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Cotton GhLAC4 coding gene, cotton disease-resistant module miR397-LAC4 and application

A technology of mir397-lac4 and ghr-mir397, applied in cotton GhLAC4 encoding gene, cotton disease resistance module miR397-LAC4 and application fields, can solve the problems of disease resistance function and its mechanism that have not yet been reported, and achieve enhanced resistance to Verticillium wilt disease performance, and the effect of improving the resistance to verticillium wilt

Pending Publication Date: 2021-12-31
九圣禾种业股份有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, miR397 and LAC4 are involved in lignin synthesis, but there is no report about its function of regulating plant disease resistance and its mechanism

Method used

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  • Cotton GhLAC4 coding gene, cotton disease-resistant module miR397-LAC4 and application
  • Cotton GhLAC4 coding gene, cotton disease-resistant module miR397-LAC4 and application
  • Cotton GhLAC4 coding gene, cotton disease-resistant module miR397-LAC4 and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] This embodiment specifically discloses the cloning and expression analysis of the cotton GhLAC4 coding gene, and the process is as follows:

[0046] 1. Extraction of total RNA and synthesis of cDNA first strand

[0047] The cotton seeds that have been develored by sulfuric acid are soaked in water, placed in an incubator at 37°C overnight, and transferred to an incubator on the next day, and germinated in a dark place while keeping the seeds moist. The seeds that germinated consistently were transferred to a culture box containing Hoagland's nutrient solution, and cultivated in a 28°C greenhouse with a photoperiod of 16h light / 8h dark incubator. After the cotton seedlings grew true leaves, the root, stem and leaf samples of the cotton seedlings were picked respectively, and the cotton RNA was extracted using the column plant total RNA isolation and purification kit. The synthesis of the first strand of cDNA was carried out according to the instructions of the EasyScrip...

Embodiment 2

[0064] This example specifically discloses that ghr-miR397 regulates the target gene GhLAC4 after transcription, and the specific steps are as follows:

[0065] 1. Construction of PCAMBIA1300-miR397 expression vector

[0066] Cotton DNA was extracted with a plant DNA extraction kit, using the extracted DNA as a template, the forward and reverse primers were (F: GGATACATGTACGTAACGCGTAATTTACTTTTCAATTGTTCCAAAGG and R: TGCTTCGAATTCATCAATGCAGCTTTGAATGAAGAACTCTT) respectively to amplify the ghr-miR397 precursor gene, and the amplification conditions were: 95°C , 5min; 95°C, 30s; 60°C, 30s; 72°C, 30s; 35 cycles. The amplified product was inserted into expression vector PCAMBIA1300 to construct PCAMBIA1300-miR397 expression vector.

[0067] 2. Construction of pBI121-GhLAC4 and pBI121-GhLAC4 mu carrier

[0068] Using cotton cDNA as a template, add forward primer GhLAC4-F (GAGAACACGGGGGACTCTAGAATGGAGATGGCACCATGGATTC), introduce Xba I restriction enzyme site and reverse primer GhLAC4-...

Embodiment 3

[0076] In order to study the disease resistance function of the miR397-LAC4 module in terrestrial cotton, this example specifically discloses the effects of ghr-miR397 and GhLAC4 gene silencing and overexpression plants on resistance to Verticillium dahliae.

[0077] 1. Cultivation of virus-induced gene silencing (VIGS) plants

[0078] 1.1 Construction of virus silencing vector TRV: STTM397

[0079] Chemically synthesized by Beijing Qingke Biotechnology Co., Ltd., STTM397 (GA GGATCC TAACTCACGTGACCGCAACTTGTTGTTGTTGTTATGGTCTAATTTAAATATGGTCTAAAGAAGAAGAATTAACTCACGTGACCGCAACTACTT GGTACC CTC), the restriction sites BamH I and KpnI were added to the 5' end and 3' end of the STTM397 synthetic fragment respectively. The STTM397 synthetic fragment was digested and inserted into the viral vector pYL156 to construct the silencing vector TRV:STTM397 vector.

[0080] 1.2 Construction of viral overexpression vector TRV:OE-miR397

[0081] Using cotton DNA as a template, the forward prim...

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Abstract

The invention discloses a cotton GhLAC4 coding gene, a cotton disease-resistant module miR397-LAC4 and application. The cotton GhLAC4 coding gene and the cotton disease-resistant module miR397-LAC4 can be used for cultivating verticillium wilt resistant cotton. Compared with the prior art, the GhLAC4 coding gene provided by the invention can improve the verticillium wilt resistance performance of the cotton, and the provided miR397-LAC4 participates in the resistance response of upland cotton to verticillium dahliae by regulating and controlling lignin synthesis pathway genes and JA pathway genes, so that the verticillium wilt resistance performance of the cotton is enhanced.

Description

technical field [0001] The invention relates to the field, in particular to a cotton GhLAC4 coding gene, a cotton disease resistance module miR397-LAC4 and applications thereof. Background technique [0002] Cotton is a dicotyledonous plant and the only crop that produces fiber from seeds. As an important textile raw material, cotton is one of the most important economic crops in the world and one of the important agricultural products in my country. The harm of cotton verticillium wilt has caused the decline in cotton yield and quality, and has repeatedly broken out in major cotton-producing areas in my country, with an annual occurrence area of ​​nearly 3 million hm 2 The annual economic loss is about 1.2 billion yuan, which has become the main problem facing the sustainable development of cotton in my country. Cotton Verticillium wilt is a serious vascular disease caused by Verticillium; the main pathogen in my country is Verticillium dahliae, which penetrates through th...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/113C12N15/82A01H5/00A01H6/60
CPCC12N9/0061C12N15/1137C12N15/8282C12Y110/03002C12N2310/141
Inventor 吴家和魏太平胡广陈爱民姜辉闻甜权永刚常宝学蔺怀龙陈微林朱清武晓刚田亚强刘殷杨恒超
Owner 九圣禾种业股份有限公司
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