CN102492695A - Cabbage TT10 gene family and its application - Google Patents

Abstract

The invention discloses a cabbage TT10 gene family which includes BoTT10-1 gene (SEQ ID No.7-8) and BoTT10-1pse pseudogene (SEQ ID No.9). The gene family provided by the invention can be applied in molecular breeding of brassica crop seed trait.

Description

Wild cabbage TT10 gene family and application thereof

The application is that application number is 201010281910.3, 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 TT10 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) TT10 (TRANSPARENT TESTA 10, transparent kind of skin 10; Claim LAC15 again, promptly LACCASE 15, laccase 15) 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.

(AtTT10, At5g48100) gene is positioned on the 5th karyomit(e) of Arabidopis thaliana Arabidopis thaliana (Arabidopsis thaliana) TT10, is that (TRANSPARENT TESTA TT) identifies among the two mutants tt10 from transparent kind of skin for method through candidate gene.Tt10 seed kind skin when results is light brown, is Vandyke brown in Chalazal Region, and after storage 6～12 months, the kind skin of two mutants reverts to the Vandyke brown of wild-type kind skin gradually.Compare with wild type seeds, the brown stain that tt10 two mutants seed shows as in the growth course postpones.AtTT10 genes encoding laccase 15 (AtLac15), (proanthocyanidin, PA) oxypolymerization becomes the kind skin pigment of brown, participates in also that the oxidation of xylogen (lignin) monomer aggregates into xylogen in kind of the skin both to have participated in pycnogenols in kind of the skin.Rape belongs to and Arabidopis thaliana belongs to Cruciferae together, possibly have identical pigment site.Pycnogenols is the basis that forms the black seed color of swede type rape, in yellow seed kind skin, obviously reduces.Content of lignin also is one of important character of swede type rape, in the yellow seed of Cruciferae is, is starkly lower than the content in brown seed or black seed, and is closely related with the yellow seed phenotype of Cruciferae.Therefore, in rape belongs to, the TT10 gene being carried out homologous clone and Function Identification, will help to disclose the molecule mechanism of swede type rape kind skin pigment and xylogen, 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 taken place in rape family plant, and promptly rape belongs to elementary species: Chinese cabbage (AA group; 529Mbp), wild cabbage (CC group; 696Mbp) and black mustard (BB group, the genome that 632Mbp) waits 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, then is equivalent to 6 times of arabidopsis gene group approximately.That is to say that the 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 tissue specificity of the number of members of TT10 gene in swede type rape and parent's species Chinese cabbage and wild cabbage, protein specificity, evolutionary relationship, expression and all do not appear in the newspapers with the relation of yellow seed proterties etc.

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 TT10 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 TT10 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 TT10 (BrTT10) gene family comprises following 3 members: BrTT10-1A gene, BrTT10-1B gene and BrTT10-2 gene; The full length cDNA sequence of said BrTT10-1A gene is shown in SEQ ID No.2, and the full length cDNA sequence of BrTT10-1B gene is shown in SEQ ID No.4, and the full length cDNA sequence of BrTT10-2 gene is shown in SEQ ID No.6;

Said wild cabbage TT10 (BoTT10) gene family comprises following 2 members: BoTT10-1 gene and BoTT10-1pse gene; The full length cDNA sequence of said BoTT10-1 gene is shown in SEQ ID No.8, and the full length cDNA sequence of BoTT10-1pse gene is shown in SEQ ID No.9;

Said swede type rape TT10 (BnTT10) gene family comprises following 3 members: BnTT10-1 gene, BnTT10-2 gene and BnTT10-3 gene; The full length cDNA sequence of said BnTT10-1 gene is shown in SEQ ID No.11, and the full length cDNA sequence of BnTT10-2 gene is shown in SEQ ID No.12, and the full length cDNA sequence of BnTT10-3 gene is shown in SEQ ID No.14.

Further, the genome sequence of said BrTT10-1A gene is shown in SEQ ID No.1, and the genome sequence of BrTT10-1B gene is shown in SEQ ID No.3, and the genome sequence of BrTT10-2 gene is shown in SEQ ID No.5; The genome sequence of said BoTT10-1 gene is shown in SEQ ID No.7; The genome sequence of said BnTT10-1 gene is shown in SEQ ID No.10, and the genome sequence of BnTT10-3 gene is shown in SEQ ID No.13.

The BrTT10-1A gene is extremely similar with the sequence of BrTT10-1B gene, each other pair of alleles.The BoTT10-1pse pseudogene is consistent with the sequence of BoTT10-1 gene, but in the coding region the single base deletion in 2 places takes place, and causes the premature termination sudden change, and the two is pair of alleles each other.Analysis according to gene order and expression pattern; BnTT10, BrTT10 and BoTT10 gene family member can be divided into two types: BnTT10-3 and BrTT10-2 gene are I type gene; The homology of they and AtTT10 is higher, possibly participate in the further oxidation of pycnogenols polymkeric substance and the polyreaction of xylogen; Other gene is an II type gene, has higher sequence homology between each member, possibly participate in the polyreaction of pycnogenols.The information biology prediction shows; 3 conserved domains and 4 cupric ion binding motif L1～L4 of having many copper oxydase family in the normal encoding protein sequence of BnTT10, BrTT10 and BoTT10 gene family; And have very high homology between other higher plant laccase albumen, predict BnTT10, BrTT10 and BoTT10 gene family coding laccase thus.The TT10 gene is mainly expressed in the seed that swede type rape, Chinese cabbage and wild cabbage are grown; Variant expression in the seed of black, yellow seed near isogenic line etap especially annesl stage in later stage, the different gene membership table reveals different black, yellow seed differential expression pattern.According to gene sequencing, tissue specific expression with at the differential expression between black, the yellow seed near isogenic line; Can infer the BnTT10-1 of swede type rape, BrTT10-1A and the BrTT10-2 gene that the BnTT10-3 gene comes from Chinese cabbage respectively, the BnTT10-2 gene of swede type rape then comes from the BoTT10-1 gene of wild cabbage.

Based on The above results; Utilize any one or more gene or gene truncated segment in BnTT10 of the present invention, BrTT10, the BoTT10 gene family; Can make up TT10 dna recombinant expression carrier and transformant, the justice expression, Antisense Suppression, RNA that is used for the TT10 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 TT10 gene family thereof.

Further, said swede type rape and parent's species Chinese cabbage thereof and the application of wild cabbage TT10 gene family in the molecular breeding of cabbage type rape yellow seed proterties.

For achieving the above object; The present invention chooses swede type rape and parent's species Chinese cabbage and the special conservative fragments BTT10A of wild cabbage TT10 gene family (nucleotide sequence is shown in the 653rd～1604 bit base among the SEQ ID No.11) thereof as the antisense fragment; Its antisense is inserted between the CaMV35S promotor and Nos terminator of pCAMBIA2301G carrier; Made up the Antisense Suppression expression vector that BnTT10, BrTT10 and BoTT10 gene family suppress altogether; And change 3 different cabbage type rape varieties over to through agriculture bacillus mediated hypocotyl infestation method; Obtained suppressing endogenous TT10 transcript effect and be 0～83% transgenic line, and the phenotype of 3 kind transgenic lines to modify effect consistent.Discover; Express in the transgenic line that is suppressed at the BnTT10 gene family; Seed painted obviously is later than contrast, shows as annesl and postpones, and plants that the solubility procyanidin content increases in the skin; Reduce and plant the skin content of lignin, prove that the BnTT10 gene family has participated in that the kind skin of swede type rape is painted, the polymerization of planting the skin procyanidin monomers and planting synthesizing of skin xylogen.The contriver has found that in early-stage Study the expression of a plurality of gene families in the cabbage type rape yellow seed material such as TT12 all have downward modulation; Therefore infer that the downward modulation of BnTT10 expression of gene participated in the formation of yellow seed proterties; But itself is not the main site of yellow seed on the source, but receives one of effector of yellow seed major gene regulation and control.The BnTT10 gene family has using value in the molecular breeding of seed properties such as the ripe annesl of kind of skin, kind skin content of lignin, but must operate simultaneously a plurality of sites that comprise BnTT10.

Beneficial effect of the present invention is: the invention provides the number of members of TT10 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 the TT10 gene has participated in planting that skin is painted, the polymerization of planting the skin procyanidin monomers and plant the synthetic of skin xylogen; Be one of effector of receiving the regulation and control of yellow seed major gene; The invention provides the TT10 gene thus and belong to the particularly application in the molecular breeding of cabbage type rape yellow seed proterties of the molecular breeding make the ripe annesl of species skin, to plant seed properties such as skin content of lignin 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 acquisition of swede type rape, Chinese cabbage and the wild cabbage first chain cDNA, and wherein M is DNA marker, and A, B, C are respectively swede type rape, Chinese cabbage, wild cabbage.

Fig. 2 is the amplification of BnTT10, BrTT10 and BoTT10 gene family member full-length cDNA; Wherein to be respectively swede type rape, Chinese cabbage and wild cabbage: M be DNA marker for a, b, c; 1～9 for adopting 9 kinds of combination of primers amplification gained PCR products respectively, and d is BrTT10-2 and BnTT10-3 full length gene cDNA.

Fig. 3 is that BnTT10, BrTT10 and BoTT10 gene family member's Southern hybridization is identified.

Fig. 4 is the proteic aminoacid sequence comparison of BnTT10, BrTT10 and BoTT10 family protein and AtTT10.

Fig. 5 is the systematic evolution tree of BnTT10, BrTT10 and BoTT10 family protein and other plant laccase, and wherein ApLAC1 is a mountain maple laccase 1 (AAB09228), and AfLAC is flavus laccase (XP_002378028); AtTT10 is Arabidopis thaliana TT10 (NP_199621), and AtLAC12 is Arabidopis thaliana laccase 12 (NP_196158), and AtLAC13 is Arabidopis thaliana laccase 13 (NP_196330); AtLAC14 is Arabidopis thaliana laccase 14 (NP_196498), and CmLAC is Chinese chestnut laccase (ACI46953), and OsLAC2 is paddy rice laccase 2 (Q8RYM9); OsLAC9 is paddy rice laccase 9 (Q6Z8L2); PtLAC1 is torch pine (AAK37823), and PtLAC2 is torch pine (AAK37824), and RcLAC is castor-oil plant (XP_002527130); S1AOX is tomato Vitamin C oxidase (AAY47050), and ZmLAC3 is corn laccase 3 (NP_001105915); The percentage of the numeral boots value check in the evolutionary tree branch (1000 repetitions).

Fig. 6 detects the overall and expression of each member in the different tissues organ of BnTT10, BrTT10 and BoTT10 gene family for RT-PCR.

Fig. 7 detects the expression of overall and each member of BnTT10, BrTT10 and BoTT10 gene family in black, yellow seed near isogenic line reproductive organ for RT-PCR.

Fig. 8 is the part drawing of BnTT10, BrTT10 and BoTT10 gene family Antisense Suppression expression vector pBTT10A.

Fig. 9 is that the PCR of transgenic brassica napus plant identifies that wherein a is transgenic Westar, and b is an oil 821 in the transgenic, and c is in the transgenic two No. 10; Last row's amplimer is FGUS+RGUS, and following row's amplimer is F35S3N+FTT10A.

Figure 10 be transgenic with contrast swede type rape seed in BnTT10 gene family and each member's expression amount detect, wherein a is T ₂For transgenic and the overall expression that contrasts BnTT10 gene in the Westar seed, b is T ₂For the overall expression of BnTT10 gene in oily 821 seeds in transgenic and the contrast, c is T ₂For the overall expression of BnTT10 gene in two tenth-seededs in transgenic and the contrast, d is T ₂For transgenic and the expression that contrasts each member of BnTT10 gene family in the Westar seed.

Figure 11 is the annesl process of transgenic and contrast swede type rape seed, and wherein a is oily 821 seeds in transgenic and the contrast (CK), and b is two tenth-seededs in transgenic and the contrast (CK).

Figure 12 is transgenic and contrast Westar T ₃Plant the solubility procyanidin content (a) and the insolubility procyanidin content (b) of skin, wherein V-10, V-12, V-13 are transgenic positive strain system, and BnTT10 genetic expression is suppressed; V-22 is transgenic positive strain system, but inhibition is not received in BnTT10 genetic expression; V-24 is contrast strain system, and BnTT10 genetic expression is normal; T ₂-P is T ₂For the transgenic positive strain be; T ₂-C is T ₁T for transgenic positive strain system ₂In generation, separated the strain system of negative strain or antisense fragment loss.

Figure 13 is the solubility content of lignin of transgenic and contrast Westar kind skin, wherein V-10, V-12, V-13, V-22, V-24, T ₂-P and T ₂The same Figure 12 of the implication of-C.

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 typical black seed strain 06K130 and Huang, black seed near isogenic line 09L597 (black seed) and 09L600 (yellow seed); The wild cabbage material all comes from kale mutation (B.oleracea var.acephala), comprises typical black seed strain 06K158 and Huang, black seed near isogenic line 09 sweet 1 (black seed) and 09 sweet 4 (yellow seeds); Brassica napus comprises typical black seed maintenance line 5B and Huang, black seed near isogenic line 09L588 (black seed) and 09L587 (yellow seed), and the land for growing field crops general planting is provided by Chongqing City's rape Engineering Technical Research Centre; Oily 821DH system is provided by Chongqing City's rape Engineering Technical Research Centre in black seed type material Westar of swede type rape and the black seed commercial variety, is provided by Inst. of Oil Crops, Chinese Academy of Agriculture for two No. 10 in the two low commercial varieties of black seed.

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; Easy-Taq archaeal dna polymerase (5U/ μ l), DL-2000 plus Marker, pEASY-T3 carrier are available from the Beijing Quanshijin Biotechnology Co., Ltd; Dna molecular amount standard DL-2000, λ-HindIII digest DNA Marker, rTaq archaeal dna polymerase, LA Taq archaeal dna polymerase (5U/ μ l) and Ex Taq ^TMHot Start archaeal dna polymerase (5U/ μ l), pMD18-T and pMD19-T carrier, RNA PCR Kit (AMV) Ver.3.0 are available from precious biotechnology (Dalian) ltd; Restriction enzyme DraI, EcoRI, EcoRV, SacI (10U/ μ l) are available from U.S. New England Biolabs company; Restriction enzyme BamHI and SacI (10U/ μ l), T ₄Dna ligase (10U/ μ l) is available from Lithuania MBI Fermentas company; MS (Murashige & Skoog medium, including vitamins) minimum medium is available from Dutch Duchefa Biochemie company; PGEM-T easy carrier is available from Promega 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 the DNA Marker of Southern hybridizing reagent, detection kit, nylon membrane, digoxigenin labeled is 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, Veriti ^TMMultiple temperature control PCR appearance is available from U.S. Applied Biosystems company; UVP HL-2000 hybridization ultraviolet interlinkage appearance is available from U.S. UVP company, and Bio-Rad Model 785 vacuum are changeed the film appearance available from U.S. Bio-Rad company, and molecular biology and engineered other conventional instrument and equipment.

One, the clone of swede type rape and parent's species Chinese cabbage thereof and wild cabbage TT10 gene family

1, the extraction of swede type rape, Chinese cabbage and wild cabbage 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.1.2% 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 swede type rape, Chinese cabbage and wild cabbage

Get flower bud (Bu), flower (Fl), the back 10 days seed (10D) of blooming, the bloom back 20 days seed (20D) and the back 30 days seed (30D) of blooming 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; And the seed of root (Ro), hypocotyl (Hy), cotyledon (Co), stem (St), leaf (Le), flower bud, flower, pod skin (SP) and the development in different stages of the swede type rape 09L587 that cultivates under the normal condition of land for growing field crops and 09L588, Chinese cabbage 09L600 and 09L597, wild cabbage 09 sweet 4 and 09 sweet 1 (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 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.1.2% agarose gel electrophoresis result shows that total RNA characteristic band of acquisition 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 swede type rape, Chinese cabbage and the wild cabbage RACE first chain cDNA

The total RNA of seed that gets flower bud, flower and 3 developmental stages of swede type rape 5B, Chinese cabbage 06K130 and wild cabbage 06K158 respectively 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 grappling simultaneously that the first chain cDNA of manual splice sequence is arranged at 3 ' end and 5 '; Carry out 1.0% agarose gel electrophoresis after PCR amplifies and detect, the result is as shown in Figure 1, and total cDNA of three species demonstrates the traction of size at 200bp～10kb; The center of gravity zone is at 1kb～4kb; Explain that reverse transcription is more complete, obtained the cDNA of better quality, it is terminal to can be used for cloning the complete cDNA of swede type rape, Chinese cabbage and wild cabbage TT10 gene family.

4, the terminal clone of swede type rape, Chinese cabbage and wild cabbage TT10 gene family 5 ' cDNA

It is right to carry out multiple ratio through 9.0 pairs of AtTT10/AtLac15 genes of Vector NTI Advance and other 16 laccases (AtLac1-AtLac14, AtLac16 and AtLac17) gene order, chooses difference site in AtTT10 gene and other Arabidopis thaliana laccase member nucleotide sequence and has designed 2 forward primers (FTT10-31 and FTT10-32) and 2 reverse primers (RTT10-51 and RTT10-52) (table 1) as amplification swede type rape, Chinese cabbage and the terminal gene specific primer (GSP) of wild cabbage TT10 gene family member cDNA.

Be template with swede type rape, Chinese cabbage, the wild cabbage first chain cDNA respectively, carry out the terminal RACE of 5 ' cDNA one with combination of primers 5 ' P+RTT10-51 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 0.5 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 25 circulations 1 minute; Last 72 ℃ were extended 10 minutes.

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

With the thing of expanding production is template, carries out the terminal RACE nest of 5 ' cDNA with combination of primers 5 ' NP+RTT10-52 and expands, and the pcr amplification system is identical with an expansion but template changes 0.1 μ l into, and the pcr amplification program is: 94 ℃ of preparatory sex change 2 minutes; 94 ℃ of sex change are 1 minute again, 43～48 ℃ of annealing 1 minute, and 72 ℃ were extended totally 30 circulations 1 minute; Last 72 ℃ were extended 10 minutes.The PCR product carries out 1.0% agarose gel electrophoresis and detects; Adopt glue recovery test kit recovery target fragment in a small amount, (pMD18-T or pMD19-T) is connected with the T carrier, again transformed into escherichia coli (DH5 α or JM109) competent cell; 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 increasing bacterium and cultivate with the LB liquid nutrient medium that contains Amp, is got bacterium liquid and is carried out PCR and identify; The positive colony sublist reveals tangible length polymorphism as a result, and each species is selected 8～9 representative mono-clonal and entrusted the Shanghai English to complete biotechnology ltd to check order.Sequencing result shows: the BnTT10 gene family obtain length be respectively 405 (2 kinds of sequences), 409,374 and 379bp [all do not comprise poly (A); 55 ' cDNA down together] are terminal; NCBI BLASTn analysis revealed they and AtTT10 gene (NM_124184) have very high homology, possibly represent 2 kinds of 5 ' different cDNA terminal respectively; The BrTT10 gene family obtain length be respectively 346,357,374,379,405 (2 kinds of sequences), 406 and 85 ' cDNA of 409bp terminal, show as two types of obvious difference, but all have higher homology with the AtTT10 gene; The BoTT10 gene family obtain length be respectively 379 (2 kinds of sequences), 398,405,409 and 65 ' cDNA of 411bp terminal, all higher homology is arranged with the AtTT10 gene, possibly represent 2 kinds of different 5 ' cDNA ends.

5, the terminal clone of swede type rape, Chinese cabbage and wild cabbage TT10 gene family 3 ' cDNA

Be template with Chinese cabbage, wild cabbage, the swede type rape first chain cDNA respectively, carry out the terminal RACE of 3 ' cDNA one with combination of primers 3 ' P+FTT10-31 and expand.The pcr amplification system expands identical with program with the terminal RACE one of 5 ' cDNA.

With the thing of expanding production is template, carries out the terminal RACE nest of 3 ' cDNA with combination of primers 3 ' NP+FTT10-32 and expands, and the pcr amplification system is identical with the RACE nest expansion of 5 ' cDNA end with program.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.Sequencing result shows: the BnTT10 gene family obtain length be respectively 430 (4), 407 and 63 ' cDNA of 387bp terminal; These 3 ' cDNA of NCBI BLASTn analysis revealed are terminal to have very high consistence with the AtTT10 gene; Show that they are terminal for 3 ' cDNA of BnTT10 gene family really; But the fragment sequence of these different lengthss is consistent, possibly represent with a kind of 3 ' cDNA end, and fragment length difference is to be caused by variable poly (A) tailing site; The BrTT10 gene family obtain length be respectively 448 (2), 449 (2), 403,390 and 7 the 3 ' cDNA of 377bp terminal, these 3 ' cDNA are terminal to have very high consistence with the AtTT10 gene, possibly represent 2 kind of 3 ' cDNA terminal; The BoTT10 gene family obtain length be respectively 430,430,388 and 43 ' cDNA of 390bp terminal, all higher with the AtTT10 gene identity, possibly represent 2 kind of 3 ' cDNA end.

6, swede type rape, Chinese cabbage and wild cabbage TT10 gene family member Full Length cDNA Cloning

According to the BnTT10 that is obtained, BrTT10, BoTT10 gene family 5 ' and 3 ' cDNA end sequence; 3 forward primers (FBNTT10, FBRTT10, FBOTT10) and 3 reverse primers (RBNTT10, RBRTT10, RBOTT10) (table 2) have been designed; All forward primers and reverse primer are matched in twos, form 9 kinds of combination of primers.Be template with swede type rape, Chinese cabbage, the wild cabbage first chain cDNA respectively, adopt above-mentioned combination of primers and 50 μ l standard Taq pcr amplification systems, amplification swede type rape, Chinese cabbage, each member's of wild cabbage TT10 gene family full-length cDNA; The pcr amplification program is: 94 ℃ of preparatory sex change 2 minutes, and 1 minute, 57 ℃ annealing of 94 ℃ of sex change were extended 2 minutes for 1 minute, 72 ℃ again, totally 35 circulations, last 72 ℃ were extended 10 minutes; PCR product such as preceding method be said carries out electrophoresis detection (Fig. 2), glue recovery, T carrier cloning, transformed into escherichia coli competent cell, the screening of positive colony, bacterium liquid PCR identifies and order-checking.The result obtains 3,3 and 8 full length cDNA sequences of BnTT10, BrTT10, BoTT10 gene family respectively; Merge identical gene order; And design special detection primer; After screening, checking and replenishing the clone, with different separate gene (unigenes) called after BnTT10-1, BnTT10-2, BrTT10-1A, BrTT10-1B, BoTT10-1 and BoTT10-1pse respectively.Through analyzing and comparison; One 5 ' the cDNA end that discovery BrTT10 gene family obtains does not increase and obtains corresponding 3 ' cDNA end and full length cDNA sequence; To this tip designs experimental program: the first chain cDNA is a template with Chinese cabbage; With this 5 ' cDNA terminal corresponding primers F BRTT10 and GeneRacer 3 ' NP pairing, use Ex Taq ^TMHot Start increases, and obtains corresponding full-length cDNA, called after BrTT10-2; Design terminal primer RBRTT10-I of corresponding 3 ' cDNA and FBRTT10 pairing again, the corresponding cDNA of amplification BnTT10, BoTT10 gene family obtains BnTT10-3, and does not obtain corresponding gene in the wild cabbage.Because gene order and the AtTT10 gene order of BrTT10-2, BnTT10-3 have higher homology, therefore this genoid is defined as I type gene, other gene then is defined as II type gene.The analytical results of multiple sequence comparison shows that the full length cDNA sequence of these genes and AtTT10 gene have very high homology, and all exists 3 ' and the 5 ' RACE corresponding with them terminal.

Table 2BnTT10, BrTT10, BoTT10 gene family member's full-length cDNA and genome sequence amplimer

7, the clone of swede type rape, Chinese cabbage and wild cabbage TT10 gene family member genome sequence

The 5th exon according to swede type rape, Chinese cabbage and wild cabbage TT10 gene family; Primers F TT10A and RTT10A (table 2) in the middle of the design; Use FTT10A and reverse primer (RBNTT10, RBRTT10, RBOTT10 and RBRTT10-I) pairing respectively; RTT10A and forward primer (FBNTT10, FBRTT10, FBOTT10) pairing; Be template with swede type rape, Chinese cabbage and wild cabbage genome DNA respectively, 3 ' and 5 ' terminal sequence fragment of amplification BnTT10, BrTT10, BoTT10 gene family member genomic dna utilizes the consensus sequence of the 5th exon to splice again.Pcr amplification adopts Easy Taq archaeal dna polymerase; Amplification program is: 95 ℃ of preparatory sex change 3 minutes, and 30 seconds, 68 ℃ annealing of 95 ℃ of sex change were extended 4～6 minutes for 1 minute, 72 ℃ again, totally 35 circulations, last 72 ℃ were extended 10 minutes; 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.The final genome sequence that obtains except that BnTT10-2 and the BnTT10 the BoTT10-1pse, BrTT10, BoTT10 gene family member.

Two, the analysis of swede type rape and parent's species Chinese cabbage thereof and wild cabbage TT10 gene family

Sequence alignment, ORFs (ORF) search and translation, the basic parameter of protein etc. are carried out on Vector NTI Advance 9.0-10.3 software.The secondary structure prediction of nucleotide sequence carries out in DINAMelt Server (http://frontend.bioinfo.rpi.edu/applications/hybrid/quikfold.ph p) website, and the microRNA structure prediction carries out in PMRD (http://bioinformatics.cau.edu.cn/PMRD/) website.The BLAST of nucleic acid and protein sequence analyzes, the conserved structure domain search of protein sequence carries out in NCBI (http://www.ncbi.nlm.nih.gov) website, and the information biology on-line analysis software that proteinic conserved domain, motif, post transcriptional modificaiton prediction, topological framework prediction, secondary structure prediction and tertiary structure prediction mainly provide through Expasy (http://www.expasy.org) website carries out.The multiple ratio of the protein sequence of other plant laccase that obtains on BnTT10, BrTT10 and BoTT10 and the GeneBank is to being carried out by Cluastal X 1.83; Use MEGA 4.0 The software adopted adjacent methods (Neighbor-Joining Method) constructing system to set then, the safety of tree is through 1000 Bootstrap replicates checks.

1, the nucleic acid sequence analysis of swede type rape, Chinese cabbage and wild cabbage TT10 gene family

The constructional feature of swede type rape, Chinese cabbage and wild cabbage TT10 gene family nucleotide sequence is as shown in table 3.

The constructional feature of table 3BnTT10, BrTT10 and BoTT10 gene family nucleotide sequence

I type gene member's (BnTT10-3 and BrTT10-2) full-length cDNA is 1793bp, and genome sequence is 2417bp, and 5 ' UTR, 0RF and 3 ' UTR are 42,1683 and 68bp respectively; II type gene member's (BnTT10-1, BrTT10-1A, BrTT10-1B, BnTT10-2 and BoTT10-1) full-length cDNA is between 1889～1896bp; Genome sequence owing to the difference of the 4th intron length appear 3662～5475bp than large span, 5 ' UTR, ORF and 3 ' UTR are respectively 46,1680～1692 and 151～170bp.All introns are all followed the GT-AG montage border characteristics of standard.The distinguishing feature of BnTT10, BrTT10, BoTT10 gene family genome sequence is that the length variation of the 4th intron is bigger.I type gene member's the 4th intron length is merely 207bp, and II type gene member's the 4th intron not only on length, demonstrate 994～2807bp than large span, and have complicated nucleic acid secondary structure.Sequencing result shows that there is the irregular splicing form of 2 kinds of introns in the full-length cDNA of BrTT10-2, shows as that the 4th exon is cut and the 5th intron keeps, with them called after BrTT10-2M1 and BrTT10-2M2 respectively.BnTT10-2M1 as intron, together cuts the 4th exon with the 3rd intron and the 4th intron, and the ORF contraction in length is 1554bp, and coding has only the BnTT10-2M1 albumen of 517aa.BrTT10-2M2 has kept the 5th intron, causes ORF to increase 18bp, the BrTT10-2M2 albumen of coding 566aa.

The RACE end sequencing is the result show, at the T of BnTT10-2 ₁₈₈₉, T ₁₈₆₆And G ₁₈₄₅There is 3 variable poly (A) tailing site in the right side, at the C of BrTT10-1A ₁₈₂₄, T ₁₈₃₈, G ₁₈₅₁And T ₁₈₉₆Also there is 4 variable poly (A) tailing site in the right side.BnTT10-2, BrTT10-1A and BrTT10-1B have 4 alternative transcription initiation site (G ₃, A ₇, A ₃₃And A ₃₈), BoTT10-1 also has 4 alternative transcription initiation site (C ₁, G ₃, A ₇And A ₃₃), BrTT10-2 has 3 alternative transcription initiation site (A ₁, T ₅₁And A ₆₂) and 2 alternative transcription initiation site (T ₅₁And A ₆₂) all after its initiator codon ATG.I type gene BnTT10-3 and BrTT10-2 have normal tailing signal A at 3 ' end of its sequence ₁₇₇₇ATAAA ₁₇₈₂, and in II type gene, do not have normal tailing signal.

Utilize Vector NTI 10.3.0 that BnTT10, BrTT10, BoTT10 gene family member and the AtTT10 gene Nucleotide consistence on mRNA, ORF, 5 ' UTR, 3 ' UTR and each intron level has been carried out comparison in twos.The result shows that BnTT10, BrTT10, all members of BoTT10 gene family all have higher homology with the AtTT10 gene, on the full length mRNA level, have 79.1～81.2% consistence, reach 82.5～83.6% consistence in the ORF level; But at non-coding region; The consistence of II type gene member and AtTT10 gene is merely 57.4% (5 ' UTR) and 52.7～57.3% (3 ' UTR); And the consistence of I type gene member and AtTT10 gene is 71.4% (5 ' UTR) and 67.1～68.4% (3 ' UTR), is higher than II type gene member; The consistence of coding region is higher than the conservative property that non-coding region has proved the coding region far away.On the full length mRNA level, has 83.3～100.0% consistence between BnTT10, BrTT10, the BoTT10 gene family member; Apparently higher than the consistence of they and AtTT10 gene, explain that the sibship between swede type rape, Chinese cabbage and the wild cabbage TT10 gene is more more approaching than the relation of they and AtTT10 gene.I type gene member's (BnTT10-3 and BrTT10-2) full length mRNA sequence has 99.7% consistence; 5 ' UTR sequence is identical; 3 ' UTR sequence has 98.5% consistence; The ORF sequence has 99.8% consistence, and the identical protein sequence of encoding infers that thus BnTT10-3 derives from BrTT10-2.II type gene member (BnTT10-1, BrTT10-1A, BrTT10-1B, BnTT10-2 and BoTT10-1) has 5 ' identical UTR sequence; But can find by sequence alignment; Characteristics according to its nucleotide sequence; II type gene member can be divided into two groups again: BnTT10-1, BrTT10-1A and BrTT10-1B are one group, and BnTT10-2 and BoTT10-1 are one group.BrTT10-1A and BrTT10-1B are respectively 99.5%, 98.8% and 99.5% at the sequence identity of full length mRNA, 3 ' UTR and ORF; The consistence of coding protein sequence is 99.3%; Belong to two genes of height homologous; Just have certain difference, infer that thus BrTT10-1B is a BrTT10-1A heterozygosis allelotrope in the 4th intron zone.3 ' the UTR sequence of BnTT10-1 and BrTT10-1A, BrTT10-1B has 98.8% and 97.6% consistence respectively; The full length mRNA sequence has 99.8% and 99.3% consistence respectively; The ORF sequence has 99.6% and 99.2% consistence respectively; Nearer with sequence identity and the expression pattern of BrTT10-1A comparatively speaking, infer that thus BnTT10-1 derives from BrTT10-1A.BnTT10-2 and BoTT10-1 have identical full length mRNA sequence; Coding region and non-coding area sequence are in full accord, and coding protein sequence is also identical, because this research adopts PCR method not obtain the genome sequence of BnTT10-2; Therefore also unclear at present two members are in the difference in intron zone; But from resulting genome sequence except that the 4th intron, two members infer thus that in genomic level also no significant difference BnTT10-2 derives from BoTT10-1.Sequence identity in full length mRNA, 3 ' UTR and ORF zone between two group memberships of II type gene is respectively 94.6～94.7%, 85.3～86.5% and 94.1～94.5%.Show comparatively evident difference between I type and the II type gene member.They are higher in the consistence in full length mRNA and ORF zone, reach 83.3～84.6%, 83.8～85.5% respectively, and differ greatly at non-coding region, are respectively 61.9%, 55.9～63.2% in the consistence of 5 ' UTR and 3 ' UTR.

2, the number of members of swede type rape, Chinese cabbage and wild cabbage TT10 gene family detects

Multiple ratio according to swede type rape, Chinese cabbage and the wild cabbage TT10 gene family member sequence of cloning is right, and the 5th exon sequence of choosing conservative relatively and suitable length is as Southern hybridization probe sequence.With the BnTT10-2 full-length cDNA is template; Adopt combination of primers FTT10-32 and RTT10-50 amplification purpose fragment (probe), and probe is carried out digoxin-dUTP (digoxigenin (DIG)-dUTP) mark with PCR method (PCR DIG Probe Synthesis Kit); The PCR program of probe mark is: 94 ℃ of preparatory sex change 2 minutes, and 1 minute, 60 ℃ annealing of 94 ℃ of sex change were extended 1.5 minutes for 1 minute, 72 ℃ again, totally 30 circulations, last 72 ℃ were extended 10 minutes.Choose do not have recognition site in the probe sequence restriction enzyme DraI, EcoRI and EcoRV respectively enzyme cut swede type rape, Chinese cabbage and wild cabbage genome DNA; Carry out 0.8% agarose gel electrophoresis, alkaline denaturation and neutralization then, DNA is transferred on the positively charged nylon membrane with the vacuum transfer printing.Mark is good probe and nylon membrane are hybridized (DIG Easy Hyb) at 42.6 ℃ 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 3; The hybridization band of Chinese cabbage genome DNA after EcoRI and EcoRV enzyme are cut is 2;, the DraI enzyme only shows 1 thicker hybridization band after cutting, hybridize; 3 different BrTT10 members have been cloned in Yin Ben research from Chinese cabbage; Infer that in view of the above BrTT10-1A has eclipsed Southern hybridization band with BrTT10-1B owing to highly similar, and BrTT10-2 is owing to having significant sequence difference property with them and can being separated by the Southern detection zone.The hybridization band of wild cabbage genomic dna after DraI, EcoRI and EcoRV enzyme are cut is 1; Though from wild cabbage, be cloned into 2 different BoTT10 genes; But these 2 members' sequence height is similar; Full length mRNA only differs 8 bases (consistence 99.6%), should have eclipsed Southern hybridization band (correspondingly, the hybridization band is also dense).The hybridization band of swede type rape genome DNA after the DraI enzyme is cut obviously has 4; And after EcoRI and EcoRV enzyme are cut, hybridized 2 tangible bands and 2 unconspicuous blanking bars appear all; Owing to all there is not the recognition site of these 3 kinds of restriction enzymes in probe area; Be illustrated in the genome of swede type rape and possibly have 4 TT10 genes; And 3 different B nTT10 members that this research is cloned into possibly only represent 3 hybrid belts on the Southern hybridization figure, and 1 hybrid belt possibly represented another BnTT10 member of temporarily not being cloned in addition.Gene clone and Southern hybridization have all proved the Chinese cabbage really of swede type rape and the allotrtraploid species of wild cabbage, and it has the summation of Chinese cabbage and wild cabbage TT10 gene.

3, swede type rape, Chinese cabbage and wild cabbage TT10 gene family are inferred analysis of protein

It is as shown in table 4 that swede type rape, Chinese cabbage and wild cabbage TT10 gene family are inferred proteic essential property.Because the mRNA sequence of BoTT10-1pse and BoTT10-1 are much at one, and 2 base deletions in its nucleotide sequence cause can not the efficient coding functional protein, so do not comprise BoTT10-1pse in the analysis of protein.

Table 4 swede type rape, Chinese cabbage and wild cabbage TT10 gene family are inferred proteic essential property

Can know by table 4; BnTT10-1, BrTT10-1A and BrTT10-1B albumen size are 559aa; BnTT10-2 and BoTT10-1 albumen size are 563aa, and BnTT10-3 and BrTT10-2 (standard montage) albumen size is 560aa, all is slightly less than AtTT10 albumen (565aa).BnTT10, BrTT10, BoTT10 family protein molecular weight are all between 63.26～63.60kDa.With AtTT10 albumen deflection neutral different in kind; The aminoacid sequence neutral and alkali amino acid ratio (8.75～9.12%) of BnTT10, BrTT10, BoTT10 family protein is higher than acidic amino acid ratio (6.79～7.16%); Therefore its iso-electric point is 9.01～9.09, obviously deflection alkalescence.And charged amino acid, polare Aminosaeren and hydrophobic amino acid ratio are all less with AtTT10 albumen difference, and BnTT10, BrTT10, BoTT10 family protein are also very similar each other.On amino acid was formed, BnTT10-1, BrTT10-1A and BrTT10-1B albumen were all the highest with proline(Pro) (Pro) content, and Serine (Ser), Threonine (Thr), leucine (Leu) and Xie Ansuan (Val) relative content are higher in addition; The highest with proline(Pro) (Pro) content equally in BnTT10-3 and the BrTT10-2 albumen, secondly be Threonine (Thr), Serine (Ser), leucine (Leu) and Xie Ansuan (Val) take second place; BnTT10-2 and BoTT10-1 albumen are the highest with Serine (Ser) content, secondly are proline(Pro) (Pro) and Threonine (Thr), and leucine (Leu) takes second place.

NCBI BLASTp and Vector NTI 10.3.0 compare of analysis show that BnTT10, BrTT10, BoTT10 family protein and AtTT10 albumen have higher homology, and the laccase albumen with many other plants also has higher homology simultaneously.BnTT10, BrTT10 and 7 members of BoTT10 family and AtTT10 albumen (NP_199621) are carried out the comparison in twos on the amino acid levels; The result is as shown in Figure 4; Consistence between BnTT10, BrTT10 and BoTT10 family protein is 83.8～100.0%; Similarity is 88.4～100.0%, all apparently higher than consistence (80.4%～83.0%) and similarity (86.0%～87.4%) between they and AtTT10 albumen.BnTT10-1 and BrTT10-1A, BrTT10-1B albumen have very high homology, and the consistence between them reaches 99.3～99.8%, and similarity reaches 99.5～99.8%; They and the proteic consistence of AtTT10 and similarity are respectively 80.9～81.1% and 86.2～86.5%.BnTT10-2 is identical with the BoTT10-1 protein sequence; They and the proteic consistence of AtTT10 and similarity are respectively 83.0% and 87.4%, are respectively 94.1～94.5% and 95.2～95.6% with consistence and the similarity of the BnTT10-1 that is all II type gene coded protein, BrTT10-1A and BrTT10-1B.The aminoacid sequence of I type gene coded protein BnTT10-3 and BrTT10-2 (standard montage) is also identical; They and the proteic consistence of AtTT10 and similarity are respectively 80.4% and 86.0%, and the consistence (85.5%) of itself and II type gene coded protein BnTT10-2 and BoTT10-1 and similarity (89.0%) are a little more than consistence (83.8～83.9%) and the similarity (88.4～88.8%) of itself and II type gene coded protein BnTT10-1, BrTT10-1A and BrTT10-1B.

NCBI guards domain search (NCBI Conserved Domain Search) and finds, the E in BnTT10, BrTT10, BoTT10 family protein sequence ₁₅₄-S ₃₀₅, D ₄₁₉-G ₅₄₁(BnTT10-3 and BrTT10-2 albumen are D ₄₁₉-G ₅₄₂, BnTT10-2 and BoTT10-1 albumen are D ₄₂₃-G ₅₄₅) and V ₂₈-R ₁₄₁The conserved domain pfam00394, pfam0773 and the pfam07732 that have many copper oxydase family respectively.The two kinds of irregular splicing form BrTT10-M1 of BrTT10-2 and the irregular montage of BrTT10-M2 obviously do not influence the existence in these cupric oxydase family structure territories.

The prediction of ScanProsite shows, all contains by 21 many copper that amino-acid residue is formed oxydase I type sequential label G-x-[FYW]-x-[LIVMFYW]-x-[CST]-x-[PR]-[K]-x in BnTT10, BrTT10 and the BoTT10 family protein sequence ₂-[S]-x-[LFH]-G-[LM]-x ₃-[LIVMFYW] is G in I type albumen ₅₁₇VWFMHCHFDRHLTWGMNVVF ₅₃₇, be the GVWFMHCHFDRHLTWGMKVVF (G of BnTT10-1, BrTT10-1A and BrTT10-1B protein sequence in II type albumen ₅₁₆-F ₅₃₆, BnTT10-2 and BoTT10-1 protein sequence G ₅₂₀-F ₅₄₀), wherein sequence HCHFDRHLTWGM and the II type sequence label H-C-H-x that reported ₃-H-x ₃-[AG]-[LM] matches.Kumar etc. once proposed four label L1-L4 of laccase sequence, in the clone's of this institute BnTT10, BrTT10 and BoTT10 gene family encoded protein sequence, and sequence H ₇₇WHGVEQPRNPWSDGPEYITQCPI ₁₀₀Type sequence label (fungal laccase label H-the W-H-G-x that meets L1 fully ₉-D-G-x ₅-QCPI and plant laccase label H-W-H-G-x ₉-D-G-P-x ₃-T-Q-C-P-I).The H of BnTT10-1, BnTT10-3, BrTT10-1A, BrTT10-2 and BrTT10-1B protein sequence ₄₅₉-F ₄₆₆, BnTT10-2 and BoTT10-1 protein sequence H ₄₆₃-F ₄₇₀(HPMHLHGF) meet L3 sequential label H-P-x-H-L-H-G-H fully.T in the II type protein sequence ₁₁₇TVWWHAH ₁₂₄With A in the I type protein sequence ₁₁₇TVWWHAH ₁₂₄Part meets L2 sequence label (fungal laccase label G-T-x-W-Y-H-S-H-x ₃-Q-Y-C-x-D-G-L-x-G-x-FLIM and plant laccase label G-T-L-x-W-H-A-H).Conserved sequence GVWFMHCHFDRHLTWGMNVVF and GVWFMHCHFDRHLTWGMKVVF partly meet L4 sequence label (plant laccase label G-V-W-[FLI]-[FML]-H-C-H-[FMLI]-[DE]-X-H-X2-W-G-L-X-M-X-[WF]).Therefore, can predict BnTT10, BrTT10, BoTT10 gene family coding laccase.Because base mutation causes BoTT10-1pse protein translation premature termination, only comprise the L1 sequence label in its sequence, do not have complete and cupric ion bonded active site, so it possibly not have the function of complete laccase.

The systematic evolution tree of BnTT10, BrTT10 and BoTT10 family protein and other plant laccase is as shown in Figure 5, and BnTT10-1 and BrTT10-1A flock together, and has constituted first branch with BrTT10-1B; BnTT10-2 and BoTT10-1 have constituted second branch; BnTT10-3 and BrTT10-2 have constituted the 3rd branch; Relation between first branch and second branch is more more approaching than them and the 3rd ramose relation; Then, BnTT10, BrTT10, BoTT10 family protein (not comprising BoTT10-1pse) form the Cruciferae TT10 albumen monoid that is closely connected with AtTT10 albumen cluster.They are nearer with the AtLAC14 relation of Arabidopis thaliana the 4th monoid that coexists; At last, they form bigger monoid with the higher plant laccase of other homology such as rice Os LAC9, Chinese chestnut CmLAC, castor-oil plant RcLAC etc. again.In this research with fungal laccase flavus AfLAC and plant Vitamin C oxidase tomato SlAOX albumen as outer crowd; TT10 albumen monoid and other plant laccase are obvious and plant Vitamin C oxidase relation is more approaching; Verified that TT10 is earlier by plant laccase evolution and next, and far away with the relation of fungal laccase.

4, the tissue and organ specificity detection of expression of swede type rape, Chinese cabbage and wild cabbage TT10 gene family

Adopt sxemiquantitative RT-PCR to detect swede type rape, Chinese cabbage and wild cabbage TT10 gene family overall expression and each member's the specifically expressing in the different tissues organ.Get seed total RNA of totally 12 organs of root, hypocotyl, cotyledon, stem, leaf, flower bud, flower, pod skin and the different developmental phases of swede type rape 09L587, Chinese cabbage 09L600, wild cabbage 09 sweet 4 respectively; Adopting RNA PCR Kit (AMV) Ver.3.0 reverse transcription is total cDNA, as the template of pcr amplification.According to the sequence of Arabidopis thaliana 26SrRNA, design primers F 26S and R26S (table 5) are with the conservative fragments of the house-keeping gene Bn26S gene 534bp in amplification swede type rape, Chinese cabbage and the wild cabbage, the cDNA concentration that is used to detect and regulate reverse transcription; Pcr amplification adopts Easy TaqDNA polysaccharase; 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 to cloning the multiple compare of analysis of the swede type rape, Chinese cabbage and the wild cabbage TT10 gene family member sequence that obtain; Difference site according to each member and other member; 6 forwards and 6 reverse special primers (table 5) have been designed respectively; Be used for each member is carried out the specifically expressing that the detection of specifically expressing: combination of primers FRT10S13+RRT10S1 is used to detect BnTT10-1 and BrTT10-1A gene; FOT10S13+ROT10S13 is used to detect the specifically expressing of BnTT10-2 and BoTT10-1 gene; FTT10S3+RTT10S3 is used to detect the specifically expressing of BnTT10-3 and BrTT10-2 gene, and FRT10S13+RRT10S3 and FOT10S3/ROT10S3 are respectively applied for the specifically expressing that detects BrTT10-1B and BoTT10-1pse gene.With swede type rape, Chinese cabbage and wild cabbage genome DNA is template, carries out 52-68 ℃ grads PCR with above-mentioned each combination of primers, and the highest annealing temperature that obtains effective amplification of above-mentioned each combination of primers is respectively 61,61,61,60 and 60 ℃.Be template with the sub-plasmid of the mono-clonal of 8 member's full-length cDNAs respectively, adopt above-mentioned each combination of primers to increase simultaneously, prove almost to intersect between each member and increase in above-mentioned effective annealing temperature.Combination of primers FTT10A+RTT10A is used to detect the overall expression of TT10 gene family.The pcr amplification program is: 94 ℃ of preparatory sex change 2 minutes, and 1 minute, 55/60/61 ℃ annealing of 94 ℃ of sex change was extended 1.5 minutes for 1 minute, 72 ℃ again, totally 30 circulations, last 72 ℃ were extended 10 minutes.

Detected result is as shown in Figure 6, and BrTT10, BnTT10 and BoTT10 gene family member mainly express in the seed of etap.In the organs such as root, stem and pod skin that rape belongs to, all do not detect the TT10 expression of gene, have only BnTT10-3, BrTT10-2 and BrTT10-1B in spending, to detect faint expression.BoTT10-1pse since in the nucleotide sequence disappearance of 2 bases cause phase shift mutation and can not encode functional protein, in all organs, all do not detect its expression.And in the seed of 4 different developmental phases; It is the highest that BnTT10 and each member of BrTT10 gene family have different expression characteristics: BnTT10-1, a BnTT10-2 and BrTT10-1B expression level in seed in mid-term (spending back 25～30 days); In the seed of later stage (spending back 40～45 days) and Veraison (spending back 50～55 days), stronger expression is arranged also, in the seed (spending back 10～15 days) faint expression is only arranged in early days; BrTT10-1A equally in the mid-term seed expression amount the highest, the expression of certain level is also arranged in the Veraison seed, then only detect faint expression in early days with in the seed in later stage; BoTT10-1 expression amount in the later stage seed is the highest, and the expression of higher level was also arranged in the seed in mid-term, and is lower at the expression level of Veraison, and also only detects the expression of lower level in the seed in early days; I type gene member BnTT10-3 and the BrTT10-2 expression level basically identical in mid-term, Veraison and later stage seed, the trend that expression amount slightly rises along with the growth of seed, they in early days the expression level in the seed be higher than other TT10 member.

Interior mark and the special detection primer of table 5 swede type rape, Chinese cabbage and wild cabbage TT10 gene family RT-PCR

5, swede type rape, Chinese cabbage and the wild cabbage TT10 gene family differential expression between black, yellow seed near isogenic line detects

Adopt sxemiquantitative RT-PCR to detect swede type rape, Chinese cabbage and wild cabbage TT10 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 seed total RNA of totally 6 Main Reproductive Organs of flower bud, flower, the different developmental phases of swede type rape 09L587, Chinese cabbage 09L600, wild cabbage 09 sweet 1 respectively, 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 as shown in Figure 7; In the seed of, yellow each etap of seed near isogenic line black swede type rape and Chinese cabbage; The overall expression in black seed of BnTT10 and BrTT10 gene family is a little more than the expression in yellow seed, and especially the difference of seed maturity is the most obvious.But BnTT10 reveals different expression patterns with each membership table of BrTT10 gene family: I type gene member BnTT10-3 and BrTT10-2 and the II type gene member BrTT10-1B expression in black seed is higher than the expression in yellow seed, and II type gene member BnTT10-1 and the BrTT10-1A expression in black seed is starkly lower than its expression in yellow seed; In addition, the expression of BnTT10-3 a little less than the Hua Zhongyou of cabbage type rape yellow seed system then do not detect expression in the spending of black seed system, and on the contrary, the expression of BrTT10-1B a little less than the Hua Zhongyou of black seed system but do not detect expression in the spending of yellow seed system.The differential expression pattern of BoTT10-1 in black, yellow seed is different from BnTT10 and BrTT10: in the black seed of wild cabbage system; BoTT10-1 expression amount in the seed of Veraison (spending back 55 days) is the highest; Next is the seed of mid-term (spending back 30 days), and expression amount is lower in the seed of later stage (spending back 45 days); And in the yellow seed of wild cabbage system, the expression amount of BoTT10-1 in the later stage seed is the highest, secondly is the seed of mid-term and Veraison; In growing early stage seed, the expression amount of BoTT10-1 in yellow seed is also is higher than the expression amount in black seed is.In any case, an identical trend of these 3 species is, is starkly lower than black seed in the transcriptional expression level of seed maturity Veraison TT10 gene in yellow seed, explains that the downward modulation of TT10 expression of gene participated in the formation of the yellow seed proterties of these 3 species.

According to gene structure and evolutionary relationship, the swede type rape that the present invention cloned, Chinese cabbage and wild cabbage TT10 gene are divided into two types.Research to gene coded protein and expression pattern shows that possibly there is the differentiation on the function in this gene of two types.At first; From expression pattern; I type gene BnTT10-3 and BrTT10-2 have the expression pattern close with the AtTT10 gene; They all have expression in the seed of 4 etap, and its transcriptional level is gradually the trend that rises along with the growth of seed, and the later stage seed behind annesl has the highest transcriptional level.In addition, identical with the proteins encoded of AtTT10, prediction shows that they are secreted into outside the born of the same parents probably after signal peptide is cut to the Subcellular Localization of BnTT10-3 and BrTT10-2 proteins encoded.According to the secretion and the pathways metabolism of pycnogenols in the Arabidopis thaliana, in the process of cell decline, along with cell rupture, after the oligopolymer of l-Epicatechol and pycnogenols is transported to outside the tenuigenin, maybe be with the TT10 reaction by further oxypolymerization.The accumulation of xylogen occurs on the secondary wall, rather than is accumulated in the born of the same parents.Predict the avtive spot of a β-glycosyl hydrolase at the proteic N-end of I type, in II type albumen, do not predict this site.β-glycosyl hydrolase is participated in multiple basal metabolism approach, comprises lignification.Therefore infer that I type gene participates in pycnogenols and the oxidative polymerization of lignin monomer in the seed development later stage kind skin probably.II type gene shows and the different expression pattern of I type gene.BnTT10-1, BrTT10-1A and BrTT10-1B have the highest transcriptional level at the seed in mid-term, and along with progressively reaching maturity of seed, their transcriptional level is downward trend gradually.According to the prediction of PSORT, the signal peptide that predicts in the II type protein sequence possibly sheared, and the Subcellular Localization that they are inferred is an endoplasmic reticulum, and colourless pycnogenols is in the vesicles that is formed by endoplasmic reticulum, to be synthesized.Therefore infer that II type gene mainly participates in the formation of the kind chrotoplast cyanine oligomer in seed development mid-term to later stage probably.

Three, the application of swede type rape and parent's species Chinese cabbage thereof and wild cabbage TT10 gene family

1, the structure of BnTT10, BrTT10 and BoTT10 gene family Antisense Suppression expression vector

According to the comparison of the BnTT10 that is cloned, BrTT10 and BoTT10 gene family full-length cDNA and genome sequence, choose the 5th conservative relatively exon of BnTT10, BrTT10 and BoTT10 gene family member as the antisense fragment that suppresses altogether.With the BnTT10-2 full-length cDNA is template, adopts combination of primers FTT10A+RTT10A amplification antisense fragment BTT10A (sequence is shown in SEQ ID No.11 the 653rd～1604 bit base) and adds that at its 5 ' end SacI restriction enzyme site, 3 ' end add the BamHI restriction enzyme site; Pcr amplification adopts the Pfu archaeal dna polymerase, and 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 30 circulations, last 72 ℃ were extended 10 minutes; 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 pMD18-T-BTT10A.

Go out antisense fragment BnTT10A with BamHI and SacI double digestion from pMD18-T-BTT10A; PCAMBIA2301G carrier with open loop behind same double digestion is connected again; Connect product and transform DH5 α competent cell, the positive single spot of the anti-kantlex of picking (Kan) is cultivated laggard performing PCR and is detected, and chooses positive colony and extracts plasmid; Carry out PCR detection and the checking of BamHI+SacI double digestion; Obtain the Antisense Suppression expression vector pBTT10A (Fig. 8) that BnTT10, BrTT10 and BoTT10 gene family suppress altogether, the reverse fragment of inserting is stopped transcribing by the Nos terminator by the CaMV35S promoters driven.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 the external source target gene.

2, the Antisense Suppression expression vector transforms agrobacterium tumefaciens

Adopt liquid nitrogen cold shock method to transform the agrobacterium tumefaciens lba4404 competent cell pBTT10A; 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.

3, agriculture bacillus mediated Antisense Suppression expression vector transforms the black seed of swede type rape system

This research is chosen three kinds of swede type rapes and is carried out transgenic research.The black seed material Westar of swede type rape has short breeding cycle, often is used as laboratory study; Oil 821 is the typical black seed production check variety of China's long-term planting in the black seed commercial variety of swede type rape, and its DH is that strain is purer, and upgrowth situation is stable; Two No. 10 is the double-low rapeseed kind that oil crops institute of the Chinese Academy of Agricultural Sciences newly authorizes in recent years in commercial kind of the black seed of swede type rape, and the seed color is black and quality is good.Select for use these three cabbage type rape varieties to carry out transgenic research simultaneously, help the Function Identification and the genetically engineered possibility of BnTT10 gene family are drawn reliable conclusion.

Frozen Agrobacterium engineering strain thawed be cultured to logarithmic phase after the activation; 5000rpm collected thalline in centrifugal 10 minutes; [MS+1.0mg/L 2 with the MSm liquid nutrient medium; The 4-dichlorophenoxyacetic acid (2,4-D)+1.0mg/L 6-benzylaminopurine (6-BA)+100 μ M Syringylethanone (AS)] regulate bacterial concentration to OD ₆₀₀About 0.3, supply to contaminate and use.Choose the swede type rape seed, soaked 1～2 hour with clear water, 95% ethanol cleaned 1 minute; Aseptic water washing 2～3 times; Used 0.1% mercuric chloride solution sterilization again 15 minutes, aseptic water washing 4～5 times is inoculated on the MS solid medium; 25 ℃ of illumination cultivation 8～10 days, the hypocotyl that cuts aseptic seedling is as genetically modified explant; Hypocotyl is cut into the long stem section of about 1cm, inserts in advance in the training substratum (MS+1.0mg/L 2,4-D+1.0mg/L 6-BA) 25 ℃ of illumination cultivation 72 hours; 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 48 hours in the substratum (MS+1.0mg/L 2,4-D+1.0mg/L 6-BA+50 μ M AS) altogether; Hypocotyl after cultivating is altogether immersed the washing sterilization 30 minutes of vibrating in the MSk liquid nutrient medium [MS+1.0mg/L 2,4-D+1.0mg/L 6-BA+500mg/L cephamycin (Cef)], repeats secondary, blots surface-moisture with aseptic thieving paper; [MS+1.0mg/L 2 to insert the callus induction substratum again; 4-D+1.0mg/L (Westar and middle oily 821 usefulness 100mg/L Kan press as screening 6-BA+500mg/L Cef+100/75mg/L Kan; In press as screening with 75mg/L Kan for two No. 10)] in illumination cultivation more than 14 days, to growing macroscopic kanamycin-resistant callus tissue; Insert division culture medium [MS+4.0mg/L 6-BA+2.0mg/L zein (ZT)+5.0mg/L AgNO again ₃+ 500mg/L Cef+100/75mg/L Kan] middle illumination cultivation is more than 14 days, and evoked callus breaks up; Insert the middle illumination cultivation of stem division culture medium (MS+3.0mg/L 6-BA+2.0mg/L ZT+500mg/L Cef+100/75mg/L Kan) again to growing little stem; Insert the middle illumination cultivation of long shoot substratum (MS+0.05mg/L 6-BA+500mg/L Cef+100/75mg/L Kan) again to growing stem and blade; Insert the middle illumination cultivation of root media [MS+2mg/L naphthylacetic acid (NAA)] again 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 and cultivation

(1) T ₁GUS dyeing for transfer-gen plant is identified

Get T ₁For the blade of transfer-gen plant (regenerated plant in the present age after the transgenic), be cut into small pieces immersion GUS dye liquor and (contain in the sodium phosphate buffer of 50mmol/L pH7.0: 0.1mol/L K ₃[Fe (CN) ₆], 0.1mol/L K ₄[Fe (CN) 6], 10mmol/L Na ₂EDTA, 0.001% (v/v) Triton X-100,0.5mg/ml X-Gluc) in, 37 ℃ of dyeing are spent the night, and immersing after decolouring is spent the night in 70% ethanolic soln again, observe coloration result.

(2) T ₁PCR for transfer-gen plant identifies

Get T ₁Blade for transfer-gen plant extracts DNA; Respectively with the forward primer F35S3N and the FTT10A pairing of the 35S promoter of pCAMBIA2301G carrier; The reverse primer RNOS5N of terminator and RTT10A pairing, and combination of primers FGUS+RGUS (detection gus gene), amplification transfer-gen plant DNA; Detect inserting fragment, the 3 pairs of primers all amplify band and consistent with expection length and confirm as transgenic positive plant (Fig. 9).

(3) T ₂And T ₃Cultivation and management for transfer-gen plant

The T of transgenic positive plant and the negative plant of contrast ₂, T ₃For planting seed and cultivate in phytotron and accomplish growth, artificial vernalization and growth, T ₃Also plant completion growth, natural vernalization and growth in the airtight greenhouse of natural illumination, all bagging selfings when blooming simultaneously for transfer-gen plant and contrast.T ₂And T ₃All extract DNA for plant, carry out PCR and detect, add up its transgenic filial generation segregation ratio, and select the representative preferably T of upgrowth situation ₂And T ₃Carry out macroscopical identification and reserve seed for planting for plant.

This research obtains regeneration plant 52 strains of anti-Kan altogether, and wherein Westar has 22 strains, middle oily 821 have 13 strains, in two No. 10 17 strains are arranged.Have only 22 strains through GUS dyeing and PCR evaluation, transplant survival and two positive plants that can normally bear pods, wherein Westar have 6 strains, middle oily 821 have 9 strains, in 7 strains are arranged pairs No. 10.

5, the inhibition effect detection of BnTT10 gene family in the transgene rape

(1) overall and each member's of TT10 gene family detection of expression in the transfer-gen plant

Adopt real-time quantitative RT-PCR to detect the overall expression of TT10 gene family in the transfer-gen plant: the T that gets transgenic positive plant and the negative plant of contrast ₂For seed, extract RNA, respectively getting 1 μ g is template, carries out reverse transcription with RNA PCR Kit Ver.3.0, the synthetic first chain cDNA.With the Bn18S gene is confidential reference items, with AlleleID 7.0 software design PCR in real time primers F BnT10Q and RBnT10Q (table 6).Adopt SYBR Premix Ex Taq to carry out pcr amplification, amplification length 92bp.The real-time fluorescence that adopts Stratagene Mx3000P quantitative real time PCR Instrument to carry out the PCR product detects, and amplification program is: 94 ℃ of preparatory sex change 2 minutes, and 30 seconds, 60 ℃ annealing of 94 ℃ of sex change were extended totally 40 circulations 30 seconds for 1 minute, 72 ℃ again; Melt curve analysis is determined as 55～95 ℃.Each sample is established 3 repetitions, carries out data analysis with MxPro QPCR software.The result shows that the integral body of TT10 gene family is expressed and received 0～83% inhibition in the transgenic seed, the transcriptional level different (Figure 10) of TT10 between the different strain systems.

Table 6 real-time quantitative RT-PCR primer

Adopt sxemiquantitative RT-PCR to detect each member's of TT10 gene family in the transfer-gen plant expression: the member's specifically expressing that detects BnTT10-1, BnTT10-2 and BnTT10-3 in the transfer-gen plant seed respectively with combination of primers FRT10S13+RRT10S1, FOT10S13+ROT10S13 and FTT10S3+RTT10S3; 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 30 circulations, last 72 ℃ were extended 10 minutes.The result shows that the conversion of Antisense Suppression expression vector pBTT10A all has close inhibition effect to 3 member BnTT10-1, BnTT10-2 and BnTT10-3 of BnTT10 gene family, does not have member's specificity (Figure 10).

(2) weight detecting of transgenic seed

Get the T of transgenic positive plant and the negative plant of contrast ₂For seed, to get 20 at every turn and weigh, data were averaged after each strain system measured 3 times, and it is heavy to calculate single seed grain then.The result shows that transgenic seed is compared with contrast, and its single seeded weight does not have significant difference.

(3) transgenic seed annesl process is observed

Respectively at spending the back to get the transgenic positive plant in 25,30,35,40 and 45 days and contrasting the T of negative plant ₂For pod, strip off pod skin, the observation kernel seed coat colour changes and takes a picture with Stereo microscope.The result is shown in figure 11, and the annesl delay phenomenon generally appears in the seed of transfer-gen plant, shows the formation in BnTT10 effect gene kind color of the leather pool.Before spending back 25 days, transgenic seed is green with the kind skin of contrast seed; Spend 30～35 days the seed in back to begin annesl in the adjoining tree, spend back 40 days seed generally to transfer redness or black to; But in the transgenic positive plant, it is painted to spend back 35 days seed just to begin minute quantity, spends back 40 days seed that the part annesl is just arranged, and spends back 45 days seed just to accomplish annesl basically; But, during results the kind color of the leather pool of genetically modified mature seed from appearance with the contrast and no significant difference, still show as black seed.

(4) planting micromicro dissolubility procyanidin content measures

Strip the transgenic positive of swede type rape Westar and the T of the negative plant of contrast ₃For the kind skin of seed, adopt butanols-salt acid system to measure the content [with reference to (2006) such as Liang, the report of Dalzell and Kerven (1998)] of pycnogenols, the kind skin that each strain system chooses 3 individual plants is as sample, and each sample replicate measurement is averaged for 3 times.The result is shown in figure 12, accumulated the solubility pycnogenols more than 3 times in contrast kind of the skin in the transgenic kind skin of Antisense Suppression BnTT10, and the inhibition degree is strong more, and the content of the solubility pycnogenols of accumulation is high more.Strain is only to keep 46% BnTT10 transcript in V-10 and the V-13 seed, and the solubility procyanidin content is respectively 3.37 times and 3.17 times that skin is planted in contrast in its kind skin.Strain is that the BnTT10 expression of gene has been suppressed 83% in the V-12 seed, and the solubility procyanidin content is the highest in its kind skin, for contrasting 3.75 times that plant skin.Strain is that V-22 is transgenic positive strain system, but the result of fluorescence quantitative RT-RCR shows that the BnTT10 expression of gene is suppressed in its seed, and significantly increasing does not appear in the content of its kind micromicro dissolubility pycnogenols.And strain is V-10T ₂The T of the negative strain after generation separates ₃The content of planting the pycnogenols in the skin also approaches contrast kind of a skin.Strain is the T of V-13 ₂For having occurred losing of target gene in the strain isolated, in its DNA, can only detect the GUS box, but detect less than the antisense fragment its T ₃The content of planting solubility pycnogenols in the skin does not increase yet.In the kind skin of positive strain that the expression of BnTT10 is not suppressed and negative strain isolated, the content of solubility pycnogenols is all less than increasing, and shows the causing owing to the BnTT10 expression of gene is suppressed really of increase of solubility procyanidin content.The T of the transgenic positive strain system that above-mentioned solubility procyanidin content increases ₃Plant in the skin; The insolubility procyanidin content of measuring the kind skin residue after the solubility pycnogenols extracts also is 1.2～1.9 times of contrast kind of skin; But the difference of insolubility procyanidin content is less than the difference of solubility pycnogenols, and do not reach significant difference.This result of study is similar with the result of study of Arabidopis thaliana tt10, and total body burden of the solubility pycnogenols that in transgenic kind skin, can measure is higher than total body burden of pycnogenols in contrast kind of the skin, explains that the polymerization of pycnogenols receives inhibition to a certain degree.

(5) planting the skin content of lignin measures

Strip the transgenic positive of swede type rape Westar and the T of the negative plant of contrast ₃Kind skin for seed; Adopt the acetyl bromide method to measure the content [with reference to the report of Morrison (1972) and Hatfield and Fukushima (2005)] that can extract xylogen in the transgenic seed; The kind skin that each strain system chooses 3 individual plants is as sample, and each sample replicate measurement is averaged for 3 times.The result is shown in figure 13, at the T of the positive strain V-10 of transgenic Westar, V-12 and V-13 ₃Plant in the skin, content of lignin has reduced by 12%, 16% and 5% respectively.And use the content of lignin of measuring cane in transgenic positive and the adjoining tree with quadrat method, and find that the two does not have significant difference, show that the TT10 expression of gene suppresses not influence the content of lignin in the plant cane.Similar with the content of pycnogenols, the kind skin content of lignin that separates negative strain and lose the segmental plant of antisense is all less than significantly reducing, and shows the causing owing to the BnTT10 expression of gene is suppressed really of reduction of content of lignin.

Comprehensive above-mentioned experimental result, the expression of Antisense Suppression BnTT10 gene family can effectively and specifically reduce kind of a skin content of lignin, and suppress kind of the polymerization of skin pycnogenols, and kind of skin annesl is postponed.Because the essential characteristics of rape yellow seed proterties is exactly reducing greatly of kind of skin pigment even disappears, and plants the skin attenuation simultaneously, plant the skin robust fibre and especially plant the minimizing of skin xylogen.Therefore; The proterties modification effect that suppresses the expression generation of BnTT10 gene family through genetically engineered is consistent on trend with the requirement of yellow seed proterties, and the BnTT10 gene family is significant and better application prospect in the molecular breeding of creating the rape yellow seed proterties through the transgenic means.The outward appearance of the ripe seed of antisense BnTT10 transgene rape black in color is still inferred that thus TT10 gene itself possibly not be that the main of rape yellow seed proterties imitated the site, but is received one of effector of these major gene regulation and control in the present embodiment.The contriver shows also that to the molecular mechanism research result who has the cabbage type rape yellow seed material yellow seed is compared with black seed early stage, at kind of skin pigment and a plurality of target spots of planting skin xylogen formation approach down-regulated expression has taken place all, and the polygene characteristics of this proterties also have been described.Therefore, must carry out gene silencing simultaneously to a plurality of metabolism target spots and the regulatory site that comprises TT10, the combined effect that transforms through multivalent genetic is hopeful to create the yellow seed material with practical value.

Need to prove; Swede type rape according to the invention and parent's species Chinese cabbage thereof and wild cabbage TT10 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 to wait other technology to mediate the down-regulated expression of endogenous TT10 gene or gene family, also can be applied to the molecular breeding of other rape genus crop seed proterties except that swede type rape.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 8 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) TT10 gene family the member; According to research method and the result of study that preferred embodiment provided; Other TT10 allelotrope sequence that comes from swede type rape, Chinese cabbage and wild cabbage; The TT10 gene order that perhaps comes from other subspecies, the ecotype or the kind of these 3 species; Perhaps the gene order with above-mentioned 8 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 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 (4)

1. wild cabbage TT10 gene family is characterized in that: comprise following 2 members: BoTT10-1 gene and BoTT10-1pse pseudogene; The full length cDNA sequence of said BoTT10-1 gene is shown in SEQ ID No.8, and the full length cDNA sequence of BoTT10-1pse pseudogene is shown in SEQ ID No.9.

2. wild cabbage TT10 gene family according to claim 1 is characterized in that: the genome sequence of said BoTT10-1 gene is shown in SEQ ID No.7.

3. the recombinant expression vector that contains any one or more gene in claim 1 or the 2 described wild cabbage TT10 gene families or gene conservative fragments.

4. claim 1 or the 2 described wild cabbage TT10 gene families application in the molecular breeding of rape genus crop seed proterties, said seed properties is ripe annesl of kind of skin and kind skin content of lignin.

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