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Application of novel cucumber gene in improving photosynthesis and promoting plant growth and autotoxicity resistance

A technology of cucumber, genetics, applied in the fields of molecular biology and genetic engineering

Active Publication Date: 2020-07-24
HENAN INST OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods have certain limitations in the research of multi-copy genes, family genes, specifically expressed genes, and conditional lethal genes (Xie Hongke et al., 2015)

Method used

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  • Application of novel cucumber gene in improving photosynthesis and promoting plant growth and autotoxicity resistance
  • Application of novel cucumber gene in improving photosynthesis and promoting plant growth and autotoxicity resistance
  • Application of novel cucumber gene in improving photosynthesis and promoting plant growth and autotoxicity resistance

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Experimental program
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Effect test

Embodiment 1

[0029] Using the existing Arabidopsis RNAi mutant library, on MS medium containing 50 mg / L hygromycin and 0.25 mM cinnamic acid (CA), about 1000 plants resistant to CA stress were screened from about 11200 RNAi transformed plants. mutant strains. In contrast, wild-type (WT) Arabidopsis plants showed obvious dwarfing and wilting symptoms on the same MS plate, and even caused some WT Arabidopsis plants to die ( figure 1 ). At the rosette stage of the mutant strains, we extracted the DNA genomes of the mutant strains of Arabidopsis, and used the specific primers NOS-60: CAACAGGATTCAATCTTAAGAAAC and Intron FW2: CATATTGCAAAGTCTGAAGACTC to amplify the interfering RNA in the transformed RNAi library of Arabidopsis thaliana by PCR. PCR products were recovered and ligated into pMD TM 19-T (TaKaRa, Code No.6013) vector, sequenced in order to obtain the mutant gene. According to BLAST sequencing results in NCBI, we found that a new Arabidopsis gene At5g16610 (NM_203062.2) with unknown...

Embodiment 2

[0034] Cloning of homologous gene of cucumber CsAt5g16610 / MTG13_5 gene and construction of pTRV-VIGS vector. In the Cucumber Genome Database ( http: / / cucurbitgenomics.org / ) in BLAST to obtain the homologous gene mRNA sequence of At5g16610 gene, design primers, and use cucumber cDNA as a template to clone the cucumber homologous gene CsAt5g16610 / MTG13_5, the coding sequence number in cucumber is Csa5G426400.1, this gene is a cucumber with unknown function new gene. At the 326-745bp of the cucumber CsAt5g16610 / MTG13_5 gene (the total length is 1905bp), the primers CsAt5g16610-pTRV2-F were designed: GCCTCCATGGGGATCCCTGGTGTGGCTGTAACTAA and CsAt5g16610-pTRV2-R: GCCTCGAPCRGACGCGTGAGCTCCTGTAGCAACCTTCTCTGT was used to amplify the DNA fragment and use GS as the template. In-FusionHD Cloning Kit (Takara, Cat.Nos.121416) ligated the recovered cucumber CsAt5g16610 gene VIGS fragment to the enzyme-cut TRV2 (restriction site is Sac I-BamH I) vector.

Embodiment 3

[0036] Preparation of cucumber pTRV-VIGS infection solution and transformation of cucumber. Transform GV3101 Agrobacterium with TRV1 and TRV2 plasmids linked with the VIGS fragment of cucumber CsAt5g16610 gene, mix them according to the ratio of TRV1:TRV2 of 1:1, and culture them at 28°C until the OD600 is 0.8, as the Agrobacterium infection solution. The prepared Agrobacterium infection solution was used to infect cucumber plants using the "cucumber cotyledon node method" to obtain cucumber pTRV-VIGS transformed cucumber plants, and WT and pTRV controls were set at the same time.

[0037] 10 days after Agrobacterium infection, WT cucumber, pTRV empty control and CsAt5g16610-VIGS cucumber plants were respectively set 0.25mM CA stress treatment and control, and the settings were as follows: (A) WT, (B) WT+CA, (C) pTRV empty control, (D) pTRV empty+CA, (E) CsAt5g16610-VIGS, (F) CsAt5g16610-VIGS+CA. After 14 days of CA treatment, photos were taken and index parameters were measu...

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Abstract

The invention belongs to the fields of molecular biology and gene engineering, and relates to a novel gene CsAt5g16610 / MTG13_5 which plays an important regulation role in improving plant photosynthesis, promoting cucumber plant growth and resisting successive cropping obstacles. A homologous gene CsAt5g16610 / MTG13_5 (Csa5G426400.1) of At5g16610 is cloned from cucumbers, a method for obtaining cucumber plants with an anti-autotoxicity effect by using an established TRV-VIGS system to silence the CsAt5g16610 / MTG13_5 gene, and the possible regulatory mechanisms of the silent CsAt5g16610 / MTG13_5 gene for improving the photosynthesis of plants, inhibiting the absorption and upward transportation of CA by root systems, maintaining the stability of the cell structure, maintaining the cell viability, promoting the growth of the cucumber plants and enhancing the autotoxicity resistance of the cucumber plants are used to provide a theoretical basis and gene resources for creating a novel cucumber germplasm with an anti-autotoxicity effect.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and genetic engineering, in particular to the application of At5g16610 gene in improving cucumber photosynthesis, promoting cucumber growth, and alleviating cucumber autotoxicity. Background technique [0002] Cucumber is a widely cultivated vegetable crop and a model vegetable crop with economic value. However, cucumbers are not resistant to continuous cropping, and the yield and quality of cucumbers are severely reduced after continuous cropping. Crop continuous cropping obstacles are closely related to the autotoxicity of autotoxic substances (Yu and Matsui, 1997; Yu et al., 2000and 2003). Chemical substances produced by plants remaining in the environment can inhibit the germination and growth of seeds of the same family or species of plants, that is, autotoxicity (Miller, 1996; Singh et al., 1999). Growth inhibition caused by continuous cropping of cucumber is related to autotoxi...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82A01H5/00A01H6/34
CPCC12N15/113C12N15/8218C12N15/8269C12N15/8261C12N2310/14Y02P60/21
Inventor 李成伟卜瑞方胡海燕王晓桠王润豪李东霄于永昂宋普文魏琦超关园园徐克东郑自超陈二永刘起丽周锋苏小佳胡平
Owner HENAN INST OF SCI & TECH
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