WO2010115297A1 - 油菜隐性核不育恢复基因BnCYP704B1及应用 - Google Patents

油菜隐性核不育恢复基因BnCYP704B1及应用 Download PDF

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WO2010115297A1
WO2010115297A1 PCT/CN2009/001095 CN2009001095W WO2010115297A1 WO 2010115297 A1 WO2010115297 A1 WO 2010115297A1 CN 2009001095 W CN2009001095 W CN 2009001095W WO 2010115297 A1 WO2010115297 A1 WO 2010115297A1
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gene
bncyp704b1
sterile
brassica napus
recessive
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PCT/CN2009/001095
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French (fr)
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涂金星
易斌
雷绍林
曾芳琴
傅廷栋
马朝芝
沈金雄
文静
李新华
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华中农业大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility

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  • the invention belongs to the technical field of rapeseed breeding. Specifically, it relates to the isolation, cloning, functional verification and application of a gene BnCYP704Bl which restores the recessive genic male sterility of Brassica napus L.
  • the recessive sterility gene /7CJ ⁇ ft ⁇ is a functionally deficient mutant of the A ⁇ F VW ⁇ gene.
  • the cloning and verification of the restorer gene BnCYP704Bl can be applied to solve the 50% fertile defect in the breeding of the rapeseed recessive nuclear male sterile line.
  • male sterility The genetic phenomenon that plants cannot produce normal pollen during sexual reproduction is called male sterility. It involves the occurrence and development of stamens in the flower, the structure of tapetum, the formation of microspores, the cracking of anthers and the external ecological environment. .
  • Male sterility can be divided into cytoplasmic male sterility (CMS) and nuclear male sterility (GMS). Cytoplasmic male sterility has been known to humans for more than 100 years and is widely used in the production of hybrids in rice, maize, and rapeseed.
  • CMS systems that can be used for the utilization of rapeseed heterosis include ogu, kos, tour and Porima.
  • Polima CMS is currently recognized as the most practical CMS system in the world, but the fertility of the Polima sterile line is unstable due to temperature, the risk of seed production is large, and its application is limited. .
  • the male sterile line of Brassica napus L. has stable fertility performance, is easy to backcross and transfer to obtain a new sterile line, and is generally not restricted by the restoration relationship. It has been widely concerned by breeders in recent years.
  • rapeseed sterility lines used at home and abroad: dominant genic male sterility, two pairs of recessive gene-controlled sterility, mutual nucleus sterility and transgenic nuclear sterility.
  • Yijia 3A a farmer's institute in Yibin, Sichuan province, China, is a typical representative of dominant genic male sterile lines. At present, there is no double-low hybrid combination that has been widely promoted.
  • S45A Pan Tao et al., 1988
  • 117A Hou Guozuo et al., 1991, Seeds, 5: 29-31
  • the approved ones are "Noisy No. 6", “Noisy No. 7", “Yuyan No. 7", “Yuyan No. 8” and "Yuyan No. 9".
  • the main drawback of this type of system is that there are 50% fertile plants in the sterile line. In the seed production, 50% of the fertile plants need to be manually removed at the initial flowering stage, which not only increases the cost of seed production, but also tends to be due to fertile plants. Incomplete removal, affecting seed purity, increases the risk of production.
  • the object of the present invention is to overcome the defects of the prior art, and specifically relates to an isolated clone, functional verification and application of a gene BnCYP704B1 for restoring recessive nuclear sterility of Brassica napus L.
  • the technical scheme of the present invention is as follows - Applicants obtained a nucleotide sequence capable of restoring recessive genic male sterility of Brassica napus L. from the reported gene BnCYP704B1, which is one of the following nucleotide sequences -
  • the present invention first uses the map cloning method to clone the gene BnCYP704Bl for restoring the recessive genic male sterile lines S45A and 117A of Brassica napus L.
  • the gene BnCYP704Bl for restoring the recessive genic male sterile lines S45A and 117A of Brassica napus L.
  • a transgenic sterile line plant and a corresponding seed for propagation of a 100% 'sterile sterility line.
  • the gene of the present invention can be transferred to other plants by sexual hybridization.
  • SEQUENCE LISTING SEQ ID NO: 1. The nucleotide sequence and corresponding amino acid sequence of the isolated Brassica napus recessive nuclear sterility recovery gene BnCYP704B1 of the present invention.
  • FIG. 1 Physical map covering the recessive nuclear sterility gene Bncy P 704Bl-l ⁇ AJCJ ⁇ TW ⁇ - ⁇ segment.
  • the open line indicates the genetic map of Bncy P 704Bl and ⁇ 7C/ 7 (?
  • the number below the molecular marker indicates the recombination exchange between the detected site and the molecular marker in the near isogenic population The number of plants per plant; the vertical dotted line connecting the molecular marker and the BAC clone indicates that the corresponding molecular marker and the BAC clone have homology by sequence comparison analysis; the solid line indicates the bacterial artificial chromosome (BAC) clone and Arabidopsis thaliana Chromosomes; Black dots on the line represent the Brassica gene predicted from the BAC sequence, and the open dots on the line represent the genes annotated on the Arabidopsis genome map in the corresponding region.
  • BAC bacterial artificial chromosome
  • Figure 3 Construction of the expression vector pBnG15 of the rapeseed functional complementation test.
  • FIG. 4 T with S45A genetic background. Comparison of the fertility phenotype and the S45A and S45B phenotypes after flowering in the genetically transformed plants. S45A genetic transformation T. The candidate gene carrying the dominant gene 67/ ⁇ ⁇ 3 ⁇ 4 ⁇ 7 was expressed as filament elongation and the fertility part was restored.
  • FIG. 1 Structure of the pDS1301 vector and the pAtG15RNAi clone.
  • RB and LB right and left border of T-DNA
  • Hp hygromycin phosphotransferase gene 35S: cauliflower mosaic virus 35S promoter
  • Adhl intron I of maize alcohol dehydrogenase gene
  • 0CS octopine synthase polyadenylation signal
  • GUS' Glucuronidase gene.
  • FIG. 6 Observation of the fertility of Arabidopsis RNAi transformed plants at flowering stage.
  • (a) As a result of microscopic examination of the transformed plant anther acetate magenta, no pollen was observed.
  • Fertile pollen can be observed in the wild type.
  • (c) Transformed plants have short pods and few seeds.
  • FIG. 1 Northern hybridization analysis of gene expression differences in S45A and S45B.
  • S and F represent S45A and S45B, respectively, and the numbers below the picture represent the flower length.
  • Figure 8 Results of multiple comparison sequencing of candidate genes.
  • the black block box represents the coding region of the gene, the thin line represents the intron, and the gray block represents the untranslated region.
  • ATG and TAG represent the translation initiation codon and the stop codon, respectively.
  • the black numbers indicate the actual sequence length of each substructure in S45B. Base substitutions, deletions or insertions are the result of comparison of other lines with the S45B sequence. Color numbers indicate bursts above lbp Change the difference. The differential position of the protein and amino acid changes are indicated on the right side of the gene.
  • FIG. 9 Microscopic anatomical structure of semi-thin sections of rapeseed recessive genic male sterile line S45A.
  • a, b, c, d, e and f are the fertile anther sporulation cell stage, the pollen mother cell stage and the meiosis stage, the tetrad stage, the single core early stage, the single core side and the second half of the pollen stage
  • Thin section micrographs g, h, i, j, k and 1 are semi-thin section micrographs of the corresponding period of sterile anthers, respectively.
  • E Newborn outer wall.
  • En Inner wall.
  • ML Middle layer.
  • T tapetum layer.
  • MMC Pollen mother cells.
  • Tds Quadrant.
  • Msp Single core pollen.
  • FIG. 10 Microscopic anatomical structure of the ultrathin section of the rapeseed recessive genic male sterile line S45A. i : Pollen inner wall e: Pollen outer wall base. Pc: protein overlay
  • Figure 11 Technical route for the use of a nuclear-sterile restorer gene for breeding 100-nuclear sterile lines.
  • the material used in this experiment is the Brassica napus recessive genic male sterile two-type S45AB (this material is from Sichuan University, see: Pan Tao et al., 1988, Chinese oil, (3): 5-8).
  • the material S45AB has been used for generations. Brother and sister (25 generations or more) saved.
  • the localized population of recessive genic male sterility genes is a near-isogenic group constructed by S45A and S45B brothers and sisters. It was planted in Wuhan in the fall of 2002 and extracted DNA of 310 individual plants in the spring of 2003 (see: Doyle JJ, Doyle J L) Isolation of plant DNA from fresh tissue. Focus, 1990, 12: 13-15 ) , for the initial positioning of the gene, and self-crossing the S45B bag. In the summer of 2004, the brothers and sisters group and the self-separated population were planted in Lanzhou, and the DNA of 1,664 single plants was extracted from the brother-sister group for the screening of recombinant plants and the fine mapping of genes; randomly selected from self-crossing populations. 72 individuals were used for the analysis of codominant markers. In the autumn of 2004, it was planted in Wuhan. In the spring of 2005, the DNA of 2,158 individual plants in the brother-sister group was extracted, further expanding the group of finely mapped genes.
  • the gene is located between two SCAR (Sequence characterized amplified region) markers SC I and SC7 in the N7 linkage group ( Yi et al, 2006, Fine mapping of the recessive genie male-sterile gene ⁇ Bnmsl) in Brassica Napus. Theor Appl Genet 113:643-650), ie the Bncyp704Bl site is located between SC I and SC7 (Fig. 2).
  • SCAR Sequence characterized amplified region
  • BAC bacterial artifical chromosome
  • SC 1F 5'- ACACGGTGATCCGGTAAGTCGT
  • SC 1R CCAGTAGGAGTCACCGAGATA
  • SC7F 5'- GGTCTAGAATAGTTGGCGAG
  • SC7R CTACCTGAGTACATCTGTGC
  • coli DH 10B purchased from Bao Bioengineering (Dalian) Co., Ltd.
  • conditions for electrical conversion capacitance 25 F, resistance 200 ⁇ , voltage 1. 8 kV ) Blue and white spot screening.
  • Positive clone plasmids were extracted and PCR amplified using universal M13-R and M13-F primers to detect insert size.
  • the primer series are as follows:
  • M13-F 5'- CGCCAGGGTTTTCCCAGTCACGAC
  • M13-R and M13-F primers and the Perkin Elmer sequencing kit (Big Dye Kit, according to the kit instructions), according to the instructions of the kit, using dideoxynucleotide end-stopping method from each Both ends of the subcloning were sequenced.
  • the sequencer is ABI3730 Sequencer from Perkin Elmer
  • the sequence was spliced using the reported SeqMen software.
  • the SeqMen software was used to automatically remove the poorly sequenced sequence and the pUC 18 vector sequence [see: Biosystems (Dalian) Co., Ltd. pUC 18 vector operating manual); the software was not removed by manual removal.
  • the BAC vector sequence and contaminating bacterial DNA sequences are removed by conventional BLAST analysis.
  • the parameters for splicing the two sequences using SeqMen software are: The overlap length (Mini Overlap) is greater than 20 bp (Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402), and the overlap of the sequences is greater than the Mini Match. 85 ? ⁇ .
  • Each base is determined by reference to multiple Shotgun fragment sequences that overlap at that location.
  • the region covered by a Shotgun fragment sequence is sequenced again to ensure base accuracy.
  • the primer walking method is used to fill the gap.
  • the sequencing results showed that the sequence length between the regions SC1 and SC7 of the target gene was 54 Kb.
  • the sequence of the tag is as follows:
  • the Blast (http://wwv.arabidopsis.org/blast) software of the model organism Arabidopsis thaliana was used to link the molecular marker SC8 and SC1 1 , and the allele dominant gene BnCYP704Bl containing Bncyp704BI was derived from rapeseed.
  • the 21.2 kb ( Figure 2) sequence of the species Tapidor was analyzed, showing that there may be four genes in this segment. Further gene annotations showed that the first, third and fourth genes were not associated with pollen development, and that the second gene, CYP704B 1, was associated with pollen façade synthesis; moreover, semi-thin sections showed that S45A abortion was characterized by missing outer walls.
  • the second candidate gene is a nuclear-sterile recovery gene, named BnC: TP704BJ.
  • the Genebank nucleic acid database was searched by CT/i ⁇ W sequence, and the result was homologous to the bacterial artificial chromosome (BAC) clone BrH012E04 sequence (Genebank ID: 199580279) 100.
  • the BAC clone was 98343 bp in length, BnCYP704Bl sequence and clone BrH012E04.
  • the complementary sequences of the sequences located at 48657-51048 are identical.
  • the gene contains 6 exons and 5 introns, encoding a protein of 519 amino acids. For detailed characteristics of the gene structure, see Sequence Listing SEQ ID NO: 1.
  • This example uses the plant expression vector CAMB 1301 (Sun et al, 2004, Xa26, a gene conferring resistance to Xanthomonas or zae pv. oryzae in rice, encoding a LRR receptor kinase-like protein. Plant J. 37: 517-527) as Brassica napus transgenic vector.
  • High-fidelity PCR method is used by analyzing the relationship between the cleavage site of the BAC clone sequence carrying the candidate gene and the candidate gene (Skerra, A., 1992, Phosphorot ioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity, Nucleic Acids Res., 20, 3551-3554, ) Amplification yielded a 3.8 kb fragment containing the E ⁇ RI and Sal I restriction sites.
  • the ORF at the beginning of the fragment was 784 bp in the upstream untranslated region, 2,392 bp in the gene interval, and 630 bp in the downstream untranslated region.
  • the amplified fragment was cloned into pMDI ST vector (purchased from Bao Bioengineering (Dalian) Co., Ltd.), and the sequencing results showed that the sequence was completely accurate, and no mismatched base was present. Extract the correct cloned plasmid, using ⁇ and &? ⁇ 3.8 kb fragment was recovered after double digestion, ligated into the expression vector pCAMBIA 1301 digested with EcoR I and Sal I, and ligated into E. coli strain DH10B, in LB medium containing 50 ug/ml kanamycin. The transformants were screened, single colony extraction plasmid was selected, and the nucleotide sequence was determined by sequencing with universal primers.
  • the transformation vector P BnG 15 was successfully constructed (see Figure 3).
  • the correct recombinant plasmid was frozen and thawed (HOLSTERS M et al. 1978, Transfection and transfonnation of Agrobacterium tumefaciens. Mol Gen Cent, 183: 181- 187. ) Introduction of Agrobacterium strain LBA4404. Genetic transformation of Brassica napus L.
  • PCR uses a 20 ⁇ l reaction system containing: 20-50 ng DNA template, 10 mM Tris-HCl, 50 mM KC1, 0.1% Triton X-100, 1.8 mM MgC12, 0.1 mM dNTP, 0.2 ⁇ Primer (CheckF: 5 -TCTAGAATGTCAACTTTGTATG-3 CheckR: 5-CTGCAGTCACTGATTAAGCACG-3) Wo 1 U Taq DNA polymerase.
  • the conditions for PCR amplification were: 94 ⁇ pre-denaturation for 4 min; 94 ° C lmin, 60. C lmin, 72 °C lmin, 34 cycles; 72'C extension for 10 min.
  • the PCR product was detected by 1% agarose gel electrophoresis.
  • the single plant of the TO generation plant was tested for positive fertility and the function of the candidate gene was verified.
  • the results showed that among the 5 transgenic T c plants, 2 showed filament elongation and complete fertility (Fig. 4).
  • the results showed that BnCYP704Bl played an indispensable role in the development of rape pollen and could complement the Brassica napus L. Sterile phenotype of sterile line S45A.
  • Two pairs of double-stranded amplification primers dsG15-5' and dsG15-3' were used to amplify a 350 bp specific fragment from Arabidopsis thaliana cDNA, and the amplified fragments did not contain Spe I, Kpn I, Sac I and B mM.
  • the restriction fragment was ligated into the pMD18-T vector, and the sequencing result showed that the sequence was completely accurate, and no mismatch base was present.
  • the cloned plasmid with the correct insert sequence was extracted, and the PMD18-T plasmid and the double-stranded RNA vector pDS 1301 were double-digested with I and SawH I, and the target fragment was separately recovered and ligated and transformed into E.
  • the positive plasmid was Kpn I and BcmM I double-digestion detection, and then the plasmid of the target clone was extracted, and the plasmid inserted into the first strand and the PMD18-T plasmid containing the gene insert were double-digested with Spe I and Sac I (purchased from Bao Bioengineering (Dalian) Co., Ltd. The company has separately recovered the target fragment and ligated and transformed E. coli DH10B.
  • the vector containing the restriction gene is the double-stranded RNA vector pAtG15R Ai of the candidate gene (Fig. 5).
  • the constructed double-stranded RNA vector pAtG15RNAi was transformed with Arabidopsis thaliana by Agrobacterium tumefaciens (see: Xu Guandong, Agrobacterium-mediated Transformation of Lat52-DTA Gene in Arabidopsis, 2000, Master Thesis, China Knowledge Network).
  • the seeds of the transformed plants were screened on LB medium containing 15 mg/L hygromycin.
  • the root length of the resistant plants was larger, the plants were larger, the color of cotyledons and true leaves was bright green; the roots of non-resistant plants were short and the plants were short. Cotyledons and true leaves are darker in color.
  • FIG. 6 shows the results of fertility observation of one of the individual plants.
  • Fig. 6a the pods are short and almost free of seeds
  • Fig. 6c fertile pollen is observed in wild-type anther acetate magenta staining
  • Fig. 6b the pods are full and the seeds develop normally
  • Fig. 6d Differential analysis of gene expression
  • the present invention extracts total RNA of the anthers of the four periods S45A and S45B (the size of the flower buds are less than lmm, lmm-2mm, 2mm-3mm and 3mm-4nim respectively), and performs Northern analysis on the candidate gene.
  • the cDNA of the candidate gene was used as a probe for hybridization, and rRNA was used as an internal standard for Northern hybridization.
  • the results showed that the total RA concentration in the four periods was basically the same, and the total RA concentration of the fertile and sterile plants was also basically the same.
  • Hybridization with the candidate gene as a probe revealed that the gene was not expressed or expressed in flower buds of less than 1 mm and 3 mm to 4 mm, and no significant hybridization signal was detected.
  • the expression level of SnC i ⁇ W in the flower buds of 2mm-3m m was higher than that in the flower buds of lmm-2mm, and there was no difference in the expression of buds between 1mm-2mm and 2mm-3mm between sterile plants and fertile plants. (See Figure 7).
  • Northern analysis showed that there was no difference in the expression level of candidate genes between sterile plants and fertile plants. It is speculated that the change in fertility may be caused by the loss of function due to changes in gene structure.
  • the sequencing primers designed for the candidate gene BnCYP704Bl are G15-F and G15-R (the sequence of the corresponding sequence amplification gene in the BAC1 clone is 2,392 bp. See the J. Sambrook et al., 2002, Guide to Molecular Cloning, for the standard PCR procedure. , Third Edition, Jin Dongyan et al. (translated), Science Press.
  • the primer sequence is as follows -
  • the sequencing results showed that the sequence of S45B in the gene structure region (2,392 bp) was identical to the sequence from BAC clone BAC1.
  • the S45A sequence has mutations at 12 sites compared to the S45B sequence, including 10 single base substitutions (2 occurrences in the intron region, 8 One occurred in the translation region) and two insertion mutations (both in the intron region, one single base insertion and one seven base insertion), of which S single base substitutions located in the gene translation region have 6 One is a synonymous mutation, and the other two single base substitutions result in a change in the two encoded amino acids (positions 179 and 297).
  • amino acid encoded by S45B at position 179 is glycine, and in S45A it becomes Arginine; the amino acid encoded by S45B at position 297 is valine, and in S45A it is changed to alanine (Fig. 8).
  • the present invention randomly selected 19 rapeseed varieties for comparative sequencing.
  • the results showed that the second mutation in the two mutation sites could be found in the normally fertile Brassica napus variety, and only the 179th glycine mutation was specific to the sterile mutant (Fig. 8), indicating P179G may be the cause of abortion.
  • the present invention performs semi-thin sectioning and ultra-thin sectioning on the recessive genic male sterile two-type S45AB anther.
  • the development of microspores and tapetum during the spore cell to the second pollen stage the differences in the development of the sterile anthers and fertile anther tapetum and microspores in different periods were compared. It was found that there was no difference between sterile anthers and fertile anthers in the stage of sporulation, pollen mother cells and meiosis.
  • the cells of sterile anthers could differentiate into normal pollen mother cells, and the tapetum also developed normally. ( Figures 8a and 8g, 8b and 8h). There was no significant difference in the tetrads of sterile anthers and fertile anthers during the tetrad period. The pollen mother cells could undergo normal meiosis to form tetrads, and the tetrads were surrounded by enamel. The tapetum structure had subtle differences. The tapetum of the sterile anther is slightly thicker than the tapetum of the fertile anther ( Figures 8c and 8i). After the microspores were released from the tetrads, the early differences in mononuclear were obvious.
  • the fertile pollen undergoes a mitosis to produce a large vegetative cell and a small germ cell, entering the second-core pollen period, and the sterile pollen remains in the mononuclear pollen period, cannot further develop, and gradually disintegrates; The difference is obvious.
  • the tapetum of the sterile anther is severely vacuolated, while the tapetum of the fertile anther is compressed into a thin layer without vacuolation (Fig. 8f and 81). Therefore, it is believed that the abortion of S45A is related to the abnormal function of tapetum.
  • the possible mechanism is that the tapetum layer can not provide the key material for the synthesis of pollen outer wall. The defects in the outer wall of pollen cause abnormal pollen development and gradually disintegrate.
  • the chemically induced expression promoter GVE VGE (Tawa, Development of a methoxyfenozide-responsive gene switch for applications in plants. Plant J, 2006, 45 (3): 457-469) and the lethal gene (Ba gene) (Kawai-Yamada, Mammalian B ax-induced plant cell death can be down-regulated by overexpression oi Arabidopsis Bax Inhibitor-1 ⁇ AtBI-1), PNAS, 2001, 98: 12295-12300), forming an inducible expression
  • the gene is ligated, and the recessive genic male sterile fertility restorer gene and the chimeric gene are ligated to the expression vector pCAMB 1301 to transform the nuclear sterile material S45A, thereby obtaining a genetically engineered fertile plant.
  • the fertile material is then crossed with the nuclear sterile material S45A, and the progeny exhibit a 1: 1 separation.
  • the fertile strain is killed by spraying the chemical inducer methoxyfenozide, thereby creating a 100% infertile population of nuclear sterility (see Figure 13 for the specific procedure).
  • This method has created a new way to utilize the heterosis of nuclear male sterility through transgenic technology. According to this design, it can be seen that the transgenic plants are not contained in the hybrid seed, because all fertile plants carrying the transgene It was killed in the seedling stage of seed production. Even a small number of unkilled individuals are removed during the flowering period, so there is little or no possibility of transgenic plants during the propagation of the hybrid.
  • Bin Yi Yulin Chen, Shaolin Lei, Jinxing Tu, Tingdong Fu. Fine mapping of the recessive genie male-sterile gene (Bnmsl) in Brassica napus. Theor Appl Genet., 2006, 1 13(4), 643-650.
  • Mammalian B ax-induced plant cell death can be down-regulated by overexpression ⁇ Arabidopsis Bax Inhibitor- 1 (AtBI-1).

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Description

油菜隐性核不育恢复基因 BnCYP704B
技术领域
本发明属于油菜育种技术领域。 具体涉及一个恢复甘蓝型油菜隐性核不育育性的基因 BnCYP704Bl的 分离克隆、 功能验证和应用。 所述的隐性不育基因 /7CJ ^ft^ 是 A^F VW^基因的功能缺陷突变体。 恢 复基因 BnCYP704Bl的克隆、验证可以应用于解决油菜隐性细胞核雄性不育系繁殖制种时存在 50%可育的缺 陷。
背景技术
植物在有性繁殖的过程中不能产生正常花粉的遗传现象称为雄性不育， 它涉及到花器中雄蕊的发生 与发育、 绒毡层结构、 小孢子形成、 花药开裂以及外部生态环境等很多环节。 雄性不育可以分为细胞质 雄性不育（CMS )和细胞核雄性不育（GMS )。 细胞质雄性不育为人类所知已有 100多年的历史， 并且在 水稻、 玉米、 油菜等作物中广泛的用于生产杂交种。 目前， 世界上可以用于油菜杂种优势利用的 CMS系 统有 ogu、 kos、 tour和波里马。 其中， 波里马 CMS 是目前国际上公认的最有实用价值的 CMS系统， 但 是波里马不育系育性受到温度的影响表现不稳定， 制种风险较大， 其应用受到了一定的限制。
甘蓝型油菜细胞核雄性不育系由于育性表现稳定， 易于回交转育获得新的不育系， 并且一般不受恢保 关系的限制， 近年来受到育种家的广泛关注。 目前国内外利用的油菜核不育系主要有以下四种类型： 显性 核不育、两对隐性基因控制的核不育、互作型核不育和转基因核不育。 中国四川省宜宾地区农科所 U972 ) 发现的宜 3A是显性核不育系的典型代表， 目前还没有选育出大面积推广的双低杂交组合。 9012A (陈凤 祥等， 1998，作物学报， 24 (4): 431-438 )和 7-7365A (huang et al., 2007, Theor Appl Genet., 1 15(1): 113-8. ) 均属于互作型核不育系， 以其为材料选育的品种中， 已审定的杂交种有"皖油 14号" （陈凤祥等， 2002, 安徽农业科学， 30 (4)： 535-537)、 "皖油 18号"（ 陈凤祥等， 2003 , 中国油料作物学报， 25 ( 1 )： 63-65 ) 和 "沪油杂 1号"（孙超才等， 2004, 中国油料作物学报， 26 ( 1 ): 63-65 ) 等组合， 该系统通过引入临保 系获得 100%的全不育系， 在油菜核不育系统研究领域取得了重大进展。 但是这个系统遗传基础复杂、 临 保系选育困难， 不利于选育强优势组合。 美国和加拿大利用烟草的 TA29基因的启动子和核糖核酸水解酶 (bamase)基因构建成融合基因转化甘蓝型油菜， 人工创造了新的核不育材料， 并通过向该材料中导入抗除 草剂基因后达到了机械化作业。 但是该系统的不足之处是必须对不育系和恢复系都进行转基因， 该系统目 前也受到专利的保护。 S45A (潘涛等， 1988) 和 117A (侯国佐等， 1991 , 种子， 5: 29-31 ) 是两对隐性基 因控制的核不育系的典型代表， 由其选育而来的杂交种中已审定的有 "蜀杂 6号" "蜀杂 7号"、 "油研 7 号"、 "油研 8号"和 "油研 9号"等。 该类系统主要缺陷是不育系中存在 50%的可育株， 在制种时需要在 初花期人工拔除其中 50%的可育株， 不仅增加了制种的成本， 而且往往由于可育株清除不彻底， 影响制种 纯度， 增加了生产的风险。
有关基因 BnCYP704Bl的序列已在 Genebank ( ID: 199580279)数据库中报道， 但是到目前为止尚未 揭示该基因的结构和编码的氨基酸序列， 也没有报道该基因具有恢复甘蓝型油菜隐性核不育育性的功能， 更没有报道该基因可以在繁殖甘蓝型油菜核不育系中应用。
发明内容
本发明的目的在于克服现有技术的缺陷， 具体涉及一个恢复甘蓝型油菜隐性核不育育性的基因 BnCYP704Bl的分离克隆、 功能验证和应用。 本发明的技术方案如下 -- 申请人从报道的基因 BnCYP704Bl中获得了一种能够恢复甘蓝型油菜隐性核不育育性的核苷酸序列， 它 是下列核苷酸序列之一-
1 ) 序列表 SEQ NO: 1中所示的 DNA序列； 或
2) 编码与 1 ) 编码的蛋白质相同的蛋白质的 DNA序列。
具体地， 本发明首先利用图位克隆方法， 克隆基因 BnCYP704Bl , 用于恢复甘蓝型油菜隐性核不育系 S45A和 117A (参见： 潘涛等， 1988, 中国油料， （3): 5-8)、 和侯国佐等， 1991， 种子， 5: 29-31 ) 的育性， 或作为甘蓝型油菜新核不育系选育的分子标记。进一步可以通过控制该基因的表达，繁殖甘蓝型油菜 100% 转基因不育系， 突破甘蓝型油菜核不育系杂交制种存在 50%可育株的障碍。
本发明的第二方面， 进一步提供利用上述基因获得转基因不育系植株和相应的种子， 用以繁殖 100%' 不育的核不育系。 可以用有性杂交的方式将本发明所述的基因转入其它的植株。
在本发明的实施例部分， 我们阐述了隐性基因 < .^7(¾^ 和显性基因 BnCYP704Bi 分离、 功能验证 和应用技术路线以及这两个基因的特点。
附图说明
序列表 SEQ ID N0: 1. 是本发明分离的甘蓝型油菜隐性核不育恢复基因 BnCYP704Bl的核苷酸序列和对应 的氨基酸序列。
图 1. 本发明鉴定、分离、克隆并验证油菜隐性核不育基因
Figure imgf000004_0001
和它的等位显性基因
功能的流程图。
图 2. 覆盖隐性核不育基因 BncyP704Bl-l \ AJCJ^TW^-^区段的物理图谱。 图中： 空心线表示 BncyP704Bl和 ^7C/ 7(? 基因精细定位的遗传图谱； 分子标记下面的数字表示在近等基因系群体中检 测到的 位点与分子标记之间发生重组交换的单株数； 连接分子标记和 BAC克隆的竖虚线表示 通过序列比较分析证实了相对应的分子标记和 BAC克隆具有同源性；实线表示细菌人工染色体（bacterial artificial chromosome, BAC) 克隆和拟南芥染色体； 线上的黑点代表根据 BAC序列预测的油菜基因， 线 上的空心点代表相应区域拟南芥基因组图谱上注释的基因。
图 3. 油菜功能互补测验表达载体 pBnG15的构建。
图 4. 具有 S45A遗传背景的 T。代遗传转化植株开花后育性表型与 S45A和 S45B表型的比较。 S45A遗传 转化 T。代植株携带显性基因 67/ ·ί¾^7的候选基因， 表现为花丝伸长， 育性部分恢复。
图 5. pDS1301载体和 pAtG15RNAi克隆的结构。 图中： RB和 LB: T-DNA的右侧和左侧边界; Hp 潮霉 素磷酸转移酶基因； 35S: 花椰菜花叶病毒 35S启动子； Adhl : 玉米乙醇脱氢酶基因的内含子 I ; 0CS: 章 鱼碱合成酶多聚腺苷酸信号； GUS'. 葡萄糖醛酸糖苷酶基因。
图 6. 开花期观察拟南芥 RNAi转化植株的育性。 （a)转化植株花药醋酸洋红染色镜检的结果， 没有观察 到花粉。 （b)野生型可以观察到可育的花粉。 （c ) 转化植株莢果短小， 几乎没有种子。 （d)野生型植株荚 果饱满， 种子发育正常。
图 7. Northern杂交分析基因在 S45A和 S45B中表达差异。 S和 F分别代表 S45A和 S45B, 图片下面的 数字代表花'曹长度。
图 8. 候选基因多重比较测序的结果。 黑色块状盒代表基因的编码区， 细线代表内含子， 灰色块状盒代表 非翻译区。 ATG和 TAG分别代表翻译起始密码子和终止密码子。黑色数字表示各亚结构在 S45B中实际的序 列长度。 碱基替换、 缺失或插入是其它品系与 S45B序列相比较的结果。 彩色数字表示超过 lbp以上的突 变差异。 蛋白质的差异位置和氨基酸变化在基因的右侧标示。
图 9. 油菜隐性核不育系 S45A半薄切片显微解剖结构。 a、 b、 c、 d、 e和 f分别是可育花药造孢细胞时 期、花粉母细胞时期与减数分裂时期、 四分体时期、 单核早期、 单核靠边期和二核花粉期半薄切片显微照 片， g、 h、 i、 j、 k和 1分别是不育花药对应时期的半薄切片显微照片。 E: 初生外壁。 En: 内壁。 ML: 中层。 T: 绒毡层。 MMC: 花粉母细胞。 Tds: 四分体。 Msp: 单核花粉。
图 10. 油菜隐性核不育系 S45A超薄切片显微解剖结构。 i ： 花粉内壁 e: 花粉外壁基座。 pc: 蛋白覆 盖层
图 11. 核不育恢复基因应用于繁殖 100核不育系的技术路线。
具体实施方式
以下实施例进一步说明本发明的内容， 但不应理解为对本发明的限制。 在不背离本发明精神和实质的 情况下， 对本发明方法、 步骤或条件所作的修改或替换， 均属于本发明保护的范围。
若未特别指明， 实施例中所用的技术手段为本领域技术人员所熟知的常规手段。 下列实施例中未注明 具体条件的实验方法， 均参照公开报道的方法或手册， 例如在分子生物学方法方面发明人参考了萨姆布鲁 克的 《分子克隆手册》 （参见： J.萨姆布鲁克， EF弗里奇， T曼尼阿蒂斯著， 黄培堂， 王嘉玺等译， 分子 克隆实验指南 （第三版)， 科学出版社， 2002版） 中所报道的方法或技术手段。 实施例 1: 构建含隐性核不育基因^^7<^«区段的物理图谱
1. 实验材料
本实验所用的材料为甘蓝型油菜隐性核不育两型系 S45AB (该材料来自四川大学， 参见： 潘涛等， 1988 , 中国油料， （3): 5-8 ) , 材料 S45AB经过多代的兄妹交 （25代以上） 保存。
隐性核不育基因的定位群体是由 S45A和 S45B兄妹交构建的一个近等基因系群体， 2002年秋播种 于武汉， 2003年春天提取 310个单株的 DNA (参见： Doyle J J, Doyle J L. Isolation of plant DNA from fresh tissue. Focus, 1990, 12: 13-15 ) , 用于基因的初步定位， 并将 S45B套袋自交。 2004年夏季在兰州播种兄妹交 群体和自交分离群体，从兄妹交群体中提取 1 ,664个单株的 DNA ,用于重组单株的筛选和基因的精细定位; 从自交群体中随机选取 72个单株用于共显性标记的分析。 2004年秋在武汉播种， 2005年春天提取兄妹交 群体中 2, 158个单株的 DNA , 进一步扩大基因精细定位的群体。
2. 筛选包含 BnCYP704Bl基因区段的 BAC克隆
根据初步定位的结果该基因位于油菜 N7连锁群的两个 SCAR( Sequence characterized amplified region) 标记 SC I和 SC7之间 ( Yi等, 2006, Fine mapping of the recessive genie male-sterile gene {Bnmsl) in Brassica napus. Theor Appl Genet 113 :643-650 ) , 即 Bncyp704Bl位点位于 SC I和 SC7之间 （图 2)。 利用油菜品种 Tapidor的细菌人工染色体 （bacterial artifical chromosome, BAC ) 文库 （Rana等， 2004， Conservation of the microstructure of genome segments in Brassica napus and its diploid relatives. Plant J. 40: 725 - 733. ) ,本发明筛 选到了 1个包含恢复基因 BnCYP704Bl的 BAC克隆（见图 2)， 该克隆包含基因两侧的 SCAR标记 SC 1和 SC7。 引物序列如下：
SC 1F: 5'- ACACGGTGATCCGGTAAGTCGT SC 1R: CCAGTAGGAGTCACCGAGATA
SC7F: 5'- GGTCTAGAATAGTTGGCGAG SC7R: CTACCTGAGTACATCTGTGC
3. 包含目标基因 BAC克隆的测序和序列拼接
釆用部分西切法 (参见： Yu J等. A draft sequence of the rice genome (Oiyzae sativa L. ssp. Indica). Science, 2002, 296:79-92 ) 构建目标 BAC克隆的 Shotgun文库。 具体步骤是： 用 Sa I处理 BAC克隆的 DNA， 通过 1 %琼脂糖凝胶电泳分离出 1一3 kb的 DNA片段，纯化后与 BamH I酶切的去磷酸化的 pUC18载体 (购 自宝生物工程 （大连） 有限公司） 连接， 电转化大肠杆菌 DH 10B ( (购自宝生物工程 （大连） 有限公司）， 电转化的条件： 电容 25 F、 电阻 200 Ω、 电压 1. 8 kV ) , 进行蓝白斑筛选。 提取阳性克隆质粒， 用通用 M13-R和 M13-F引物进行 PCR扩增， 检测插入片段大小。 引物系列如下：
M13-R: 5，- GAGCGGATAACAATTTCACACAGG
M13-F: 5'- CGCCAGGGTTTTCCCAGTCACGAC
采用 M13-R和 M13-F引物和美国 Perkin Elmer公司的测序试剂盒（Big Dye Kit,按照试剂盒的说明书 操作），根据试剂盒的使用说明以双脱氧核苷酸末端终止法分别从每个亚克隆的两端测序。测序仪为 Perkin Elmer公司的 ABI3730 Sequencer
使用报道的 SeqMen软件拼接序列。用 SeqMen软件自动去除末端测序质量较差的序列和 pUC 18载体 序列 〔参见： 宝生物工程 （大连） 有限公司 pUC 18载体操作手册）； 对软件没有去除干净的序列则用手工 删除。 BAC载体序列和污染的细菌 DNA序列则通过常用的 BLAST分析的方法剔除。 用 SeqMen软件对 两条序列进行拼接的参数是： 重叠序列长度（Mini Overlap) 大于 20 bp (Altschul等， 1997, Nucleic Acids Res. 25:3389-3402 ) , 重叠序列的一致性（Mini Match)大于 85 ? ί。 每一个碱基的确定都要参照重叠在该位 点的多个 Shotgun片段序列。 对于由一个 Shotgun片段序列所覆盖的区域要再次测序验证， 以确保碱基的 准确性。 而对于 shotgun测序后存在的 Gap区域， 则用 primer walking的方法， 填补缺口。 测序结果表明 目标基因的区域 SC1和 SC7之间的序列长度为 54Kb。 实施例 2: 精细定位核不育基因 BnCYP704Bl
利用分子标记 SC 1和 SC7在 4132个单株的近等基因系群体中分别检测到 4个和 18个交换单株， 这 些单株在标记和 位点间发生了重组 （见图 2)。 为了进一步縮小目标基因区域的范围， 根据目 标区域的序列设计了 35对特异引物， 在 S45A、 S45B之间进行多态性筛选， 发现 6个新的分子标记 SC8、 SC9、 SC 10、 SC1 SC 12和 SC13。
标记的序列如下：
SC8F 5-TTCCAGGAGCACATCATCCGCAGAG SC8R 5-AGGGGGAGAGAAAAAGATAGAAAC SC9F 5-CTGAAGAATAAGCGACCAGC SC9R 5-CTTCATGGAAGGCGAGTCTT
SC I OF 5-GGCCTACTAAGGTAGTCCTG SC 1 OR 5-CCACACGCTGAGTTCATATTGGACAC SC 1 1 F 5-GAGCTCTCACGTTGAAAGT SC 11 R 5-CGGCGTAGAAGTGA AGTCTC
SC 12F 5-CCCGAACTTCATCTTACTCG SC12R 5-CACGTGTCAAGCTCTGGTGG
SC 13F 5-ATCTACTCTTCTTTGGCTGTT SC13R 5-GAGCGTTCAAGTCAGTTCC
用新的 SCAR标记进一步分析 22个重组交换单株（参见： BinYi等 Fine mapping of the recessive genie male-sterile gene (Bnms l) in Brassica napus. Theor Appl Genet., 2006, 1 13(4), 643-650.)。 然后用新标记 SC8、 SC9、 SC 10、 SCl l , SC 12和 SC 13对交换单株进行分析， 在 SC 1检测到的 4个交换单株中， 有 2个在 SCS 与 BnCYP704BI基因间亦表现交换。 在标记 SC7检测到的 18个交换单株中， 标记 SC11、 SC 12和 SC 13 分别检测到 6个、 7个和 9个交换单株。 标记 SC9和 SC10与 AzCr t^W基因共分离。 通过以上精细定 位， 将 ^C / i S 基因定位于分子标记 SC8和 SC 11之间， 与 SC9和 SC 10共分离。 该区间在 BAC克 隆 JBnBBACI上跨度约 2 I .2-kb (图 2)。 实施例 3: 显性基因 BnCYP704Bl的分离和功能验证
1. BnCYP70 Bl候选基因的确定
本实施例中采用模式生物拟南芥网站的 Blast ( http://wwv.arabidopsis.org/blast) 软件对分子标记 SC8 和 SC1 1之间、 包含 Bncyp704BI的等位显性基因 BnCYP704Bl、 来源于油菜品种 Tapidor的 21.2 kb (图 2) 序列进行分析， 显示该区段可能存在四个基因。 进一步的基因注释显示： 第一、 第三个和第四个基因与花 粉发育不相关， 第二个基因 CYP704B 1与花粉外壁合成相关； 而且， 半薄切片显示 S45A败育的特征是外 壁缺失， 因此推测第二个候选基因基因是核不育恢复基因， 命名为 BnC:TP704BJ。 用 ? CT/ i^W序列检 索 Genebank核酸数据库， 结果与白菜细菌人工染色体 (BAC)克隆 BrH012E04序列（Genebank 登录的 ID: 199580279 ) 100 同源，该 BAC克隆全长 98343 bp, BnCYP704Bl序列与克隆 BrH012E04位于 48657-51048 的序列的互补序列完全相同。 该基因包含 6个外显子和 5个内含子， 编码 519个氨基酸的蛋白质。 基因结 构的详细特征参见序列表 SEQ ID NO: 1。
2. ^油菜转基因实验
本实施例采用植物表达载体 CAMB 1301 ( Sun 等， 2004, Xa26, a gene conferring resistance to Xanthomonas or zae pv. oryzae in rice, encoding a LRR receptor kinase-like protein. Plant J. 37:517-527 )作为甘 蓝型油菜转基因载体。 通过分析携带候选基因的 BAC克隆序列的酶切位点和候选基因的关系， 利用高保 真 PCR方法 ( Skerra, A., 1992 , Phosphorot ioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity, Nucleic Acids Res., 20, 3551-3554, )扩增获得一个 3.8 kb的片段，该片 段包含 E∞R I和 Sal I酶切位点。片段起始端包基因上游非翻译区 784 bp的序列、基因区间 2,392 bp的序 列和下游非翻译区的 630 bp的序列。 将扩增片段克隆到 pMDI S-T载体 （购自宝生物工程 （大连） 有限公 司〉 上， 测序结果表明序列完全准确， 没有错配碱基存在。 提取序列正确克隆的质粒， 用 ^^ 和&？^ 双酶切后回收 3.8 kb的片段， 连接到经 EcoR I和 Sal I双酶切的表达载体 pCAMBIA 1301上， 连接产物转 化大肠杆菌菌株 DH10B , 在含有 50ug/ml卡那霉素的 LB培养基上筛选转化子， 挑选单菌落提取质粒， 并 用通用引物测序检测核苷酸序列是否正确， 成功构建了转化载体 PBnG 15(见图 3)。 正确的重组质粒通过冻 融法 ( HOLSTERS M等. 1978， Transfection and transfonnation of Agrobacterium tumefaciens. Mol Gen Cent, 183: 181- 187. )导入农杆菌菌株 LBA4404。甘蓝型油菜遗传转化釆用常规的农杆菌转化法（陈苇等， 2006, 甘蓝型油菜 Fad2 基因的 RNA干扰及无筛选标记高油酸含量转基因油菜新种质的获得， 植物生理与分子 生物学学报， 32 (6): 665 -671 ) 转化油菜核不育系 S45A (用分子标记鉴别两型系中的 S45A和 S45B ) , 用 70 %酒精浸泡种子 15 min, 0.1 ° 升汞消毒 15min, 20 °；)〜 30 °。的次氯酸钠消毒 15min, 无菌水清洗 3次， 每次间隔 5min。灭菌的种子播于 MS基本培养基（PH=5.8， MURASHIGE, Toshio and SKOOQ Folke. 1962, A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, 15 ( 3 )： 473-497)， 25 °C暗培养 7d。 将幼苗下胚轴切成 0.5cm-0.8cm的小段， 于含有农杆菌 LBA4404 (悬浮过夜至 对数生长期） 的 MS液体培养基（PH=5.8 )浸染 90min-120 min后， 吸干液体， 于 25Ό黑暗条件下共培养 3d； 将外植体转到愈伤组织诱导培养基（ MS基本培养基 + 2, 42 D 1 . 0 mg L + KT (激动素） 0 . 3 mg/L + 蔗糖 30g L)上， 25 °C光照培养 15- 18d。将外植体转入分化培养基（MS基本培养基 + I AA (吲哚乙酸） 0 . 1 mg/L + ZT (玉米素） 1 . 0 mg/L +葡萄糖 10 . 0 g L ) 培养， 每 15- 18 d继代 1次， 直至分化出幼苗。 当 幼苗长至 2-3 cm髙时， 将幼苗转入生根培养基 （ 1 /2 MS基本培养基 + 1 BA (吲哚丁酸） 0 . 1 mg/L +蔗 糖 10 g/ 上培养。转化植株生根后， 假植于混有腐质土的细土纸杯中， 保持 70 。相对湿度， 一个月后移 入大田。
从获得阳性植株上提取叶片总 DNA ((参见： Doyle J J, Doyle J L. Isolation of plant DNA from fresh tissue. Focus, 1990, 12:13-15 ) ) ,经 PCR方法鉴定转化植株， PCR标准程序参见参见】.萨姆布鲁克等， 2002, 分子克隆实验指南， 第三版， 金冬雁等译， 科学出版社， 介绍的方法。 PCR采用 20 μ 1的反应体系， 包含： 20-50 ng DNA模板， 10 mM Tris-HCl, 50 mM KC1, 0.1% Triton X-100, 1.8 mM MgC12, 0.1 mM dNTP, 0.2 Μ 引物 （CheckF: 5-TCTAGAATGTCAACTTTGTATG-3 CheckR: 5-CTGCAGTCACTGATTAAGCACG-3 ) 禾口 1 U Taq DNA polymerase。 PCR扩增的条件为： 94Ό预变性 4min; 94 °C lmin, 60。C lmin, 72 °C lmin, 34个循环； 72'C 延伸 10min。 PCR产物用 1%琼脂糖凝胶电泳检测。 TO代植株检测为阳性的单株观察育 性表现， 验证候选基因的功能。 结果显示在 5个转基因 Tc代植株中， 有 2株表现为花丝伸长， 育性完全恢 复 （图 4)， 结果表明 BnCYP704Bl在油菜花粉发育中发挥不可或缺的作用， 能够互补甘蓝型油菜不育系 S45A的不育表型。
3. 拟南芥 RNAi实验
利用两对双链扩增引物 dsG15-5'和 dsG15-3'分别从拟南芥的 cDNA中扩增出 350bp的特异片段， 扩 增片段都不包含 Spe I、 Kpn I , Sac I和 B mM I酶切位点， 将扩增片段克隆到 pMD18-T载体上， 测序结 果表明序列完全准确， 没有错配碱基存在。 提取插入片段序列正确的克隆的质粒， 用 I和 SawH I双 酶切处理 PMD18-T质粒和双链 RNA载体 pDS 1301，分别回收目标片段后连接并转化大肠杆菌菌株 DH10B, 阳性质粒用 Kpn I和 BcmM I双酶切检测， 然后提取目标克隆的质粒， 用 Spe I和 Sac I双酶切处理插入第 一链的质粒和包含基因插入片段的 PMD18-T质粒（购自宝生物工程（大连）有限公司）， 分别回收目标片 段后连接并转化大肠杆菌 DH10B , 酶切检测准确的克隆所包含的载体就是候选基因的双链 RNA 载体 pAtG15R Ai (图 5 )。 构建的双链 RNA载体 pAtG15RNAi用农杆菌沾花的方法 （参见： 许冠东， 农杆菌 介导 Lat52-DTA基因转化拟南芥菜， 2000, 硕士学位论文， 中国知网） 转化拟南芥。 在含 15mg/L潮霉素 的 LB培养基上筛选转化植株的种子， 抗性植株根长， 植株较大， 子叶和真叶的颜色为鲜绿色； 非抗性植 株根很短， 植株矮小， 子叶和真叶颜色较深。 植株长到 3— 5片真叶后移植到营养土中培养， 得到 35个抗 性植株。 在开花期观察转化植株的育性， 用醋酸洋红染色、 镜检发现 2个植株表现为雄性不育， 图 6是其 中 1个单株育性观察的结果， 转化植株花药醋酸洋红染色没有发现花粉 （图 6a)， 荚果短小， 几乎没有种 子 （图 6c); 野生型花药醋酸洋红染色观察到可育花粉 （图 6b) , 植株荚果饱满， 种子发育正常 （图 6d)。 实施例 4: 基因的表达差异分析
本发明分别提取 S45A和 S45B四个时期花药 （花蕾的大小分别为小于 lmm、 lmm-2mm、 2mm-3mm 和 3mm-4nim) 的总 RNA, 对候选基因 ^进行 Northern分析。 转膜后分别用候选基因的 cDNA 做探针进行杂交， 以 rRNA作为 Northern杂交的内标， 结果表明四个时期总 R A浓度基本一致， 可育株 与不育株总 R A浓度也基本相同。用候选基因做探针的杂交结果显示， 基因在小于 1mm和 3mm-4mm的 花蕾中不表达或者表达量很低， 没有检测到明显的杂交信号。 在 2mm-3mm花蕾中 SnC i^W的表达量 高于 lmm-2mm花蕾中的表达量， 不育株和可育株之间在 lmm-2mm和 2mm-3mm的花蕾中表达量都没有 差异（见图 7)。 Northern分析结果表明候选基因的表达量在不育株和可育株之间没有差异， 推测育性的变 化可能是由于基因结构变化导致功能缺失引起的。
实施例 5: 候选基因 的比较测序
针对候选基因 BnCYP704Bl设计的测序引物为 G15-F和 G15-R (在 BAC1克隆中对应序列扩增基因区 间 2,392 bp的序列。 PCR标准程序参见参见 J.萨姆布鲁克等， 2002， 分子克隆实验指南， 第三版， 金冬雁 等(译)， 科学出版社介绍的方法。 引物序列如下-
G15-F 5-ATGTCTATGTGGATCGTTCTAG G15-R 5-CTATGAACGTCTGGATACAG
测序结果表明： S45B在基因结构区域的序列 （2,392 bp) 与来自 BAC克隆 BAC1的序列完全一致。 而 S45A序列与 S45B序列相比有 12个位点发生突变， 包括 10个单碱基替换 （2个发生在内含子区域， 8 个发生在翻译区） 和 2个插入突变 （都发生在内含子区域， 一个单碱基的插入和一个 7个碱基的插入）， 其中位于基因翻译区的 S个单碱基替换有 6个是同义突变,另外两个单碱基替换造成两处编码的氨基酸 (第 179位和第 2 97位） 发生改变， 在第 179位 S45B编码的氨基酸为甘氨酸， 而在 S45A 中则变为精氨酸； 在第 297位 S45B编码的氨基酸为缬氨酸， 而在 S45A 中则变为丙氨酸基 （图 8)。
为了进一步确定突变位点是否与育性相关， 本发明随机选择了 19个油菜品种进行了比较测序。 结果 表明， 2个突变位点中的第二个突变可以在正常可育的白菜型油菜品种中找到， 只有第 179位的甘氯酸突 变是是不育突变体特异的 （图 8 ) , 表明 P179G可能是导致败育的原因。
表 1 显性基因 BnCYP704Bl和隐性基因 B yp704Bl序列差异位点
Figure imgf000009_0001
序列表 SEQ ID NO: 1 (显性基因 C7P70^/ ) 所示序列位点。 实施例 6: 细胞学切片分析
为了进一步确定隐性核不育系 S45A败育的细胞学特点， 并阐明 BnCYP704Bl基因的功能， 本发明对 隐性核不育两型系 S45AB 花药进行半薄切片和超薄切片， 重点观察了造孢细胞时期至二核花粉期过程中 小孢子和绒毡层发育情况， 比较了不育花药和可育花药绒毡层和小孢子在各时期发育的差异。 比较发现- 不育花药和可育花药在造孢细胞时期、 花粉母细胞时期与减数分裂时期没有差异， 不育花药的造抱细胞可 以分化形成正常的花粉母细胞， 绒毡层也发育正常 （图 8a和 8g、 8b和 8h)。 四分体时期不育花药和可育 花药的四分体没有明显差异， 花粉母细胞可以进行正常的减数分裂形成四分体， 四分体被胼胝质包围； 绒 毡层结构有细微的差异， 不育花药的绒毡层较可育花药的绒毡层稍厚 （图 8c和 8i)。 在小孢子从四分体中 释放后的单核早期差异明显， 主耍表现在不育花粉没有外壁的合成； 可育花药的絨毡层压缩变扁， 而不育 花药的绒毡层径向扩大 （图 8d和 8j)。 在单核晚期， 可育花粉细胞质出现一个大的液泡， 细胞核靠近细胞 壁的一侧， 花粉外壁进一步加厚； 不育花粉的细胞质也液泡化， 但是花粉外壁合成一直受阻； 不育花药的 絨毡层进一步增厚（图 8e和 8k)。 随后可育花粉经过一次有丝分裂产生一个大的营养细胞和一个小的生殖 细胞， 进入二核花粉期， 而不育花粉一直停留在单核花粉期， 不能进一步的发育， 并逐渐解体； 绒毡层差 异明显， 不育花药的绒毡层液泡化严重， 而可育花药的绒毡层被压縮成很薄的一层， 没有产生液泡化 （图 8f和 81)。因此认为 S45A败育与绒毡层功能异常相关，可能的机制是绒毡层不能提供花粉外壁合成的关键 物质， 花粉外壁合成缺陷而导致花粉发育异常， 逐渐走向解体。
确定了败育的关键时期后，对单核花粉期的小孢子进行了超薄切片观察。超薄切片观察的结果进一步 确定了花粉外壁合成缺陷的是 S45A败育的原因 （图 9 ) , 因此， C P7i^8/基因的功能可能与油菜花粉 外壁合成相关， 这一结论与拟南芥 CYP704B1基因的注释吻合。 实施例 7： 利用油菜细胞核雄性不育基因 BnCYP704Bl繁殖 100%不育群体
将能够在田间应用的化学诱导表达的启动子 GVE VGE ( Tawa, Development of a methoxyfenozide-responsive gene switch for applications in plants. Plant J, 2006， 45 (3):457-469 ) 与致死基因 (Ba 基因) （Kawai-Yamada, Mammalian B ax-induced plant cell death can be down-regulated by overexpression oi Arabidopsis Bax Inhibitor-1 {AtBI-1), PNAS, 2001 , 98： 12295- 12300)连接， 形成诱导表达的嵌合基因， 再 将隐性核不育育性恢复基因与该嵌合基因连接到表达载体 pCAMB 1301上转化核不育材料 S45A , 从而获 得基因工程可育株。 然后将该可育材料与核不育材料 S45A杂交， 后代呈现 1 : 1 的分离。 苗期， 通过喷施 化学诱导剂甲氧虫酰肼 （methoxyfenozide) 杀死可育株， 从而创造细胞核不育 100%的不育群体 （具体操 作步骤见图 1 1的流程）。
该方法通过转基因技术创造了细胞核雄性不育杂种优势利用的新途径， 在转基因安全的问题上，根据 本设计， 可以看出在杂交种籽中不含有转基因植物， 因为所有携带转基因的可育株在制种的苗期被杀死。 即使少数没有被杀死的单株， 在开花期的除杂过程中， 也会被去掉， 因此， 在杂交种推广的过程中， 实际 上不含， 或极少可能性存在转基因植物。
在本发明所提及的所有文献都在本申请中引用作为参考文献，就如同每一篇文献被单独引用作为参考 那样。此外应理解，在阅读了本发明所讲述内容后，本领域的技术人员可以对本发明做各种改动或者修改， 这些等价形式同样属于本申请所附的权利要求书所限定的范围。 参考文献：
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Claims

权 利 要 求 书
1、一种恢复甘蓝型油菜隐性核不育育性的基因 BnCYP704Bl在繁殖甘蓝型油菜核不育系中 的应用， 其特征在于， 该恢复甘蓝型油菜隐性核不育育性的基因 ^C yW^是下列核苷酸 序列之一：
1 ) 序列表 SEQ NO: 1中所示的 DNA序列； 或
2 ) 编码与 1 ) 编码的蛋白质相同的蛋白质的 DNA序列。
2、 权利要求 1所述的基因在油菜育种中的应用。
3、 权利要求 1所述的基因在甘蓝型油菜育种中的应用。
4、 权利要求 1所述的基因在甘蓝型油菜核不育系育种中的应用。
PCT/CN2009/001095 2009-04-08 2009-09-28 油菜隐性核不育恢复基因BnCYP704B1及应用 WO2010115297A1 (zh)

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