CN109825512B - CottonGhTRX134Application of gene in improving drought stress tolerance of plant - Google Patents
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Abstract
The invention discloses cottonGhTRX134Application of gene in improving plant drought stress toleranceGhTRX134The gene has a nucleotide sequence shown as SEQ ID NO. 1. The invention relates to a transgenic technologyGhTRX134The tolerance of the gene arabidopsis thaliana plant to drought, salt and oxidative stress is found to be remarkably improved. Further by silencing cottonGhTRX134Gene, results show thatGhTRX134The gene plays an active role in regulating and controlling cotton drought resistance. The invention provides a new idea for cultivating high drought-resistant plants, particularly cotton varieties.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to cottonGhTRX134The application of the gene in improving the drought stress tolerance of plants.
Background
Cotton is a commercial crop grown worldwide, is a major natural fiber crop worldwide, and is also a basic source of seed oil. Along with the gradual warming of the climate, abiotic stresses such as cotton drought and the like cause serious harm to the yield and the quality of cotton. Since one of the challenges in the breeding of drought tolerant cotton varieties is the lack of drought tolerant genes and the limited tools for cotton gene manipulation, the disclosure of the drought stress resistance gene function of cotton remains very challenging. The availability of the cotton genome sequence lays a foundation for the functional genome analysis of the cotton gene in the later gene era.
Drought is one of the serious challenges facing agricultural production in China and even the world for a long time, and the loss caused by drought is almost the sum of losses caused by other natural disasters. In addition, the current global climate warming, the continuous deterioration of ecological environment, uneven distribution of precipitation and malposition of supply and demand, the drought threat of agricultural production is more serious in future, and the stress causes negative influence on the growth, development and production of plants and huge economic loss. It has been reported that cotton production areas are gradually decreasing worldwide as drought and saline areas increase (http:// www.isaaa.org /). Therefore, the research on the major gene and the expression regulation thereof in the drought-resistant process of plants deeply clarifies the sensing and signal transmission process of plants to drought signals and reveals the molecular mechanism of plant adaptation and drought tolerance, thereby furthest excavating the drought-resistant potential of plants.
Disclosure of Invention
In order to solve at least one of the above technical problems, the inventors have unexpectedly found cotton through extensive studiesGhTRX134The gene can improve the drought stress tolerance of plants, thereby completing the invention.
The invention provides cottonGhTRX134Use of a gene for increasing drought stress tolerance in a plant, wherein said gene is characterized in thatGhTRX134The gene has a nucleotide sequence shown as SEQ ID NO. 1.
In some embodiments of the invention, the nucleotide sequence shown in SEQ ID NO. 1 isGhTRX134Exon sequences of genes.
In some embodiments of the invention, the plant is a transgenic plantGhTRX134The expression level of the gene is used for improving the drought stress tolerance of the plant.
In some embodiments of the invention, said increasing is in a plantGhTRX134The expression level of the gene is realized by the following method: increasing plant endogenesisGhTRX134Expression of genes, or overexpression of foreign sources in plantsGhTRX134A gene.
In a particularly desirable embodiment of the present invention, said overexpression source is exogenousGhTRX134The gene refers toGhTRX134The gene is transferred into the plant for expression by agrobacterium mediation by using the plant expression vector.
Further, theGhTRX134The gene is introduced into a plant cell, tissue or organ by a plant expression vector.
Further, the plant expression vector drives the expression vector through a constitutive or inducible promoterGhTRX134Expression of the gene.
Still further, the constitutive promoter is a 35S promoter.
In the present invention, the plant is cotton, corn, rice, wheat or Arabidopsis.
The invention has the advantages of
The invention relates to a transgene obtained by transgenic technologyGhTRX134The tolerance of the gene arabidopsis thaliana plant to drought, salt and oxidative stress is found to be remarkably improved. Further by silencing cottonGhTRX134Gene, results show thatGhTRX134The gene plays an active role in regulating and controlling cotton drought resistance. This provides a new idea for cultivating high drought-resistant plants, especially cotton varieties.
Drawings
FIG. 1 showsGhTRX134The expression specificity of the gene in cotton tissue.
FIG. 2 showsGhTRX134Expression pattern of gene under drought stress. A:GhTRX134induced expression levels at 2 and 24 hours after drought treatment; b: comparing the plant heights of the wild type plants and the transgenic line plants under drought stress; c: phenotype of wild type plants and transgenic line plants under drought stress; d: comparison of the Dry/Wet ratio of wild-type and transgenic plants under drought stress.
FIG. 3 showsGhTRX134Overexpression in Arabidopsis results in increased salt resistance in Arabidopsis. A:GhTRX134inducible expression level under salt stress; b: plant height of wild type plant and transgenic line plant under salt stressComparing; c: phenotype of wild type plants and transgenic line plants under salt stress; d: comparison of fresh weight of wild-type and transgenic plants under salt stress.
FIG. 4 showsGhTRX134Overexpression in Arabidopsis results in increased oxidative stress tolerance in Arabidopsis. A:GhTRX134inducible expression under oxidative stress; b: phenotype of wild type plants and transgenic line plants under oxidative stress; d: comparison of survival rates of wild-type and transgenic plants under oxidative stress.
FIG. 5 showsGhTRX134The influence on improving the drought stress tolerance of cotton. A:GhTRX134the phenotype of the gene silencing cotton plant under drought stress; b:GhTRX134gene silencing cotton plant under drought stressGhTRX134The level of expression of the gene.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments.
Examples
The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Example 1GhTRX134Gene cloning and tissue expression specificity analysis
GhTRX134Is cloned from true leaves of upland cotton, and has the following sequence overall length (SEQ ID NO: 1):
ATGGCTGCAATCACTAATTTCTTAACTAAGCCACCTACTTCCGATCTTCACTTCACCCCAAAACTCACTTCTCTTTGCTCTCATGCTTCTTTTCTACCATCCCTTGCGACTAGGCCCAGATCTTTTTCAACAAAGCTAGGAACAAGCACAAGCTCTCGCTTCATTACCAAGGCTACAGCTGCTCCTGGGACCAAGAATGCTCCGAGTGATGAGCGAGTTCAGAAGGTTCACAGCATTGAGGAATTCGATGAAGCTCTCAGGAAGGCTAAAAACAGGCTTGTTGTGGTGGAATACGCCACTAGCTTCAGCTATCATAGCAGTAAAATTTACCCGTTTATGGTAGACCTTAGCAGGACATGCAACGATGTCGAATTCATCCTCGTGATGGGTGATGAATCGGACGAGACTAGGGAGCTTTGCAAAAGAGAGAAAATTACGAAAGTCCCGCACTTTAGCTTCTACAAGAGCATGGAGAAAATCCATGAAGAAGAAGGGATCGGACCGGACCAGCTGATGGGTGATGTGTTGTACTACGGTGACAGCCATTCGGGTGTGGTTCAGCTTCACAGCAGGGAGGACGTTGAGAAGCTCATCGAAGACCACAAGCTTGACCATAAGCTGATTGTTCTGGACGTGGGGCTCAAACATTGTGGGCCATGTGTCAAGGTGTACCCGACGGTGATTAAGCTGTCAAGGCAAATGGATTCGGTGGTTTTCGCGAGGATGAACGGTGATGAGAACGACAGCTGCATGCAGTTCCTCAAGGACATGGACGTGGTGGAGGTGCCGACGTTTTTGTTCATCAGGGACGGAGAGATATGTGGAAGGTACGTAGGATCTGGGAAAGGAGAGCTTATTGGGGAGATTCTAAGATACCAAGGCGTTCGAGTTACTTATTAG
based on the information of the genome, it is possible to identify,GhTRX134has an ORF length of 900 and comprises 1 exon.
By RT-PCR analysis, it was found thatGhTRX134High expression in true leaves and low expression in cotyledons and young leaves (as shown in FIG. 1).
Example 2GhTRX134Expression patterns under drought stress
To study forGhTRX134In response to drought stress, wild type arabidopsis and transgenic arabidopsis were treated with 20% PEG 6000. The method adopts a carrier with 35S:GhTRX134the constructed agrobacterium strain LBA4404 soaks wild type col-0 arabidopsis thaliana to obtain transgenic arabidopsis thaliana. T0 generation transgenic plant seeds were screened using a solid medium for the immunological antibiotic, 50U/ml, and using a solid medium for the immunological antibiotic, and the antibody was isolated. After 10 days of light culture, the seedlings were transplanted into the soil and placed in a culture room of the same environment. Heavy loadSowing again and harvesting until obtaining T3 transgenic Arabidopsis plants, identifying by PCR to obtain positive plants, using 35S promoter and gene (GhTRX134) The sequence is based. In transgenic lines (L1, L2, L4)GhTRX134The induced expression levels were higher than wild type (Wt) at both 2 and 24 hours after drought treatment (fig. 2A). Phenotypically, the plant height of the wild type plant (Wt) was much lower under drought stress than the transgenic lines (L1, L2, L4) (fig. 2B, C). Furthermore, the dry/wet ratio of the transgenic lines (L1, L2, L4) was lower than that of the wild type plant (Wt) (fig. 2D), while a high dry-wet ratio indicates a more severe water loss.
Example 3GhTRX134Overexpression in Arabidopsis results in increased salt resistance in Arabidopsis
For the study ofGhTRX134Biological function under salt stress, the inventors treated 2-week-old transgenic seedlings and wild-type plants (Wt) with 200mmol/L NaCl. After the salt stress is carried out for 48 hours,GhTRX134the induction expression amount in the transgenic plants (L1, L2 and L4) is higher than that in the wild plants (Wt), which indicates thatGhTRX134Expression can be induced by salt stress in Arabidopsis (FIG. 3A). The plant height of the transgenic plants (L1, L2 and L4) under the salt stress is reduced by a lower extent than that of wild plants (Wt) (fig. 3B and C). In addition, the fresh weight of the transgenic plants (L1, L2, L4) is obviously higher than that of the wild type plants (Wt) (FIG. 3D), and the results show that the salt tolerance of Arabidopsis is improved by over-expressing the gene.
Example 4GhTRX134Overexpression in Arabidopsis leads to increased oxidative stress tolerance in Arabidopsis
For discussingGhTRX134In response to oxidative stress, the inventors treated transgenic arabidopsis thaliana (L1, L2, L4) and wild-type arabidopsis thaliana (Wt) with methyl viologen MV. The results show that the method has the advantages of high yield,GhTRX134the expression of the gene in the transgenic plants (L1, L2, L4) was not significantly different from that of the wild type (Wt) (FIG. 4A). Phenotypically, after methyl viologen MV treatment, plants showed stress symptoms and became progressively more severe as the plants developed, while the wild type (Wt) was more damaged than the transgenic lines (fig. 4B). This is achieved byIn addition, the survival rate of the transgenic lines (L1, L2, L4) was significantly higher than that of the wild type plant (Wt) (fig. 4C).
Example 5GhTRX134Has positive regulation and control function in cotton drought resistance
The inventors analyzed using the Virus-induced Gene silencing (VIGS) System (pCLCrVA-pCLCrVB) (Gu et al, A versatil system for functional analysis of genes and microRNAs in cotton, Plant Biotechnology Journal (2014)12, pp. 638-GhTRX134Function in cotton under drought stress. Three carriers are respectively inoculated to cotton seedlings:pCLCrVA, PDS, pCLCrVA and pCLCrVA:Gh TRX134as indicator, control and infested plants, respectively.
Phenotypic analysis showed that leaves of plants stressed by 20% PEG6000 drought showed severe wilting, while control plants remained fresh (fig. 5A). When indicating a plant (pCLCrVA:PDS) When the leaves show albino phenotype, qRT-PCR detection is usedGhTRX134The expression level of (a).GhTRX134In infected plants (pCLCrVA：GhTRX134) The expression level in (a) is lower than that in a control plantpCLCrVA:GhTRX134) (FIG. 5B). These results show thatGhTRX134Plays an active role in regulating and controlling the drought resistance of cotton.
This example illustrates from another aspect, improvement in cottonGhTRX134The expression level of the gene can improve the drought stress tolerance of cotton.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Cotton research institute of Chinese academy of agricultural sciences
Application of cotton GhTRX134 gene in improvement of plant drought stress tolerance
<130> XY-2019-1-W-015
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 900
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atggctgcaa tcactaattt cttaactaag ccacctactt ccgatcttca cttcacccca 60
aaactcactt ctctttgctc tcatgcttct tttctaccat cccttgcgac taggcccaga 120
tctttttcaa caaagctagg aacaagcaca agctctcgct tcattaccaa ggctacagct 180
gctcctggga ccaagaatgc tccgagtgat gagcgagttc agaaggttca cagcattgag 240
gaattcgatg aagctctcag gaaggctaaa aacaggcttg ttgtggtgga atacgccact 300
agcttcagct atcatagcag taaaatttac ccgtttatgg tagaccttag caggacatgc 360
aacgatgtcg aattcatcct cgtgatgggt gatgaatcgg acgagactag ggagctttgc 420
aaaagagaga aaattacgaa agtcccgcac tttagcttct acaagagcat ggagaaaatc 480
catgaagaag aagggatcgg accggaccag ctgatgggtg atgtgttgta ctacggtgac 540
agccattcgg gtgtggttca gcttcacagc agggaggacg ttgagaagct catcgaagac 600
cacaagcttg accataagct gattgttctg gacgtggggc tcaaacattg tgggccatgt 660
gtcaaggtgt acccgacggt gattaagctg tcaaggcaaa tggattcggt ggttttcgcg 720
aggatgaacg gtgatgagaa cgacagctgc atgcagttcc tcaaggacat ggacgtggtg 780
gaggtgccga cgtttttgtt catcagggac ggagagatat gtggaaggta cgtaggatct 840
gggaaaggag agcttattgg ggagattcta agataccaag gcgttcgagt tacttattag 900
Claims (8)
1. The application of the cotton GhTRX134 gene in improving the drought stress tolerance of plants is characterized in that the nucleotide sequence of the GhTRX134 gene is shown as SEQ ID NO. 1, wherein the plants are arabidopsis thaliana.
2. The use according to claim 1, wherein the nucleotide sequence shown in SEQ ID NO. 1 is an exon sequence of the GhTRX134 gene.
3. Use according to any one of claims 1-2, characterized in that: the expression level of the GhTRX134 gene is increased in the plant so as to improve the drought stress tolerance of the plant.
4. The use of claim 3, wherein the increase of the expression level of GhTRX134 gene in the plant is achieved by: increasing the expression of a plant endogenous GhTRX134 gene, or over-expressing an exogenous GhTRX134 gene in the plant.
5. The use of claim 4, wherein the overexpression of the exogenous GhTRX134 gene is achieved by agrobacterium-mediated transformation of the GhTRX134 gene into a plant using a plant expression vector.
6. The use of claim 5, wherein the GhTRX134 gene is introduced into a plant cell, tissue or organ via a plant expression vector.
7. The use according to claim 6, wherein the plant expression vector drives the expression of the GhTRX134 gene by a constitutive or inducible promoter.
8. Use according to claim 7, wherein the constitutive promoter is a 35S promoter.
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| 桑树MaCDSP32基因转化烟草的抗旱性研究;孙红梅 等;《中国蚕学会2018年学术年会论文集中国蚕学会会议论文集》;20181011;第20页 * |
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