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Soybean Salt Tolerance Gene gmchs5 and Its Application

A soybean, salt-tolerant technology applied in the field of genetic engineering

Inactive Publication Date: 2020-04-28
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But soybean salt-tolerant gene report is less at present, and wherein comparatively common is soybean salt-tolerant gene GmCIPK2 (CN104726476A) and soybean salt-tolerant gene GmCBL3 (CN104726479A), but these two kinds of genes are specifically aspect improving the salt tolerance of Arabidopsis Have good biological function

Method used

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  • Soybean Salt Tolerance Gene gmchs5 and Its Application
  • Soybean Salt Tolerance Gene gmchs5 and Its Application
  • Soybean Salt Tolerance Gene gmchs5 and Its Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Cloning of GmCHS5

[0083] 1.1 Extraction of soybean total RNA:

[0084] 1. Pour liquid nitrogen into the mortar in advance, cut 100-500mg of fresh root samples from soybeans and put them directly in the grinder to grind them fully, move the powder to the 1.5ml Ep tube prepared in advance, add 600ml of Kangwei Century RLC or RL (pre-added 1% volume of β-mercaptoethanol).

[0085] 2. Vortex for several minutes to fully lyse the sample. Let stand on ice for 5 min to fully lyse the sample.

[0086] 3. Slowly transfer the above lysate to a 2mL Shredder Spin tube, centrifuge at 12000rpm for 2min at 4°C; (if the volume is too large, transfer it in two times; if the sample particles in the lysate are large, cut off the tip of the pipette by 0.5cm )

[0087] 4. Prepare a new 1.5ml RNase-Free centrifuge tube, transfer the filtrate in step 3, add 0.5 times the volume of absolute ethanol (the filtrate is very dark, you can add more appropriately), and mix quickly.

[0088] 5....

Embodiment 2

[0143] Construction of eukaryotic expression vector containing GmCHS5 gene

[0144] 2.1 Overexpression vector construction

[0145] 2.1.1 The vector pDL28-HA-RFP (containing the CAM35S promoter) was digested with BamHI, electrophoresis, and the 10k fragment was recovered to obtain the digested product; the enzyme digestion system was vector 1ug, SacⅠ2ul, KpnⅠ2ul, 2ul 10×Kbuffer, 2ul 0.1% BSA, make up to 20ul with double distilled water; enzyme digestion condition is 37°C for 2h.

[0146] 2.1.2 The cloning vector GmCHS5T vector was single-digested with BamHI, electrophoresis, and a fragment of about 0.8k was recovered to obtain the digested product; the enzyme digestion system was the cloning vector 1ug, SacⅠ2ul, KpnⅠ2ul, 2ul10×Kbuffer, 2ul 0.1% BSA, double distilled water Make up to 20ul; enzyme digestion condition is 37°C for 2h.

[0147] 2.1.3 Mix the digested products in 2.1.1.1 and 2.1.1.2, and ligate overnight at 16°C to obtain the plasmid pDL28-HA-GmCHS5-RFP, transform...

Embodiment 3

[0171] soybean hairy root transformation

[0172] 1. Streak activation of Agrobacterium and germinate soybean seeds. Streak activated Agrobacterium k599 on the LB plate containing kan; configure the mixed solution according to the formula of bleaching water: sterile water = 1:2, add it to a 50ml centrifuge tube containing about 50 complete and plump soybeans (completely submerged), Put it on the side for 5 minutes, wash it with sterile water for 5 times, and put it on the side for 5 minutes for the fourth wash. After washing, spread the soybeans flat in a square dish with filter paper and an appropriate amount of sterile water, place 15-20 soybeans in each of the upper and lower layers, and then culture in the incubator at 25-28°C for 48 hours in dark;

[0173] 2. Soybean infection. Preparation of FM medium → high temperature and high pressure sterilization → cooling and pouring plate → root cutting → cutting the cotyledon node in half along the cotyledon direction → scratch...

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Abstract

The invention provides soybean salt-resistant protein GmCHS5 and an encoding gene of the soybean salt-resistant protein GmCHS5, belongs to the field of gene engineering, and provides an expression carrier pDL28-HA-GmCHS5-GFP containing the encoding gene of the soybean salt-resistant protein GmCHS5. The soybean salt-resistant protein GmCHS5 has the advantages that by utilizing the plant gene engineering technique and an agrobacterium tumefaciens-induced method, the gene is transferred into the soybean, so that the salt-resistant ability of the transgenic soybean with excessive expression of GmCHS5 is obviously improved; after the transgenic soybean is treated by the 200mM of high-concentration salt water, the fresh weight of the transgenic soybean root is equal to 1.20 times of the fresh weight of a wild salt treatment group, and is equal to 2.08 times of the fresh weight of a gene silent group, so that the salt-resistant ability of the transgenic soybean with excessive expression of GmCHS5 is obviously improved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to soybean salt-tolerant gene GmCHS5 and its application. Background technique [0002] Soil salinization is a global ecological problem. Nearly 20% of irrigated soils in the world are threatened by salinization, and the situation of cultivated land salinization is becoming more and more serious. Among them, about 34.6 million hectares of land in my country suffer from salinization, accounting for about 25% of the country's arable land. High concentrations of salinity in soil lead to slowed plant growth and even death, and are one of the main abiotic stress factors limiting crop yield. [0003] How to alleviate the impact of salt stress on agricultural production, so that large areas of saline-alkali land, desertified land and abundant salt water resources can be used for agricultural production is an important issue for future agricultural development. Sal...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/54
CPCC07K14/415C12N15/8273
Inventor 皮二旭赵沁怡李洋洋朱成敏黄盈盈杜立群
Owner HANGZHOU NORMAL UNIVERSITY
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