CN1273594C - Method, special plasmid and function nucleotide segment for obtaining male sterile wheat - Google Patents
Method, special plasmid and function nucleotide segment for obtaining male sterile wheat Download PDFInfo
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- CN1273594C CN1273594C CN 03130630 CN03130630A CN1273594C CN 1273594 C CN1273594 C CN 1273594C CN 03130630 CN03130630 CN 03130630 CN 03130630 A CN03130630 A CN 03130630A CN 1273594 C CN1273594 C CN 1273594C
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- 241000209140 Triticum Species 0.000 title claims abstract description 31
- 235000021307 Triticum Nutrition 0.000 title claims abstract description 29
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- 239000002773 nucleotide Substances 0.000 title abstract description 5
- 125000003729 nucleotide group Chemical group 0.000 title abstract description 5
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- 239000012634 fragment Substances 0.000 claims description 8
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- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 4
- 235000005822 corn Nutrition 0.000 claims description 4
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present invention discloses a method for obtaining male sterile wheat and a special plasmid and function nucleotide segment. The method aims to provide a cDNA segment which is relevant to wheat pollen development and a code polypeptide sequence. The cDNA segment which is relevant to wheat pollen development and is provided by the present invention is named WSK1, and is one of the following nucleotide sequence: (1) SEQ ID No. 1 in a sequence table, (2) SEQ ID No. 2 polypeptide sequence polyribonucleotide in the code sequence table, (3) a DNA sequence which has more than 80% of autoploidy with the DNA sequence limited by SEQ ID No. 1 in the sequence table and (4) a DNA sequence which has more than 80% of autoploidy with a non-coding region of a DNA sequence limited by the SEQ ID No. 1 in the sequence table. The second purpose of the method is to provide a method for obtaining male sterile wheat. In the method, an RNAi double base expression vector is mediated by an agrobacterium, and T-DNA is inserted and integrated to a chromosome of wheat to obtain a transformant. The method of the present invention has very important application value for culturing necessary sterility seeds of wheat breeding.
Description
Technical field
The present invention relates to a kind of method that obtains male sterile wheat in the genetically engineered field, realize used special-purpose plasmid of this method and the purpose functional gene that suppresses with this method to express.
Background technology
Hybrid vigour is a kind of universal phenomenon of organic sphere.Utilize hybrid vigour can significantly improve crop yield, improve crop quality, in agriculture production, occupy an important position.The seed selection of crop male sterile line is the key link of heterosis utilization, but utilize conventional breeding method to come the seed selection sterile line to have cycle length, take effect slowly, sterile gene is single, to problems such as such environmental effects sensitivities, far can not satisfy the needs of production development.In recent years, create male sterility line of plants by genetically engineered and on some crops, obtained success, for new prospect has been started in the utilization of crop heterosis.Utilizing genetically engineered to create male sterile strategy at present mainly is to utilize the specific promoter of pollen development and foreign gene chimeric, construction of expression vector, thereby the conversion plant is blocked the process of pollen development and reaches male sterile purpose (Zhang Aimin etc., 2000, Chinese science fund 3,132-136; Clarke et al., 1991, Plant Cell 3,431-433).For example utilize the tapetum specific promoter in crop flower pesticide, to express Barnase gene (Wang Yingchun etc., 2000, biotechnology progress 3,132-136; Mascarenhas, 1990, Annu.Rev.Plant Physiol.Mol.Biol.41 317-338), destroys the pollen tapetum; Transform with pathogenic genes involved destroy callosity whetstone matter wall (Curtis et al., 1996, Plant Science 113,113-119) and utilize antisense gene technique to transform tapetum enzyme gene (Yan Longfei etc., 1999, Science Bulletin 44,2471-2475; Meer et al., 1990, Plant Biol.15,95-109) etc.But above-mentioned technology all exists poor stability, to shortcomings such as temperature sensitives.
In recent years, in plant reduction division research, obtained bigger progress.People such as Ming (Ming Y et al., 1999, Proc.Natl.Acad.Sci.USA 96,11416-11421) confirmed that the disappearance of ASK1 gene can make pollen mother cell homologous chromosomes in the reduction division process separate unusually in the Arabidopis thaliana, cause pollen abortion at last, objectively caused the male sterile of plant, yet the disappearance of this gene does not have influence on the growth of megaspore.This gene belongs to same gene family skp1 in comprising the biology of Arabidopis thaliana, its proteins encoded participated in ubiquitin protein ligase complex body E3 in the multiple biology formation (Feldman et al., 1997, Cell 91,221-230; Skowyra et al., 1997, Cell 91,209-219).
It is a kind of effective means that is used to study gene function of rising in recent years that RNA disturbs (RNAi) technology, its cardinal principle is to utilize mixes or expresses double-stranded RNA (ds-RNA) in vivo, make the endogenous and special mRNA degraded of double-stranded RNA nucleotide sequence homologous in the organism, thereby obtain phenotype (the Scott et al. behind the goal gene afunction (loss-of-function), 2001, Nature 2,110-119; Phillip, 2001, Genes ﹠amp; Development 15,485-490; Bryan, 2002, Nature Immunology 3,597-599; Fagard etal., 2000, Annu.Rev.Plant Physiol.Plant Mol.Biol 51,167-194).This technology is compared with the mutant that inactivation (gene knock-out) is inserted in screening, have simple and easy to do, the characteristics that workload is little; And compare with simple antisence RNA, having again destination gene expression is suppressed significantly, the conversion individuality of acquisition often can present the phenotype consistent with mutant.
Summary of the invention
CDNA 3 ' the end fragment and the encoded polypeptides thereof that the purpose of this invention is to provide a gene relevant with the wheat pollen development.
The cDNA fragment called after WSK1 of provided by the present invention and wheat pollen development related gene is one of following nucleotide sequences:
1) the SEQ ID № in the sequence table: 1;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 peptide sequences;
3) with sequence table in SEQ ID №: the dna sequence dna that 1 dna sequence dna that limits has 80% above homology.
4) with sequence table in SEQ ID №: the dna sequence dna that the non-coding region of 1 dna sequence dna that limits has 80% above homology.
With wheat pollen development proteins associated WSK1, be the polypeptide that comprises sequence 2 aminoacid sequences in the sequence table, or the amino acid residue sequence of sequence 2 is passed through replacement, disappearance or the interpolation of one or several amino-acid residue and has identical active by sequence 2 polypeptides derived with the amino acid residue sequence of sequence 2.
The dna sequence dna of sequence 1 is by 528 based compositions in the sequence table, the polypeptide of sequence 2 in the 3rd～236 of the front the alkali yl coding sequence table wherein, and sequence 2 is made up of 78 amino acid.237 292 later non-coding regions that base is this cDNA of sequence 1.Contain SEQ ID № in the ordered list: 1 plasmid and clone all belong to protection scope of the present invention.
Second purpose of the present invention provides a kind of RNAi of utilization technology and obtains applied novel plasmid in the male sterile wheat.
Plasmid provided by the present invention is the RNAi binary expression vector, contains promotor, WSK1 justice sequence-intron-WSK1 antisense sequences, the terminator of corn ubiquitin protein (Ubiquitin) gene.
In order to make transformant be convenient to screening, also contain antibiotic-screening mark and/or reporter gene on the described plasmid.
The 3rd purpose of the present invention provides a kind of method that obtains male sterile wheat effectively, easily.
A kind of method that obtains male sterile wheat, be with the RNAi binary expression vector through agriculture bacillus mediated, T-DNA inserts also and is incorporated on the chromosome of wheat, obtains transformant.
Described RNAi binary expression vector contains promotor, WSK1 justice sequence-intron-WSK1 antisense sequences, the terminator of corn ubiquitin protein gene.
Use method of the present invention, when the RNAi expression vector through agriculture bacillus mediated, after T-DNA inserts and is incorporated on the chromosome of wheat, the ds-RNA that generation that can be stable in the wheat body is consistent with the WSK1 sequence, thereby guiding plant inherent PTGS (PTGS, Post-Transcriptional Gene Silencing), makes the mRNA degraded of source code WSK1 in the wheat, but obtain the wheat male sterility mutant of the genetic stability that causes unusually owing to reduction division.In cultivating the essential sterile strain of wheat breeding, will be used widely.
The present invention is further elaborated below in conjunction with specific embodiment.
Description of drawings
Fig. 1 is the structure collection of illustrative plates of RNAi plant expression vector
Embodiment
Test materials of the present invention is winter wheat capital winter No. one.
The segmental clone of embodiment 1.WSK1cDNA
The wheat capital winter No. 1 (Triticum aestivum L.cv Jingdong No 1) with chamber planting is a material, and when treating young spike length 2.1cm, microscopy shows and is in meiophase the life history.The Xiao Hua that gets this period is material construction wheat cDNA meiophase library.Library construction is selected the SMART cDNA Library ConstructionKit (Catalog #:k1051-1) of CLONTECH company for use, carries out according to the test kit specification sheets.The most double-stranded cDNA of this test kit is inserted on the carrier pTriplEx2.
Utilize the W3712 fragment that has obtained to do screening by hybridization for probe in the library, obtaining 3 ' complete length is the cDNA fragment of 528bp, and sequence is the sequence 1 in the sequence table.With the unnamed gene of this cDNA is WSK1.
The structure of embodiment 2.RNAi plant expression vector
Utilize the WSK1cDNA sequence that has obtained, design two primers: primer I (5 '-
ATC GAT GGT ACCACC TCAAGA ACT GGG ACG CCG A-3 ') and primer I I (5 '-
TCT AGA CTC GAGTAA ACA AGC AAA GCATTC CAC T-3 ') and these two primers 5 ' end added ClaI/KpnI and XbaI/XhoI site (line part) respectively, be that template is carried out pcr amplification according to following response procedures to contain the segmental plasmid of WSK1: 94 ℃ of 4min; 94 ℃ of 1min, 61 ℃ of 30s, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min.
After reclaiming the PCR product, be inserted on two multiple clone site of plasmid pHANNIBAL in the mode of justice and antisense respectively through double digestion digestion.To include the fragment cutting-out of " justice-intron-antisense " structure again with XhoI, insert on the XhoI site of intermediate carrier pTriplEx2 plasmid, adopt partially digested method to obtain to have " WSK1 justice-intron-WSK1 antisense " fragment of KpnI and SacI sticky end, this segment is inserted into the downstream of corn ubiquitin protein promotor Ubi the most at last, structure has been finished the plant expression vector of Ubi::sense-intron-antisense, called after pUNiwsk, the structure collection of illustrative plates as shown in Figure 1.
Embodiment 3, agriculture bacillus mediated T-DNA transform
The mode that electricity consumption transforms changes plasmid pUNiwsk over to Agrobacterium EHA105, by the Agrobacterium-mediated Transformation WHEAT CALLUS, through tentatively obtaining resistant calli containing on the substratum of Totomycin screening, differentiate tissue cultured seedling through subculture on division culture medium, obtain the positive seedling of wheat by the gus reporter gene detection, therefrom screen unusual, the acarpous transformant of reduction division after the transplanting.
Sequence table
<160>2
<210>1
<211>528
<212>DNA
＜213〉Triticum wheat (Triticum aestivum L.)
<400>1
acctcaagaa?ctgggacgcc?gagttcgtca?aggtcgacca?ggccaccctc?ttcgacccca 60
tcctggctgc?caactacctg?aacatcaagg?ggctgctgga?cctgacctgc?cagactgttg 120
ctgacatgat?caagggcaag?accccagagg?agatccgcaa?gactttcaac?atcaagaacg 180
actttacgcc?cgaggaggag?gaggagatcc?gcagggagaa?ccagtgggcc?tttgagtaga 240
ggagcatcta?ggcaggcgat?gccgccgccg?cgttgaatga?acgaacaacg?cttagtattc 300
gtcatgtctg?catttcagcg?ctcttcttta?tgtctgtcgt?aatgggttgt?aattatcctg 360
gagtctatga?ggtggtgtcg?gtgaacatgt?tcggtcagtg?gttatgtttg?cgacaacaag 420
aaaaacctat?cgtggtcagt?ggtcatcact?tgtcaaactg?aatcttaatc?gtcaagtgga 480
atgctttgct?tgtttaccaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaa 528
<210>2
<211>78
<212>PRT
＜213〉Triticum wheat (Triticum aestivum L.)
<400>2
Leu?Lys?Asn?Trp?Asp?Ala?Glu?Phe?Val?Lys?Val?Asp?Gln?Ala?Thr
1 5 10 15
Leu?Phe?Asp?Pro?Ile?Leu?Ala?Ala?Asn?Tyr?Leu?Asn?Ile?Lys?Gly
20 25 30
Leu?Leu?Asp?Leu?Thr?Cys?Gln?Thr?Val?Ala?Asp?Met?Ile?Lys?Gly
35 40 45
Lys?Thr?Pro?Glu?Glu?Ile?Arg?Lys?Thr?Phe?Asn?Ile?Lys?Asn?Asp
50 55 60
Phe?Thr?Pro?Glu?Glu?Glu?Glu?Glu?Ile?Arg?Arg?Glu?Asn?Gln?Trp
65 70 75
Ala?Phe?Glu
78
Claims (5)
1, with the cDNA fragment WSK1 of wheat pollen development related gene, be the SEQ ID № in the sequence table: 1.
2, contain the segmental expression vector pUNiwsk of the described cDNA of claim 1.
3, expression vector according to claim 2 is characterized in that: described carrier is the RNAi binary expression vector, contains corn ubiquitin protein gene promoter, WSK1 justice sequence-intron-WSK1 antisense sequences and terminator.
4, a kind of method that obtains male sterile wheat, be with RNAi binary expression vector pUNiwsk through agriculture bacillus mediated, T-DNA inserts also and is incorporated on the chromosome of wheat, obtains transformant.
5, contain the segmental clone of the described cDNA of claim 1.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 03130630 CN1273594C (en) | 2003-05-06 | 2003-05-06 | Method, special plasmid and function nucleotide segment for obtaining male sterile wheat |
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| CN 03130630 CN1273594C (en) | 2003-05-06 | 2003-05-06 | Method, special plasmid and function nucleotide segment for obtaining male sterile wheat |
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| CN1273594C true CN1273594C (en) | 2006-09-06 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101417255B (en) * | 2007-10-24 | 2011-02-02 | 沈阳黎明航空发动机(集团)有限责任公司 | Rutile mineral aggregate processing technique |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101845087A (en) * | 2010-05-21 | 2010-09-29 | 北京市农林科学院 | Protein TaPaO associated with plant senescence induction and male sterility, coding gene and application thereof |
| CN103290026B (en) * | 2013-04-27 | 2014-09-10 | 中国科学院遗传与发育生物学研究所 | Mitochondrial gene inducing plant male sterility, and applications thereof |
| CN109355293B (en) * | 2013-09-16 | 2021-06-01 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | Application of male nuclear sterile gene and mutant thereof in crossbreeding |
| GB2552657A (en) * | 2016-07-29 | 2018-02-07 | Elsoms Dev Ltd | Wheat |
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2003
- 2003-05-06 CN CN 03130630 patent/CN1273594C/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101417255B (en) * | 2007-10-24 | 2011-02-02 | 沈阳黎明航空发动机(集团)有限责任公司 | Rutile mineral aggregate processing technique |
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