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Method for cultivating highly noduled transgenic plants based on RNA interference technology

A technology of transgenic plants and RNA interference, applied in the field of genetic engineering, can solve the problems that have not yet been seen

Active Publication Date: 2019-04-30
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this process, ABI5 is an important factor of ABA. It has been proved that ABI5 plays an important role in seed dormancy, germination, transcription and drought response, but there is no information about the role of ABI5 in soybean root and root nodulation and symbiotic nitrogen fixation. report

Method used

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  • Method for cultivating highly noduled transgenic plants based on RNA interference technology
  • Method for cultivating highly noduled transgenic plants based on RNA interference technology
  • Method for cultivating highly noduled transgenic plants based on RNA interference technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1. Analysis of the expression pattern of GmABI5L gene in response to ABA, drought and salt treatment.

[0035] 1) Material acquisition: The material used in the experiment is Williams 82 (abbreviated as W82); the material is carried out according to the following process: soybean seeds are sterilized with 70% alcohol for 30 seconds, germinated in B5 medium, 16h light / 8h dark, light intensity 7000LUX, temperature 26℃, relative humidity 70%. After 5 days, the following methods were used: ABA treatment, 0μM ABA or 50μM ABA treatment for 6 hours; drought treatment: 200mM mannitol treatment for 6 hours; salt treatment: 100mM NaCl treatment for 6 hours. Pour out the vermiculite, and sample the site from 2cm below the rhizome junction to the root tip.

[0036] 2) Isolation of mRNA: the Trizol method is used to extract total soybean RNA. ① Put the tissue into the grinding and grind it with liquid nitrogen 3 times, add 0.1-0.2g of the ground tissue to a 1mL centrifuge tube a...

Embodiment 2

[0042] Example 2. Analysis of the expression pattern of GmABI5L gene in response to Rhizobium infection.

[0043] 1) Material acquisition: The material used in the experiment is Williams 82 (abbreviated as W82); the material is carried out according to the following process: Soybean seeds are sterilized with 70% sprinkling for 30 seconds, and sown in a vermiculite matrix soaked in a low nitrogen nutrient solution. Cultivate in the culture room, 16h light / 8h dark, light intensity 7000LUX, temperature 26℃, relative humidity 70%. 9 days after germination, the soybeans are inoculated with rhizobia, and each strain is inoculated with slow-growing rhizobia USDA110 bacterial solution 600 =0.08) 30 mL, and take the soybean roots at 0, 14, 21 and 28 days after inoculation.

[0044] 2) Isolation of mRNA: the Trizol method is used to extract total soybean RNA. ① Put the tissue into the grinding and grind it with liquid nitrogen 3 times, add 0.1-0.2g of the ground tissue to a 1mL centrifuge tu...

Embodiment 3

[0050] Example 3. Using RNAi vector to down-regulate and express GmABI5L gene to cultivate transgenic soybean with high nodulation and nitrogen fixation ability.

[0051] 1) Construct a recombinant expression vector.

[0052] (1) Cloning of GmABI5L gene fragment:

[0053] According to the coding sequence of GmABI5L (SEQ ID NO:1), a primer pair was designed for RNAi vector construction. According to the multiple cloning site on the vector pTCK303, the primer ends were introduced with KpnI, SpeI, BamHI, and SacI restriction sites; The cDNA of the sequenced variety W82 was used as a template for PCR, and the amplification primer sequence was:

[0054] Forward primer: GGGGTACCAC TAGTTGGGAA GACACCAGCA AATAA (SEQ ID NO: 2);

[0055] Reverse primer: CGGGATCCGA GCTCCTCTCA CAACACCAGC TCTCAC (SEQ ID NO: 3);

[0056] The amplification procedure is: 95°C 5min; 95°C 30sec, 56°C 30sec, 68°C 2min, 30 cycles; 72°C 70sec;

[0057] The PCR amplified product was subjected to 1% agarose gel electrophoresis,...

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Abstract

The invention discloses a method for cultivating highly noduled transgenic plants based on an RNA interference technology. Firstly, an RNAi vector is constructed by a gene fragment shown in SEQ ID NO:1, then the transgenic plants are constructed by the obtained RNAi vector, and the transgenic plants with higher nodulation / nitrogen fixation ability as compared with normal plants are obtained. The transgenic plants are obtained by transforming receptor soybean plants with the constructed RNAi vector, can significantly improve the nodulation and nitrogen fixation ability of soybean under abioticstress, and have great significance for improving soybean yield.

Description

Technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for cultivating high-nodulation / nitrogen-fixing plants by using RNAi technology. Background technique [0002] Soybean is an important economic crop of grain and oil, and also a crop with high nitrogen demand. Soybeans can convert nitrogen in the air into ammonia nitrogen that can be absorbed and utilized by plants through root nodules, thereby providing essential nitrogen nutrients for soybean growth. The symbiotic nitrogen fixation process mediated by root nodules not only affects the normal growth and yield of soybeans, but also helps to save energy and reduce environmental chemical pollution. At present, improving the efficiency of symbiotic nitrogen fixation has become one of the important ways to increase soybean yield and ensure the sustainable development of agriculture. [0003] The occurrence of nodules is the result of the sequential expression of genes...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/113C12N15/11A01H5/00A01H6/54
CPCC12N15/113C12N15/8205C12N15/8261C12N2310/14
Inventor 王志娟宋姗姗许世敏李霞
Owner HUAZHONG AGRI UNIV
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