Soybean gene GmCYS20 and application

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

Active Publication Date: 2018-06-29
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 gene GmCYS20 and application
  • Soybean gene GmCYS20 and application
  • Soybean gene GmCYS20 and 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 gene GmCYS20 of a cysteine protease inhibitor for regulating and controlling symbiotic nodulation of leguminous plants, and the application of the gene in regulating and controlling number of root nodules of leguminous plant (crowtoe, astragalus sinicus, alfalfa, soybean, peanut and the like). The length of the gene is 291bp; the gene is positioned at the 8475315-8478532site of a No.20 chromosome of a soybean genome, and contains two exons and one intron. The gene has the advantages that a reverse transcription PCR (polymerase chain reaction) technique is applied toseparate the gene GmCYS20 of the cysteine protease inhibitor in the root tip of the soybean, and the gene is proofed to be a gene of the cysteine protease inhibitor; an over-expression genetic transformation method is utilized to verify that the gene participates, regulates and controls the nodulation of the leguminous plant; the regulating and controlling effect of the gene GmCYS20 of the cysteine protease inhibitor on the symbiotic nodulation of the leguminous plant is proofed for the first time, and the application prospect on the symbiotic nitrogen immobilizing of the leguminous plant is realized.

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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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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