CN102140138A - Plant drought-resistant associated protein TaNAC2742, coding genes and application thereof - Google Patents
Plant drought-resistant associated protein TaNAC2742, coding genes and application thereof Download PDFInfo
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- CN102140138A CN102140138A CN 201110071474 CN201110071474A CN102140138A CN 102140138 A CN102140138 A CN 102140138A CN 201110071474 CN201110071474 CN 201110071474 CN 201110071474 A CN201110071474 A CN 201110071474A CN 102140138 A CN102140138 A CN 102140138A
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Abstract
The invention relates to the field of genetic engineering, in particular to a plant drought-resistant associated protein TaNAC2742, coding genes and application thereof. The amino acid sequence of the protein is as shown in SEQ ID No. 1; and the gene sequence is as shown in SEQ ID No.2. The plant drought-resistant associated protein and the coding genes thereof have extremely important theoretical and practical significance on improving and reinforcing the tobacco stress resistance, improving the yield, accelerating breeding process of stress resistance molecules and effectively saving water resources.
Description
Technical field
The present invention relates to the genetically engineered field, particularly, the present invention relates to the relevant protein TaNAC 2742 of a kind of plant drought salt and encoding gene and application.
Background technology
Environment stress such as arid, saline and alkaline is that limiting plant growth is grown and the main abiotic stress factor of crop yield.According to statistics, world's arid, semiarid zone account for 1/3rd of global land area, and China's arid, semiarid zone account for 1/2nd of national land area; Also has 3.6 * 107hm
2The saltings remain open up wasteland to be utilized.Wheat occupies important status as one of China's important crops in national economy.Yet the annual underproduction that wheat is caused because of adverse circumstance China such as arid, saline and alkaline reaches hundred million kilograms of 700-800, is having a strong impact on the yield and quality of wheat, is restricting the fast development of China's wheat grain-production.Therefore, the resistance of improvement farm crop, seed selection is drought-enduring, the farm crop new product, has become the important topic of improvement of crop cultivar.
In recent years, along with going deep into of plant stress-resistance molecular biology research, tentatively disclosed plant to the environment stress signal reply, signal conduction and gene expression regulation equimolecular mechanism, for the resistance of utilizing genetic engineering technique improvement crop provides scientific basis.At present, many drought resistings, salt tolerant, low temperature resistant relevant gene are imported in the plant, improve the existing correlative study report of resistance of plant by genetic engineering technique.
In recent years, the research emphasis of adversity gene engineering progressively turns to the regulatory gene relevant with environment stress.Transcription factor is mostly relevant with the expression regulation of adversity gene under biology and the abiotic stress, has become one of emphasis of environment stress signals-modulating network research.AP2/ERF class transcription factor extensively is present in plant, has found 145 members in Arabidopis thaliana, generally is divided into AP2, RAV, DREB, an ERF and AL0793495 subtribe.Wherein, the ERF subtribe comprises 65 genes, mostly participates in the conduction of biological and abiotic stress signal, the expression of regulation and control downstream functional gene.For example, separation such as Cao obtain 3 paddy rice ERF class transcription factor OsBIERF1, OsBIERF3 and OsBIERF4, and its expression is subjected to stress-inducings such as pathogenic bacteria, salt, low temperature, arid.Tang etc. have improved the salt resistance and the frost resistance of Arabidopis thaliana with JcERF gene overexpression in Arabidopis thaliana of Cortex jatrophae.Nearest studies show that, the overexpression of potato StEREBP1 gene can induce the downstream resistant gene to express, and improves the tolerance of transgenic Rhizoma Solani tuber osi to high salt and low temperature stress.A series of Physiology and biochemistries can take place and change in plant when suffering environment stress.Long-term salt stress will cause salt accumulation in the blade, increase the activity of plant chlorophyll enzyme.
In sum, AP2/ERF class transcription factor plays crucial effects regulating the adverse circumstance reaction of plant in the resistance of raising plant.Overexpression AP2/ERF transcription factor gene has improved the anti-adversity ability of plant, and transfer-gen plant is grown and do not produced any negative effect, and this produces huge pushing effect and economic benefit to degeneration-resistant breeding and agriculture production meeting.Therefore, utilize degeneration-resistant relevant AP2/ERF class transcription factor gene improvement and the resistance of raising crop to have extremely important theory and practice meaning.Utilize drought resisting wheat germplasm resource, excavate, filter out the resistance of good gene related to drought tolerance improvement wheat, provide important adversity gene resource for cultivating food crop drought resisting new variety such as wheat, aspects such as the improvement in salinification soil and improvement are all had important practical usage.
Summary of the invention
The purpose of this invention is to provide a kind of plant drought associated protein TaNAC 2742.
A further object of the present invention provides the encoding gene of the above-mentioned plant drought associated protein TaNAC 2742 of coding.
Another object of the present invention provides the recombinant vectors that comprises said gene.
Another object of the present invention provides the application of above-mentioned plant drought associated protein TaNAC 2742 and gene thereof.
Drought resistant correlative protein TaNAC2742 provided by the present invention derives from the wheat capital winter 17, and its aminoacid sequence is shown in SEQ ID NO.1.
SEQ?ID?NO.1：
MSEASVLNQAEVEDAAAAAGLDLPPGFRFHPTDEEIISHYHTPKALDHRFCSGVIG
EVDLNKCEPWHLPSKAKMGEKEWYFFCHKDRKYPTGTRTNRATESGYWKATGK
DKEIFRGRGVLVGMKKTLVFYLGRAPRGEKTGWAMHEFRLEGKLPHPLPRSAKD
EWAVSKVFNKELTATNGAMAAAEAGIERVSSFGFIGDSLDSGELPPLMDSPLGGDV
DEVIDFKSTSAYATGAHSGLQVKMEQHMPQQPPHMMYSSPYFSLPAANSGDMSPA
IRRYCKAEQVSGQTSALSPSRETGLSTDPNAAGCAEISSAATPSSQNQDFLDQFDE
YPALNLADIWKY
According to the above-mentioned albumen of TaNAC2742 genes encoding of the present invention, can have nucleotide sequence shown in SEQ ID NO.2, TaNAC2742 open reading frame (ORF) length is 1029bp.
SEQ?ID?NO.2：
atgtctgagg?cgtcggtttt?aaaccaggcg?gaggtggagg?acgcggcggc?ggcggccggg 60
ctggacctgc?cgccgggctt?ccggttccac?cccacggacg?aggagatcat?ctcgcactac 120
cacaccccca?aggcgctcga?ccaccgcttc?tgctccggcg?tcatcggcga?ggtcgacctc 180
aacaagtgcg?agccatggca?tctcccaagc?aaggcgaaga?tgggagagaa?ggagtggtac 240
ttcttttgcc?acaaagaccg?caagtacccg?acggggacga?ggacgaaccg?cgccaccgag 300
agcggctact?ggaaggccac?cggcaaggac?aaggagatct?tccgggggag?gggcgtcctt 360
gtcggaatga?agaagacgct?cgtcttctac?ctaggccgcg?ccccccgcgg?cgagaagacc 420
ggctgggcca?tgcacgagtt?ccgcctcgag?ggcaagctcc?cccacccgct?cccgcgctcc 480
gccaaggacg?agtgggccgt?gtccaaggtg?ttcaacaaag?agctcacggc?caccaacggg 540
gcaatggcag?cggcggaggc?cgggatcgag?cgagtcagct?ccttcggctt?catcggtgac 600
tcccttgact?ctggggagct?gccgcccctc?atggactctc?ccttgggcgg?cgacgtcgac 660
gaagtcatcg?atttcaagtc?cacctctgcc?tacgccaccg?gtgcccattc?cggactgcag 720
gttaagatgg?aacagcacat?gccgcagcag?ccgccgcaca?tgatgtactc?gagcccgtac 780
ttctctctgc?cggccgctaa?ctccggcgac?atgtcgccgg?cgatccggag?gtactgcaag 840
gcggagcagg?tctcggggca?gacgtctgcg?ctcagcccgt?cacgcgagac?cgggctgagc 900
accgacccca?acgccgccgg?ttgcgcggag?atctcgtcgg?cggcgacacc?gtcgtctcag 960
aatcaagact?tccttgacca?attcgacgag?taccccgccc?tgaacctcgc?cgacatttgg 1020
aagtactga 1029
In addition, drought stress is handled to express to test and is shown that the TaNAC2742 gene is subjected to drought-induced strong expression.Simultaneously, make up the overexpression carrier of this gene and change TaNAC2742 over to tobacco by agriculture bacillus mediated leaf disc transformation method, PEG coerces to handle to test and shows that changeing the TaNAC2742 genetic tobacco has drought resistance, proved TaNAC2742 gene successful expression in tobacco, and improved the drought-resistant ability of tobacco, can be used as the high-quality candidate gene of tobacco drought resisting breeding.
Another object of the present invention provides a kind of method of cultivating drought-resistant plant.
The method of cultivation drought-resistant plant provided by the present invention is that the above-mentioned recombinant expression vector that any contains the TaNAC2742 gene is imported in the vegetable cell, obtains plant with adverse resistance.
Described plant stress tolerance specifically can be the resistance of reverse to abiotic stress, as the resistance of reverse to arid or salt stress.
The present invention is an experiment material with drought resisting, strong wheat capital winter 17 (Triticum aestivum L.) of salt tolerance, has obtained drought resisting, salt tolerance TaNAC2742 gene, and it is imported tobacco, has significantly improved the drought resistance of plant.Drought resistant correlative protein of the present invention and encoding gene thereof to improvement, strengthen stress resistance of plant, improve output, quicken degeneration-resistant molecular breeding process, and effectively save water resources and have important theoretical and practical significance.
Description of drawings
The expression pattern analysis of Fig. 1 TaNAC2742 gene under adverse circumstance is induced, wheat seedling is taken out the moisture that blots on the root from soil, place on the exsiccant filter paper, extract RNA sampling in 0,1,2,5,10,24 hour respectively, carry out the analysis of drought stress expression characterization, A is the sxemiquantitative PCR result of gene of the present invention behind the drought stress, and B is the sxemiquantitative PCR result of gene of the present invention behind the drought stress of internal reference in contrast.
Figure 23 5S-TaNAC2742 transgene tobacco drought tolerance identifies that 35S-TaNAC2742 transgene tobacco and contrast (W) are at 2 months growing states of processing that contain on the 5%PEG substratum.
Embodiment
Make the experimental methods of molecular biology specify in following examples, all carry out, perhaps carry out according to test kit and product description with reference to listed concrete grammar in " molecular cloning experiment guide " (third edition) J. Sa nurse Brooker one book.
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.
The clone of embodiment 1, TaNAC2742 gene and sequence motifs analysis
1, wheat capital winters 17 plant of 25 ℃ of illumination cultivation about 2 weeks carried out drought stress and handle, take a sample behind 0h, 1h, 2h, 5h, 10h, 24h respectively, place liquid nitrogen freezing rapidly ,-80 ℃ of preservations are standby.Extract total RNA extraction that test kit (sky, Beijing root biochemical technology company limited) is finished the plant leaf tissue according to RNA, be template with the total RNA that extracts afterwards, with the AP sequence is primer, and (M-MLV) carries out reverse transcription with ThermoScript II, obtains being used for the cDNA template of subsequent experimental.Design a pair of primer:
P1：atgtctgaggcgtcggtttt
P2：agtcatgaaggtttacagc
The present invention obtains a gene TaNAC2742, TaNAC2742 open reading frame (ORF) length 1029bp, and 342 amino acid of encoding, compare of analysis on NCBI, the highest consistence is the highest only 79%, proves new gene
Embodiment 2, environment stress are handled wheat TaNAC2742 expression of gene feature down
With the wheat seed kind in basin, grow and get seedling after 3 weeks and carry out drought stress and handle, capital winters 17 wheat seedling is taken out the moisture that blots on the root from soil, place on the exsiccant filter paper, respectively arid is handled 0,1,2,5,10,24 hour the total RNA of sample extraction in back, with TaNAC2742DNA is probe (sequence 1 in the sequence table), carries out quantitative fluorescence analysis.
Semi-quantitative analysis the results are shown in Figure 2.
The result shows: peak in drought stress TaNAC2742 genetic expression in 1 hour, illustrate that this gene is subjected to the drought stress abduction delivering.
Embodiment 3, commentaries on classics TaNAC2742 gene plant drought tolerance are identified
1, the structure of recombinant expression vector
The cDNA that obtains with total RNA reverse transcription of wheat is a template, carries out pcr amplification with the special primer that contains SmaI and XbaI joint sequence; SmaI and XbaI double digestion PCR product reclaim then, enzyme are cut between the CaMV 35S promoter SmaI and XbaI enzyme cutting site afterwards of product forward insertion carrier pBI121, obtain recombinant vectors 35S-TaNAC2742.
Primer sequence is as follows:
TaNAC2742[SmaI]5’-GCGCCCGGG?atgtctgaggcgtcggtttt-3’
TaNAC2742[XbaI]5’-TGCTCTAGA?agtcatgaaggtttacagc-3’
2, the acquisition of transgene tobacco and evaluation
The recombinant expression vector 35S-TaNAC2742 of above-mentioned structure is transformed agrobacterium tumefaciens EHA105 with freeze-thaw method respectively, to be integrated with the agrobacterium tumefaciens EHA105 transformation of tobacco W38 of 35S-TaNAC2742 more respectively with leaf dish method, carry out 2 with the MS substratum that contains the 100mg/L kantlex and take turns screening, the every wheel screened 10-15 days, obtained positive transfer-gen plant.The positive transfer-gen plant that screening obtains is done further evaluation and screening with PCR, and the used a pair of primer of PCR is P3 and P4.
P3：atgtctgaggcgtcggtttt
P4：agtcatgaaggtttacagc
The 35S-TaNAC2742 transgene tobacco carries out PCR and identifies that positive transfer-gen plant can obtain 1000bp left and right sides band through pcr amplification, and the result obtains to change 40 strains of 35S-TaNAC2742 tobacco.
Simultaneously the pBI121 empty carrier is imported tobacco W38, method is the same, in contrast, obtains commentaries on classics empty carrier tobacco (the transgene tobacco T that screening obtains of 10 strain systems
1Representative is shown).With T
1In generation, changeed 35S-TaNAC2742 genetic tobacco seed and T
1Generation change the empty carrier adjoining tree 3 age in week seedling root system move into respectively and carry out drought stress in the substratum that contains 2%PEG and handled 60 days, observe phenotype and also take pictures.The result shows: coerce and handle after 60 days, the 35S-TaNAC2742 transfer-gen plant can normal growth under the drought stress condition, and well developed root system, leaf color are dark green; All empty carrier rotaring gene plant blade poor growths.Transgene tobacco drought tolerance qualification result proof TaNAC2742 gene can improve the drought tolerance of plant.
Claims (7)
1. the plant drought associated protein TaNAC 2742, it is characterized in that, it has the aminoacid sequence shown in SEQ ID NO.1.
2. plant drought genes involved TaNAC2742 is characterized in that, the described albumen of coding claim 1.
3. plant drought genes involved TaNAC2742 as claimed in claim 2 is characterized in that it has the base sequence shown in SEQ ID NO.2.
4. the recombinant vectors that comprises claim 2 or 3 described plant droughts, salt-resistant related gene TaNAC2742
5. the application of the described plant drought associated protein TaNAC 2742 of claim 1.
6. the application of the described plant drought genes involved of claim 2 TaNAC2742.
7. method of cultivating drought-resistant plant, described method comprise that the recombinant expression vector that will contain the described TaNAC2742 gene of claim 2 imports in the vegetable cell, obtains the step of drought-resistant plant.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015180479A1 (en) * | 2014-05-26 | 2015-12-03 | 中国科学院遗传与发育生物学研究所 | Use of tanac2 protein and encoding gene thereof |
| CN110343153A (en) * | 2019-05-28 | 2019-10-18 | 华南农业大学 | Oryza officinalis OoMYB3 albumen and its encoding gene and application |
Citations (1)
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| CN101899103A (en) * | 2010-07-23 | 2010-12-01 | 北京市农林科学院 | Plant drought-resistance and salt-tolerance associated protein TaNAC, and encoding gene and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101899103A (en) * | 2010-07-23 | 2010-12-01 | 北京市农林科学院 | Plant drought-resistance and salt-tolerance associated protein TaNAC, and encoding gene and application thereof |
Non-Patent Citations (3)
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| 《中国优秀硕士学位论文全文数据库》 20091015 冶晓芳 植物抗逆相关基因的分离及功能分析 全文 1-7 第2009卷, 第10期 * |
| 《中国优秀硕士学位论文全文数据库》 20110115 刘美英 小麦NAC类转录因子的克隆和分子特征研究 1-7 第2011卷, 第1期 * |
| 《中国烟草学报》 20101231 刘美英 TaNAC提高转基因烟草的抗旱功能 第82-88页 1-7 第16卷, 第6期 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015180479A1 (en) * | 2014-05-26 | 2015-12-03 | 中国科学院遗传与发育生物学研究所 | Use of tanac2 protein and encoding gene thereof |
| CN110343153A (en) * | 2019-05-28 | 2019-10-18 | 华南农业大学 | Oryza officinalis OoMYB3 albumen and its encoding gene and application |
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Application publication date: 20110803 |