gene family, and FBoTT8S+RBoTT8S2, FBrTT8S+RBrTT8S1 are respectively applied for the specifically expressing (table 7) that detects BnTT8-1, BnTT8-2.For fear of the amplification of the intersection between height homologous member, adopt clone's that contains each member's genome sequence to be template during the special detection of gene family member, carry out grads PCR, filter out the minimum annealing temperature that can only amplify corresponding gene.Simultaneously, use total cDNA to be template, carry out 55～65 ℃ grads PCR, find out the more consistent annealing temperature of each combination of primers amplification efficiency with above-mentioned each combination of primers.The result is through being the grads PCR of template with mono-clonal with total cDNA, and the highest annealing temperature that the BrTT8-1 that obtains increasing, BrTT8-2 gene specific detect primer is respectively 58 ℃ and 56 ℃; The highest annealing temperature that amplification BoTT8-1, BoTT8-2 gene specific detect primer is respectively 60 ℃ and 61 ℃; The highest annealing temperature that amplification BnTT8-1, BnTT8-2 gene specific detect primer is respectively 59 ℃ and 60 ℃; The PCR cycle number is 35.

Interior mark and the special detection primer of table 7 Chinese cabbage, wild cabbage and swede type rape TT8 gene family RT-PCR

Detected result is as shown in Figure 9; Interior mark 26S rRNA is behind 20 round-robin pcr amplifications; In 10～11 histoorgans of Chinese cabbage, wild cabbage and swede type rape, all amplify the more consistent band of brightness; Initial total RNA amount, reverse transcription efficient, PCR efficient etc. that each organ is described are more consistent, and it is believable that the tissue and organ specificity that carries out is on this basis expressed comparative result.Can know that by figure the overall expression amount of BnTT8 gene family in the seed of back 10 days, 20 days and 30 days of blooming is the highest, secondly is flower bud and pod skin, then is root and leaf, and more weak expression is also arranged in hypocotyl, cotyledon, stem.The expression amount of BnTT8-1 gene in the seed of spending back 10 days, 20 days is the highest, secondly is flower bud and flower, then is root, hypocotyl, stem, leaf and pod skin, has only trace expression at cotyledon with spending in the back 30 days seed; The BnTT8-2 gene expression amount in the seed of spending back 10 days, 20 days equally is the highest, secondly is colored, and extremely weak expression is arranged in stem, leaf, flower bud and pod skin, in other organ, does not detect expression.The overall expression amount of BrTT8 gene family in the seed of blooming back 10 days, 30 days is the highest, and the expression of trace is arranged hypocotyl, cotyledon, stem, leaf, flower bud, in spending, and in root and pod skin, does not detect expression.The expression amount of BrTT8-1 gene in the seed of blooming back 10 days, 30 days is the highest, and the expression of trace is arranged root, hypocotyl, cotyledon, flower bud, in spending, and in stem, leaf and pod skin, does not detect expression; The BrTT8-2 gene expression amount in the seed of blooming back 10 days, 30 days equally is the highest, and the expression of trace is arranged flower bud with in spending, and in other organ, does not detect expression.The overall expression of BoTT8 gene family does not have tissue specificity, at leaf, flower bud, flower, in the seed of spending back 10 days, 30 days and the pod skin abundant expression is arranged all, and the expression level of a great deal of is also arranged in root, hypocotyl, cotyledon and stem.The BoTT8-1 gene all has expression in 10 histoorgans, but the expression amount in the back 10 days seed of blooming is the highest, and the expression level in other 9 histoorgans is suitable.The BoTT8-2 gene also all has expression in 10 histoorgans, but the expression amount in leaf, flower bud, flower and pod skin will be higher than the expression amount in other histoorgan, and the expression in other histoorgan is roughly suitable.

5, Chinese cabbage, wild cabbage and the swede type rape TT8 gene family differential expression between black, yellow seed near isogenic line detects

Adopt sxemiquantitative RT-PCR to detect Chinese cabbage, wild cabbage and swede type rape TT8 gene family overall expression and each member's the specifically expressing in yellow seed material Main Reproductive Organs, and compare with the detected result of aforementioned black seed material.Get Chinese cabbage 06K124, wild cabbage 06K165 respectively, (swede type rape is got the seed of 10D, 20D and 30D to the seed of the flower bud of swede type rape L1 and L2, flower and development in different stages; Chinese cabbage and wild cabbage are got the seed of 10D and 30D) etc. total RNA of Main Reproductive Organs, adopting RNA PCR Kit (AMV) Ver.3.0 reverse transcription is total cDNA, as the template of pcr amplification.Primer and amplification program are with aforementioned black seed material.

Detected result is shown in figure 10; Totally being expressed in of BnTT8 gene family exists evident difference in swede type rape black seed system and the yellow seed system: in 5 histoorgans that black seed is, expression is arranged all; Expression amount in the back 20 days seed of blooming is the highest, and the expression amount in other 4 histoorgans is suitable; But in yellow seed system, only flower bud with spend middle expression, in the seed of development in different stages, do not detect expression.The black seed of the Chinese cabbage system that totally is expressed in of BrTT8 gene family is consistent with expression trend during yellow seed is, all is that the expression in the back 30 days seed of blooming is the highest, secondly is than the precocity seed in period; But, no matter be overall expression, or each member express, yellow seed system is that tangible downward modulation is arranged than black seed all, but the participation between two members is variant, and BrTT8-1 closes fully, and BrTT8-2 only is suitable downward modulation.The overall expression of BoTT8 gene family in wild cabbage black seed system and yellow seed system do not have difference, in 4 histoorgans, the expression than high abundance arranged all; The expression of two members of BoTT8 gene family in deceiving seed system and yellow seed system do not have the difference of essence yet, just on the expression abundance, has limited difference.The above results shows that TT8 gene family down-regulated expression is relevant with the yellow seed proterties of Chinese cabbage and swede type rape, and the TT8 gene family is not the major reason of the yellow seed proterties of wild cabbage.

Three, the application of swede type rape and parent's species Chinese cabbage thereof and wild cabbage TT8 gene family

1, the structure of BnTT8, BrTT8 and BoTT8 gene family justice plant expression vector and Agrobacterium engineering strain

Full length cDNA sequence with BrTT8-1, BrTT8-2, BoTT8-2 and BnTT8-1 gene inserts between the MCS BamHI and SacI of pCAMBIA2301G carrier respectively; Just plant expression vector pBrTT8-1, pBrTT8-2, pBoTT8-2 and pBnTT8-1 have been made up; Insert fragment by the CaMV35S promoters driven, stop transcribing by the Nos terminator.The pCAMBIA2301G carrier by Chai Yourong make up (cloning and expression of the plant Chinese People's Anti-Japanese Military and Political College beautiful Verticillium acceptor proteinoid gene and seminose binding lectin gene. Agricultural University Of Southwest; 2003); It contains three expression of plants boxes that started by CaMV 35S; One is the NPTII expression casette; Two other is the gus gene expression cassette, can realize the screening of the active double-tagging of Kan and GUS, wherein with NPTII expression casette gus gene expression cassette in the same way in gus gene can be replaced by external source target gene (Figure 11).

Respectively 4 constructed just plant expression vectors are transformed the agrobacterium tumefaciens lba4404 competent cell; Coat on the YEB flat board that contains 100mg/L Kan, 40mg/L Rifampin (Rif) and 20mg/L Streptomycin sulphate (Str); Be inverted for 28 ℃ and cultivated 2 days; Picking resistance bacterium colony is inoculated in to contain in the aforementioned identical antibiotic YEB liquid nutrient medium and cultivates, and gets bacterium liquid and carries out the PCR detection; The correct bacterium liquid of detected result in-80 ℃ of preservations, promptly gets the Agrobacterium engineering strain that contains pBrTT8-1, pBrTT8-2, pBoTT8-2 and pBnTT8-1 respectively with glycerine.

2, agriculture bacillus mediated just arabidopsis thaliana transformation tt8 two mutants has complementary functions

The seed of Arabidopis thaliana tt8 two mutants (available from the international Arabidopis thaliana Biological resources center of Ohio State Univ-Columbus USA) was soaked 10 minutes with chlorine bleach liquor's (dilution in 1: 8) sterilization; After washing 4～5 times with distilled water, be tiled on the MS flat board 4 ℃ of vernalization 2 days; Cultivated about 10 days down in 21 ℃, 16h illumination/8h dark condition again; Treating that seedling grows moves into behind the true leaf in the Nursery (with nutrition soil and vermiculite is 1: 1 mixing by volume, promptly gets in the little alms bowl of packing into after mixing thoroughly with the Hoagland nutritive medium), treats that elementary inflorescence length is to about the 5cm; Wipe out the above inflorescence of first axillalry bud of elementary inflorescence top, induce the growth of secondary inflorescence.

The Agrobacterium engineering strain that contains pBrTT8-1, pBrTT8-2, pBoTT8-2 and pBnTT8-1 respectively is inoculated at 2mL contains in the YEB liquid nutrient medium of microbiotic (Kan 100mg/L+Str 20mg/L+Rif 40mg/L), 28 ℃ of jolting overnight cultures are got 200 μ L cultures again; Adding to 20mL contains in the corresponding antibiotic YEB liquid nutrient medium; 28 ℃ of joltings were cultivated about 9 hours, added 200mL penetrating fluid (containing 20g sucrose and 100 μ L Silwet L-77) mixing again, and the Arabidopis thaliana tt8 two mutants plant inflorescence of full-bloom stage is immersed in this mixed solution; Contaminated 30～60 seconds; Inhale residual liquid on the defoliation sheet with thieving paper, encase with preservative film and preserve moisture, secretly cultivated 24 hours; Contaminate once more after one week, collect T after about one month ₁For seed.

With the T that obtains ₁For planting seed on moistening sandy soil and at sandy soil surface coverage layer of transparent film to keep sandy soil moisture, put 4 ℃ of vernalization 2 days, put into the greenhouse again and cultivate; When treating that plant grows to 2～3 true leaves; With concentration is that the Kan solution (contain massfraction be 0.1% polysorbas20) of 500mg/L is sprayed on the blade and covers layer of plastic film and preserved moisture 1 day, every later at a distance from sprinkling in 5 days 1 time, sprays altogether 3～4 times; The observation leaf color changes; Blade bleaches and dead gradually is non-transgenic plant (not having the Kan resistance), and blade still is transfer-gen plant (having the Kan resistance) for green, it is transferred in the nutrition soil cultivate.Since Arabidopis thaliana tt8 two mutants plant growing way a little less than, influenced transformation efficiency, through after the Kan resistance screening, obtained BnTT8-1 transfer-gen plant (Figure 12) in the first batch.

With the Kan resistance Arabidopis thaliana T that obtains ₁Further doing GUS dyeing for plant identifies: with T ₁Being immersed in the GUS dye liquor for the blade of plant (contains in the sodium phosphate buffer of 50mmol/L pH7.0: 0.1mol/L K ₃[Fe (CN) ₆], 0.1mol/LK ₄[Fe (CN) 6], 10mmol/L Na ₂EDTA; 0.001% (v/v) Triton X-100,0.5mg/ml X-Gluc) in, 37 ℃ of incubated overnight; Using volume(tric)fraction again is that 75% ethanol decolorization 2～3 times to negative contrast blades present white, on blade, can observe the transfer-gen plant that is that blue GUS expresses the site.Resistant plant all has blue occur (Figure 13) as a result, explains that the resistant plant that is filtered out is transfer-gen plant, explains that simultaneously foreign gene successfully obtains expressing in transfer-gen plant.

Do the positive and negative control respectively with Arabidopis thaliana wild-type and tt8 two mutants, to transgenic positive T ₁Investigate for apparent proterties such as the form of plant, growing ways.The result shows, transgenic positive T ₁On growth potential, plant leaf proterties, size, leaf color, there is not difference for plant basically.

Do the positive and negative control respectively with Arabidopis thaliana wild-type and tt8 two mutants, to transgenic positive T ₁T for the plant generation ₂Kernel seed coat colour recovery situation for seed is investigated.The result finds most of transgenic positive T ₁T for the plant generation ₂Kernel seed coat colour for seed reverts to Vandyke brown, and few part reverts to light brown, and similar with wild-type (Figure 14) prove that the TT8 gene of rape genus such as swede type rape has the function of answering with Arabidopis thaliana TT8 gene pairs.

3, the structure of BnTT8, BrTT8 and BoTT8 gene family antisense plant expression vector and Agrobacterium engineering strain

According to the comparison of the BnTT8 that is cloned, BrTT8 and BoTT8 gene family full-length cDNA and genome sequence, choose the conservative relatively section of BnTT8, BrTT8 and BoTT8 gene family member as the antisense fragment that suppresses altogether.Total cDNA is a template with swede type rape 5B first chain; With combination of primers FTT8A+RTT8A amplification BnTT8A fragment (nucleotide sequence is shown in the 837th～1653 bit base among the SEQ ID No.10); PCR product such as preceding method be said carries out electrophoresis detection, glue recovery, T carrier cloning, transformed into escherichia coli competent cell, the screening of positive colony, bacterium liquid PCR identifies and order-checking, recombinant vectors pMD 18-T-BnTT8A.(Figure 15 a) and reclaim to adopt XbaI+SacI double digestion in pMD 18-T-BnTT8A to go out the BnTT8A fragment of 829bp; Adopt the XbaI+SacI double digestion from the pCAMBIA2301G carrier, to excise the gus gene of about 1.9kb simultaneously, reclaim the carrier framework (Figure 15 b) of about 12kb.Gained BnTT8A fragment is connected with carrier framework; Connect product transformed into escherichia coli competent cell, carry out the Kan resistance screening, positive colony carries out PCR through combination of primers FTT8A+RTT8A and identifies (Figure 15 c); Obtain antisense plant expression vector pBnTT8A; The reverse fragment of inserting is stopped transcribing by the Nos terminator by the CaMV35S promoters driven, simultaneously the chain respectively expression cassette (Figure 16) that reporter gene GUS and selection markers gene nptII are arranged in the T-DNA border.

Adopt liquid nitrogen freezing to transform the agrobacterium tumefaciens lba4404 competent cell antisense plant expression vector pBnTT8A; 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 picking resistance bacterium colony 2 days; Be inoculated in to contain in the aforementioned identical antibiotic YEB liquid nutrient medium and cultivate; Get bacterium liquid and carry out composite PCR and detect, the correct bacterium liquid of detected result in-80 ℃ of preservations, promptly gets the Agrobacterium engineering strain with glycerine.

4, agriculture bacillus mediated antisense plant expression vector transforms black seed swede type rape

Frozen Agrobacterium engineering strain thawed is cultured to logarithmic phase after the activation, and 5000rpm collected thalline in centrifugal 10 minutes, with MS liquid nutrient medium (pH5.4) adjusting bacterial concentration to OD ₆₀₀About 0.3, supply to contaminate and use.Choose the seed of full swede type rape typical black seed kind Hunan oil 15, with 75% ethanol surface sterilization after 1 minute, aseptic water washing 3 times; Soaked 20 minutes with 5% chlorine bleach liquor again; Aseptic water washing is clean, is inoculated on the MS solid medium 26 ℃ of dark cultivations 2 days; Be to carry out the photoperiod under 2000Lux, 16 hours the condition of illumination every day to cultivate in intensity of illumination again, the hypocotyl that cuts 5 days aseptic seedling of seedling age is as genetically modified explant; With hypocotyl segment, insert in advance in the training substratum [MS+1.0mg/L6-benzylaminopurine (6-BA)+1.0mg/L 2,4 dichloro benzene ethoxyacetic acid (2,4-D)] and cultivated 2 days in advance; Hypocotyl after cultivating is in advance immersed in the aforementioned Agrobacterium engineering bacteria liquid of getting ready and contaminated 10 minutes, inhales with aseptic thieving paper and removes unnecessary bacterium liquid, inserts and trains 23 ℃ of dark cultivations 3 days in the substratum [MS+2.0mg/L 6-BA+0.5mg/L naphthylacetic acid (NAA)] altogether; Hypocotyl after cultivating was altogether soaked 30 minutes with the sterilized water that contains 500mg/L cephamycin (Cef), blotted surface-moisture with sterilization filter paper; Insert screening culture medium [the MS+2.0mg/L 6-BA+0.5mg/L NAA+7.5mg/L Kan+300mg/L Pyocianil (Carb)+5mg/L AgNO that contains 7.5mg/L Kan again ₃] in illumination cultivation, about 2 all subcultures 1 time, treated that the green regenerating bud grows after, insert again and continue in the screening culture medium contain 12.5mg/L Kan to cultivate; The regrowth that obtains inserts root media (MS+0.1mg/L indolylacetic acid+300mg/L Carb+7.5mg/L AgNO ₃) in illumination cultivation to growing flourishing root system.

Cut the blade that transforms green seedling and be used for the GUS histochemical stain, identify the seedling stabilized expression of exogenesis genes situation that transforms.GUS histochemical stain method: the Eppendorf that the blade of Hunan oil 15 transgenic seedling to be measured and contrast (unconverted seedling) is cut into pieces the 1.5ml that packs into manages, and adds suitable GUS dye liquor, 37 ℃ of incubated overnight, and 70% alcohol decolouring back is observed.582 hypocotyl explants of experiment cotransformation; Obtain 24 resistance seedlings (Figure 17) at last; GUS dyeing has the blade of 13 strain seedling can produce blue the reaction, and contrast produces blue reaction, shows to have at least 13 strains conversion success and reporter gene can express (Figure 18).

Transgenic is consistent with offspring's tree characteristics the present age, explains that the transgenic proterties can genetic stability.The color of transfer-gen plant seed is compared obviously shoal (Figure 19) with contrast, though do not reach the golden yellow proterties of standard, relatively near yellow seed.Subject form characteristic of resulting antisense BnTT8 transgenic progeny plant and not genetically modified adjoining tree no significant difference, but the general plumpness of transgenic seed descends, particle shape departs from sphere, and thousand seed weight drops to 2.54 grams by 3.15 grams of contrast.Explain that rape belongs to the TT8 gene except participation regulation and control kind of skin pigment is synthetic, possibly also participate in some other seed properties such as seed sizes, it is carried out the genetically engineered operation can modify proterties such as kernel seed coat colour, seed size.

Need to prove; Swede type rape according to the invention and parent's species Chinese cabbage thereof and wild cabbage TT8 gene family; Except above-mentioned employing Antisense Suppression technology is applied to the molecular breeding of swede type rape seed properties; Also can adopt RNA to disturb and wait the down-regulated expression of other technology endogenous TT8 gene of mediation or gene family to plant the skin pigment accumulation to reduce; Also can carry out accumulation even the increase seed size of overexpression, also can be applied to the molecular breeding of other rape genus crop seed proterties except that swede type rape through just transgenic to increase pycnogenols.Even adopt the Antisense Suppression technology, in preferred embodiment, the used pCambia2301G carrier, also can adopt other carrier to make up the Antisense Suppression expression vector; Gained Antisense Suppression expression 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 6 of disclosed swede type rape and parent's species Chinese cabbage (coming from the turnip type rape subspecies) thereof and wild cabbage (coming from the kale mutation) TT8 gene family the member; According to research method and the result of study that preferred embodiment provided; Other TT8 allelotrope sequence that comes from swede type rape, Chinese cabbage and wild cabbage; The TT8 gene order that perhaps comes from other subspecies, the ecotype or the kind of these 3 species; 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 according to the invention or effect in the molecular breeding of rape genus crop seed proterties.In addition, swede type rape according to the invention and parent's species Chinese cabbage thereof and wild cabbage TT8 gene family can also be applied to the regulation and control of Kuromanine proterties such as cauline leaf colour.

In a word, above embodiment is only in order to illustrating technical scheme of the present invention, and is not to be limited to this.Although through 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 form with on the details, and the spirit and scope of the present invention that do not depart from appended claims and limited.

Claims

1. Chinese cabbage TT8 gene family is characterized in that, comprises following 2 members: BrTT8-1 gene and BrTT8-2 gene; The full length cDNA sequence of said BrTT8-1 gene is shown in SEQ ID No.2, and the full length cDNA sequence of BrTT8-2 gene is shown in SEQ ID No.4.

2. Chinese cabbage TT8 gene family according to claim 1 is characterized in that, the genome sequence of said BrTT8-1 gene is shown in SEQ ID No.1, and the genome sequence of BrTT8-2 gene is shown in SEQ ID No.3.

3. the recombinant expression vector that contains any one or more gene in claim 1 or the 2 described Chinese cabbage TT8 gene families or gene conservative fragments.

4. recombinant expression vector according to claim 3; It is characterized in that: said recombinant expression vector is just plant expression vector, is the genome sequence of any gene in the Chinese cabbage TT8 gene family or full length cDNA sequence justice are inserted between CaMV35S promotor and the Nos terminator of pCAMBIA2301G carrier and obtained.

5. recombinant expression vector according to claim 3; It is characterized in that: said recombinant expression vector is the antisense plant expression vector, is that all or part of encoding sequence antisense with any gene in the Chinese cabbage TT8 gene family inserts between CaMV35S promotor and the Nos terminator of pCAMBIA2301G carrier and obtains.

6. the transformant that contains each said recombinant expression vector of claim 3 to 5, said transformant are mikrobe.

7. claim 1 or the 2 described Chinese cabbage TT8 gene families application in the molecular breeding of rape genus crop seed proterties, said seed properties is kernel seed coat colour and seed size.

8. application according to claim 7 is characterized in that: it is swede type rape that said rape belongs to crop.