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Soybean chalcone reductase gene CHR2 and applications thereof

A CHR2, reductase technology, applied in the field of genetic engineering, can solve the problems related to unclear synthesis ability and so on

Inactive Publication Date: 2013-10-02
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is only one soybean reductase gene CHR1 whose function can be confirmed in GENEBANK in the United States
So far, there is no conclusion about the type and quantity of chalcone reductase (CHR) in soybean, and it is not clear that different types of chalcone reductase (CHR) are related to the synthesis ability of daidzein in plants

Method used

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  • Soybean chalcone reductase gene CHR2 and applications thereof
  • Soybean chalcone reductase gene CHR2 and applications thereof
  • Soybean chalcone reductase gene CHR2 and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Cloning of Soybean Chalcone Reductase Gene CHR2

[0021] The soybean variety "Jinong 17" (seeds provided by the Biotechnology Center of Jilin Agricultural University) was planted in the field. When the plants grew to the flowering stage, the leaf tissue was selected and ground with liquid nitrogen. The total RNA was extracted using the kit RNAiso Plus. Formaldehyde denaturing gel electrophoresis was used to identify the quality of RNA, and then the concentration of RNA was measured with a spectrophotometer. Then the obtained total RNA was used as a template, and the first strand of cDNA was synthesized by reverse transcription. The primers GSP1 and GSP2 for specifically amplifying the full-length gene were designed and synthesized according to the two-terminal sequences of the new gene CHR2. Then the full-length gene fragment was amplified by PCR reaction. The PCR conditions were as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 40 s...

Embodiment 2

[0025] Example 2 Construction of Plant Expression Vector PBI121-CHR2 Gene

[0026] The gene sequence of the open reading frame was predicted by the special analysis software on the website of the National Center for Biological Information (NCBI), and the predicted result was a fragment with a length of 969 bp. Specific primers were designed according to the analysis results, and the sequence fragment of the open reading frame was amplified by PCR reaction using soybean cDNA as a template. The cDNA sequence of soybean chalcone reductase CHR2 gene was analyzed by DNAMAN software, and the connecting primers were designed with restriction enzyme cutting sites according to the plant expression vector, and the XbaⅠ restriction site was added upstream, and the BamHI restriction site was added downstream Point, upstream primer: GGG TCTAGA ATGGCAGGAAAGAAAATC, downstream primer: TTT GGATCC TTAAACGTCTCCATCCC, using the cDNA obtained in Example 1 as a template for PCR amplification, the...

Embodiment 3

[0028] Example 3 Cultivation of Transformed CHR2 Gene Tobacco

[0029] Then the recombinant expression vector pBI121-CHR2 was transformed into competent Agrobacterium DH105 by heat shock method. Tobacco explants were prepared for transformation with Agrobacterium. Select tobacco seeds with full grains, sterilize them with 70% alcohol and 5% sodium hypochlorite solution, wash them with sterile deionized water, inoculate them on the tobacco germination medium, and cultivate them in a bright place for 15 days; transform Agrobacterium in YEP The liquid medium was cultured with shaking at 28°C until the OD600 value was 0.6-0.8, and the bacteria were collected by centrifugation and resuspended with MS liquid medium. Cut off the sprouted tobacco 1cm 2 Leaves of the same size and punched a number of holes, put them into the MS medium containing transformed Agrobacterium and suspended them for 8 minutes, then placed the leaves on the common medium of tobacco, and cultivated them in...

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Abstract

The invention discloses a soybean chalcone reductase gene CHR2 and applications thereof. CHR2 gene is a novel chalcone reductase gene in soybean. Tests have testified that the expression product of the CHR2 gene is capable of catalyzing to synthesize isoliquiritigenin. CHR2 is originated from soybean, and has optimized codon, which is suitable for dicotyledons such as soybean. CHR2 gene engineering acceptor is mainly suitable for dicotyledons such as soybean, tobacco, cotton, and the like, and is also suitable for monocotyledons such as paddy rice, wheat, corn, and the like. CHR2 gene can be induced into plants as a target gene to catalyzed synthesis of isoliquiritigenin, which is a necessary precursor substance for biological synthesis of daidzein, therefore daidzein content is increased in the plants. Daidzein is a plant defensin, which can improve the disease resistance of plants, has prevention and curative functions on a plurality of human diseases, and is also a very good health-care product.

Description

technical field [0001] The invention relates to soybean chalcone reductase gene CHR2 and its application in genetic engineering. It belongs to the field of genetic engineering and specifically relates to the biosynthesis of plant secondary metabolite daidzein. technical background [0002] Rapid amplification of complementary DNA ends (RACE technology) is a molecular biology technique that completely separates and clones the 3' and 5' ends of incomplete genes. So far, with the rapid development of library construction technology and high-throughput sequencing technology, more and more nucleic acid fragments have been isolated and sequenced. However, the problem is that a lot of experimental work is still needed to determine the integrity and functionality of the analyzed nucleic acid fragments, and continue to explore and discover genetic resources that are beneficial to human practical use. [0003] Daidzein is one of the main components of soybean isoflavones, accounting...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/82A01H5/00
Inventor 张卓王丕武王雨鑫马建付永平刘思言张超魏洪波吴楠
Owner JILIN AGRICULTURAL UNIV
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