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Cotton nematode-resistant gene GhNtR1 and application thereof

A cotton and gene technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of cotton damage, reduce cotton yield and quality, etc. Effect

Active Publication Date: 2014-09-17
聊城云购通信息技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cotton diseases such as bacterial wilt, fusarium wilt, verticillium wilt and root-knot nematodes cause serious harm to cotton production, reducing cotton yield and quality

Method used

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  • Cotton nematode-resistant gene GhNtR1 and application thereof
  • Cotton nematode-resistant gene GhNtR1 and application thereof
  • Cotton nematode-resistant gene GhNtR1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Cloning and Sequence Analysis of Cotton GhNtR1 Gene

[0025]Degenerate primers were designed according to the P-loop and GLPL amino acid conserved regions of the NBS domain. The primer sequence is 5'-GGNGGNAT(T / C / A)GGIAA(A / G)ACIAC-3', 5'-NA(G / A)NGCIA(G / A)IGGIA(G / A)ICC-3' . A double primer amplified band of about 500 bp was obtained by PCR amplification, which was recovered and sent to multiple clones for sequencing. As a result, multiple EST sequences containing NBS domains were obtained, and these sequences had extremely high homology. Select one of the ESTs to amplify the whole sequence by Tail PCR technology. N-terminal specific primers are 5'-TGCCTTGTAAAAAGTTCCTCAGCT-3', 5'-CGTCCATGACAATGAGATATCTC-3'. The C-terminal specific primers were 5'-GGTGTAGAAGAAACTCAACTATGG-3', 5'-CTCAAAGAATGTGGCTTCACTGG-3'. (Paterson AH, Brubaker CL, Wendel JF.A rapid method for extraction cotton (Gossypium spp.) Genomic DNA suitable for RFLP or PCR analysis. Plant Mol Biol Rep1993, 11...

Embodiment 2

[0027] Obtaining Transformant Strains of Tobacco Overexpressing GhNtR1

[0028] In order to study the function of GhNtR1, primers were designed according to the sequence of SEQ ID NO.1, and SalI and XbaI endonuclease recognition sites, 5'-GTCGACAAGGCAAATTAGTTTTCTGAGTGA-3' and 5'-TCTAGACCTTCAATAAAGAAGCCACTCTTC-3' were added to the primers. The cDNA against cotton was amplified with this pair of primers. The PCR amplified product and PCAMBIA2301 were simultaneously digested with SalI and XbaI, and the digested product was recovered and ligated with T4ligase. The recombinant vector obtained above was used to transform the recombinant vector into Agrobacterium LBA4404 by freeze-thawing method. Transformation of common tobacco with Agrobacterium-mediated method (Zhang Baolong, Yang Yuwen, Ni Wanchao, Hou Jibo. Studies on tobacco transfected with H5 avian influenza virus M2e gene. Jiangsu Agricultural Journal, 2010, 01:51-54). The T0 generation individual plants were extracted by ...

Embodiment 3

[0030] Analysis of Nematode Resistance in Tobacco Overexpressing GhNtR1

[0031] Preliminary identification of the resistance of the transgenic plants was carried out, and 9 transgenic tobacco lines were treated with the root-knot nematode incognita provided by the School of Plant Protection, Nanjing Agricultural University. Tobacco seedlings were cultivated in a light incubator at 25°C (day, 12h) / 16°C (night, 12h), and the cultivation soil was sterilized. When the seedlings have 4-5 true leaves, inoculate with 2nd instar larvae. The inoculation method is to use a glass rod to make holes around the tobacco root system (3 holes per pot), inject the collected 2nd instar larvae into the hole, the inoculation amount is 2000 larvae per plant, and then cover with soil about 1 cm, and only inject the same larvae into the control plant. volume of water without inoculating nematodes. The inoculation number of each transgenic line was >16. After 45 days of inoculation, it was found t...

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Abstract

The invention relates to a plant nematode-resistant gene GhNtR1 and an application thereof, belonging to the field of biotechnology. The gene GhNtR1 is a CC-NBS-LRR gene obtained from a verticillium wilt-resistant material, namely gossypium hirsutum L. and coding protein of the gene contains 861 amino acids; the similarity of the gene GhNtR1 to a listed R gene is relatively low and the content of castor RPPR8 with maximum similarity to the gene GhNtR1 is also only 27%. A plant overexpression vector of the gene GhNtR1 is built and diseased general tobaccos are converted by adopting an agrobacterium-mediated method. The resistance of 5 lines in 9 transgenic lines is remarkably improved through inoculation by meloidogyn incognita; in addition, the expression quantity of defense response genes PR1 and PR3 of a transgenic plant after inoculation of nematodes is higher than that of a non-transgenic plant and more callosities are accumulated in the transgenic plant; the cloning and the functional analysis of the gene GhNtR1 deepen the understanding of resistance mechanisms and related signal paths of the gene and lay a foundation for obtaining nematode-resistant genetically engineered plants.

Description

technical field [0001] The invention relates to plant gene cloning and functional analysis, provides a cotton anti-nematode related gene, and belongs to the field of plant genetic engineering. It is used to improve plant disease resistance and other beneficial production traits through plant genetic engineering technology. Background technique [0002] Root-knot nematode (Meloidogyne spp) is an important pathogen that harms crops. Currently, there are more than 80 species of root-knot nematode reported in the world, and 29 species of root-knot nematode reported in my country. More than 90% of crop losses caused by root-knot nematodes are caused by southern root-knot nematode (M.incognita), peanut root-knot nematode (M.arenaria), java root-knot nematode (M.javanica), northern root-knot nematode M.Hapla) caused by four main root-knot nematodes. Plant root-knot nematodes have caused great harm to agricultural production, especially with the large-scale promotion of greenhouse...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82C12N1/21A01H5/00
Inventor 张保刚岳继承张灿
Owner 聊城云购通信息技术有限公司
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