Soybean gmcys20 gene and its application

A technology of genes and leguminous plants, applied in the fields of application, genetic engineering, plant genetic improvement, etc., to achieve the effect of increasing the number, strengthening the nitrogen fixation ability, and increasing fertility

Active Publication Date: 2021-03-12
XINYANG NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on the function of these genes in the symbiotic nodulation process of legumes

Method used

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  • Soybean gmcys20 gene and its application
  • Soybean gmcys20 gene and its application
  • Soybean gmcys20 gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] A method for preparing a cysteine ​​protease inhibitor gene GmCYS20 involved in legume symbiotic nodulation, the steps of which are:

[0032] After surface sterilization of W82 soybean seeds, spread them on sterile wet filter paper with the hilum facing down, and cultivate them in the dark at 28°C until they germinate. Fresh soybean root tip tissues were collected and quick-frozen in liquid nitrogen. According to the instructions of the RNA extraction kit (Invitrogen, USA), the total RNA of soybean root tip tissue was extracted. The first strand of cDNA was obtained according to the instructions of the reverse transcription kit of TIANGEN Company. According to the soybean GmCYS20 gene (Glyma.20g045500) sequence published on https: / / www.soybase.org / search / website, primers F-GmCYS20 and R-GmCYS20 (Table 1) were designed to amplify the GmCYS20 target gene by PCR (attached figure 1 shown). The target fragment was recovered, connected to the T vector, and sent to Nanjing...

Embodiment 2

[0038] A kind of application of the GmCYS20 gene involved in regulating the symbiotic nodulation of legumes in regulating the number of root nodules of legumes (Lotus japonicus, milk vetch, alfalfa, soybean and peanut, etc.), the steps are:

[0039] In order to study the biological function of GmCYS20 in the leguminous plant Lotus japonicus, the present invention explores the biological function of GmCYS20 through the method of overexpression (attached image 3 shown). It mainly involves the construction and genetic transformation of the overexpression vector, and the specific implementation steps are as follows:

[0040] Construction of the overexpression fusion vector: design primers F-OX and R-OX (see Table 1) that introduce restriction sites EcoR I and Sma I, and insert the sequenced correct target gene into the plant expression vector p1302G (Gus gene as a screening marker Gene), construct the p1302G-GmCYS20 recombinant plasmid, and transfer it into Agrobacterium rhizoge...

Embodiment 3

[0067] Embodiment 3 is basically the same as Embodiment 2, the difference is that

[0068] In concrete implementation step (1), plant material cultivation is to germinate alfalfa 5 days in advance respectively; In step (7), rhizobium inoculation is alfalfa Rhizobium Sinorhizobium meliloti 1021 (this bacterial strain is the bacterial strain that has announced in the world, Juan etc., Comparison of Developmental and Stress-Induced Nodule Senescence in Medicagot runcatula. Plant Physiol, 152:1574-1584, 2010).

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PUM

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Abstract

The invention discloses a cysteine ​​protease inhibitor GmCYS20 gene involved in the regulation of symbiotic nodulation in leguminous plants, with a length of 291 bp, located at positions 8475315-8478532 of chromosome 20 of the soybean genome, containing 2 exons and 1 Intron. It also discloses the application of the gene in regulating the number of root nodules of leguminous plants (radish root, milk vetch, alfalfa, soybean and peanut, etc.). The present invention utilizes reverse transcription PCR technology to isolate a cysteine ​​protease inhibitor gene GmCYS20 in the soybean root tip, and confirms that the gene is a cysteine ​​protease inhibitor gene; The method confirmed that the gene is involved in and positively regulates the nodulation of legumes. This is the first confirmation that the soybean cysteine ​​protease inhibitor gene GmCYS20 positively regulates symbiotic nodulation in legumes, and has a promising application in symbiotic nitrogen fixation in legumes.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a cysteine ​​protease inhibitor GmCYS20 gene involved in the regulation of symbiotic nodulation in leguminous plants and its application. Background technique [0002] Cysteine ​​protease inhibitors (cystatins) exist widely in organisms, and are a class of proteins that can specifically bind to cysteine ​​proteases and inhibit their proteolytic activity. Cystatin protein was first reported to be involved in the regulation of plant development, including strictly controlling the degradation of proteins in plants, and participating in physiological processes such as the deposition and transport of storage proteins. In addition, cystatin protein can delay the leaf senescence process by regulating the activity of cysteine ​​protease. In recent years, studies have found that cystatin genes play an important role in improving plant resistance to abiotic stress. Rice...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N1/21C07K14/81A01H5/00A01H6/54C12R1/01
CPCC07K14/8139C12N15/8261
Inventor 柯丹霞舒勇彭昆鹏
Owner XINYANG NORMAL UNIVERSITY
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