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RNA (ribonucleic acid) interference vector and application thereof in culture of high nodulation nitrogen-fixing plants

A technology of RNA interference and carrier, applied in the field of RNA interference carrier

Inactive Publication Date: 2015-06-03
中国科学院遗传与发育生物学研究所农业资源研究中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there are no reports about ARF8 regulating leguminous root nodule development

Method used

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  • RNA (ribonucleic acid) interference vector and application thereof in culture of high nodulation nitrogen-fixing plants
  • RNA (ribonucleic acid) interference vector and application thereof in culture of high nodulation nitrogen-fixing plants
  • RNA (ribonucleic acid) interference vector and application thereof in culture of high nodulation nitrogen-fixing plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1, Analysis of the expression pattern of GmARF8L1 gene in early response to rhizobia infection

[0024] 1) Material acquisition: The material used in the experiment was Williams 82 (W82 for short); the material was carried out according to the following process: soybean seeds were sterilized with 70% alcohol for 30 seconds, and sowed in vermiculite perlite soaked in low-nitrogen nutrient solution (3 :1) In the mixed medium, culture in the culture room, 16h light / 8h dark, light intensity 7000LUX, temperature 26°C, relative humidity 70%. Seven days after sowing, each plant was inoculated with Bradyrhizobium USDA110 bacterial solution (OD 600 : 0.08) 30ml, take soybean roots respectively at 0, 1, 3, 6, 12 and 24 hours after inoculation;

[0025] 2) Isolation of mRNA: Use the Trizol method to extract soybean total RNA. ①Put the tissue into a grinder and grind it with liquid nitrogen for 3 times, add 50-100 mg of the ground tissue into a 1.5 mL centrifuge tube, then...

Embodiment 2

[0031] Example 2, Analysis of the expression pattern of GmARF8L1 at different stages of root nodule development

[0032] 1) Material acquisition: The material used in the experiment was Williams 82 (W82 for short); the material was carried out according to the following process: soybean seeds were sterilized with 70% alcohol for 30 seconds, and sowed in vermiculite perlite soaked in low-nitrogen nutrient solution (3 :1) In the mixed medium, culture in the culture room, 16h light / 8h dark, light intensity 7000LUX, temperature 26°C, relative humidity 70%. Seven days after sowing, each plant was inoculated with Bradyrhizobium USDA110 bacterial solution (OD 600 :0.08) 30ml, the soybean root and leaf tissues were taken respectively at 0, 1, 3, 5, and 10 days after inoculation;

[0033]2) Isolation of mRNA: Use the Trizol method to extract soybean total RNA. ①Put the tissue into a grinder and grind it with liquid nitrogen for 3 times, add 50-100 mg of the ground tissue into a 1.5 mL...

Embodiment 3

[0039] Example 3. Using the RNA interference vector to down-regulate the GmARF8L1 gene to cultivate transgenic soybeans with high ability to nodulate and fix nitrogen.

[0040] 1) Extraction of total RNA from functional leaf tissue of Wilimas82 soybean

[0041] The mortar has been treated at 180°C for 8 hours or burned to eliminate RNase contamination; reagents such as chloroform, isopropanol, and ethanol should be freshly opened and uncontaminated; other equipment such as pipette tips, centrifuge tubes and reagents such as ultrapure water , NaAc, were all treated with 1‰ DEPC water overnight, then sterilized at 121°C for 30 minutes, and the pipette tip and centrifuge tube were dried at 65°C for later use; Trizol method was used to extract soybean total RNA:

[0042] (1) Take 50mg of material (leaf) and grind it with liquid nitrogen, add 1ml of redzol reagent, after fully homogenizing, inhale the homogenate into a 1.5ml centrifuge tube, and place it at room temperature for 5 m...

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Abstract

The invention discloses an RNA (ribonucleic acid) interference vector for regulating and controlling nodulation nitrogen-fixing capability of plants. The RNA interference vector comprises a backbone vector and a hairpin structure, wherein the hairpin structure is constituted by utilizing a gene fragment as shown in SEQ (sequence) ID (identity) NO: 2, and the gene fragment is inserted between enzyme digestion recognition sites of restriction endonucleases Kpn I / Spe I and BamH I / Sac I of a pTCK 303 vector to obtain the recombinant RNA interference vector. The obtained RNA interference vector can transform receiver plants, silence corresponding genes in the receiver plants and enable root tissues of the receiver plants to have the enhanced nodulation nitrogen-fixing capability. By utilizing the RNA interference vector constructed according to the invention to transform receiver soybean plants to obtain transgenic plants, the nodulation nitrogen-fixing capability of soybeans can be significantly improved, and the RNA interference vector further has great significance for improving the yield of the soybeans.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an RNA interference carrier for Papilionaceae and its application. Background technique [0002] Soybean is a very important crop with special economic value. The nodules on the roots of soybeans can convert nitrogen in the air into ammonia nitrogen that plants can absorb and use, thus providing the necessary nitrogen nutrients for the growth and development of soybeans. The symbiotic nitrogen fixation mediated by root nodules not only affects the normal growth and yield of soybean, but also helps to save energy and reduce environmental chemical pollution. At present, improving nitrogen fixation efficiency has become one of the important ways to increase soybean yield and ensure sustainable agricultural development. [0003] Soybean root nodulation and nitrogen fixation is a very complex biological process regulated by multiple genes, and more and more regulatory ge...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00
Inventor 李霞王幼宁陈亮李科学邹艳敏王蕊
Owner 中国科学院遗传与发育生物学研究所农业资源研究中心
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