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Application of Asian cotton GaNCED3 gene in improving drought resistance of plants

A technology of drought resistance and plants, applied in the fields of application, plant products, genetic engineering, etc., can solve the problems of limited quantity and complex mechanism of drought resistance response, and achieve the effect of enriching understanding

Pending Publication Date: 2020-12-25
COTTON RES INST HEBEI ACAD OF AGRI & FOREST SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The mechanism of plant drought resistance response is complex, involving a variety of drought resistance mechanisms and signal transduction pathways. Although some cotton drought resistance related genes have been cloned, the number is limited, and there are still many unknown important genes waiting to be discovered.

Method used

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  • Application of Asian cotton GaNCED3 gene in improving drought resistance of plants
  • Application of Asian cotton GaNCED3 gene in improving drought resistance of plants
  • Application of Asian cotton GaNCED3 gene in improving drought resistance of plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Cloning and expression analysis of GaNCED3 gene

[0049] 2.1 Total RNA extraction and cDNA first-strand synthesis

[0050] The Shixiya No. 1 seeds after velvet were sown in wet sand and cultured in an incubator at 27°C with a photoperiod of 16h light / 8h dark. After 5 days, the cotton seedlings were transferred to a hydroponic box containing 1 / 2 Hoagland nutrient solution, and the culture conditions remained unchanged. When the cotton seedlings have 3 true leaves, carry out the drought stress treatment simulated by PEG. The treatment method is to add 17% PEG6000 (w / v) to the hydroponic nutrient solution. Cut the young leaves and roots of cotton seedlings, and use the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit to extract total RNA. cDNA synthesis using reverse transcription kit PrimeScript TM RT reagent Kit, then refer to the fluorescent quantitative PCR kit TB Green TM Premix Ex Taq TM II instructions for real-time fluorescent quant...

Embodiment 2

[0062] GaNCED3 gene silencing reduces drought resistance in cotton plants

[0063] 1. Construction of GaNCED3 gene VIGS silencing vector

[0064] The cDNA of leaves treated with PEG for 2 hours was used as a template, and GaNCED3-V-F and GaNCED3-V-R were used as primers for PCR amplification, and a PCR product with a length of 300bp was obtained.

[0065] GaNCED3-V-F: 5'-GAGTAAGGTTACCGAATTCTCTTCATTTGCCTAAGCAACAATCAC-3', SEQ ID NO: 3;

[0066] GaNCED3-V-R:5'-TGAGCTCGGTACC GGATCC TAGACTCCTTGTATGCAATCCGG-3', SEQ ID NO: 4; where underlined GAATTC and GGATCC The recognition sequences of restriction endonucleases EcoRI and BamHI are indicated respectively, and GAGTAAGGTTACC and TGAGCTCGGTACC are protection bases.

[0067] The PCR product was double-digested with restriction endonucleases EcoRI and BamHI, and the digested product was recovered. The pTRV-RNA2 vector was double-digested with restriction endonucleases EcoRI and BamHI, and the vector skeleton was recovered. Liga...

Embodiment 3

[0082] Overexpression of GaNCED3 gene can improve the drought resistance of transgenic Arabidopsis

[0083] 1. Construction of recombinant plasmids

[0084] The cDNA of leaves treated with PEG for 2 hours was used as a template, and GaNCED3-F and GaNCED3-R were used as primers for PCR amplification.

[0085] GaNCED3-F:5’-GAACACGGGGGAC TCTAGA ATGGCTTCATCAACAGCAGC-3', SEQ ID NO: 6; GaNCED3-R: 5'-TGAACGATCGGGGAAATTC GAGCTC TTAGGCCTGTTTTTCCAAGTCC-3', SEQ ID NO: 7; where underlined TCTAGA and GAGCTC The recognition sequences of restriction endonucleases XbaI and ScaI are indicated respectively, and GAACACGGGGGAC and TGAACGATCGGGGAAATTC are protected bases.

[0086] The above PCR product and pBI121 vector were digested with restriction endonucleases XbaI and ScaI. Ligate the recovered enzyme-digested product and the vector backbone to construct a recombinant plasmid, and perform sequencing verification on the recombinant plasmid.

[0087] The results showed that the recomb...

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Abstract

The invention discloses an application of an Asian cotton GaNCED3 gene in improving the drought resistance of plants, and belongs to the technical field of plant genetic engineering. The invention also provides a method for cultivating a transgenic plant with improved drought resistance. According to the method, a target nucleotide sequence is introduced into a receptor plant through a plant over-expression vector pBI121-GaNCED3 to increase the activity of the GaNCED3 in the receptor plant or increase the content of the GaNCED3 in a target plant or promote the expression of a GaNCED3 encoded protein, and the drought resistance of the obtained transgenic plant is higher than that of a control receptor plant. The improvement of the drought resistance is shown in the following aspects: underthe drought stress simulated by mannitol, the seed germination rate, green plantlet frequency and root length of the transgenic plant are higher than those of the receptor plant, and under natural drought, the malondialdehyde content of leaves of the transgenic plant is smaller than that of the receptor plant, and the accumulation amount of free proline is larger than that of the receptor plant.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to the application of an Asian cotton GaNCED3 gene in improving plant drought resistance. Background technique [0002] Drought is one of the main factors affecting plant growth and development. Cotton is an important economic crop in my country. Under the extremely severe situation of national water resources, in order to maintain the sustainable development of the cotton industry and stabilize the national cotton production, it is necessary to improve the drought resistance of cotton. Breeding drought-resistant cotton materials by means of genetic engineering is an effective way to solve this problem. The mechanism of plant drought resistance response is complex, involving a variety of drought resistance mechanisms and signal transduction pathways. Although some cotton drought resistance related genes have been cloned, the number is limited, and there are still...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/82C12Q1/6895C12Q1/686A01H5/00A01H6/20A01H6/60
CPCC12N9/0069C12N15/8273C12Q1/6895C12Q1/686C12Y113/11071C12Q2563/107
Inventor 蔡肖张建宏江振兴迟吉娜刘存敬唐丽媛张素君李兴河王海涛
Owner COTTON RES INST HEBEI ACAD OF AGRI & FOREST SCI
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