Yangbi Dapao walnut ribonuclease gene jsrnase and its application

A walnut ribonucleic acid and enzyme gene technology, which is applied in the fields of application, genetic engineering, and plant gene improvement, can solve the problems of not discovering the specificity of the substrate, and achieve the effects of broad market application prospects, cost saving, and shortened breeding cycle

Active Publication Date: 2018-02-09
KUNMING UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Type II only includes T2 RNases, that is, the RNase T2 family, the optimum pH is 4.0-5.0, and the molecular weight is greater than 20 kDa, generally 24-36 kDa. This type of RNase has a wide distribution and no substrates have been found. Specificity (DeshpandeRA, Shankar V. Ribonucleases from T2 family. Critical reviews inmicrobiology. 2002, 28: 79–122.)

Method used

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  • Yangbi Dapao walnut ribonuclease gene jsrnase and its application
  • Yangbi Dapao walnut ribonuclease gene jsrnase and its application
  • Yangbi Dapao walnut ribonuclease gene jsrnase and its application

Examples

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Embodiment 1

[0021] Example 1: JsRNase Full-length cDNA cloning and sequence analysis

[0022] The walnut was inoculated with G. anthracnose, and the total RNA was extracted from the leaves 4 hours after inoculation. The treated leaves of walnut were ground into powder with liquid nitrogen, then transferred to a centrifuge tube, and treated with isothiocyanate. Total RNA was extracted by acid guanidine method. Use reverse transcriptase M-MLV (promega) to synthesize the first strand of cDNA using total RNA as a template. The reaction system and operation process are as follows: take 5 μg total RNA, add 50 ng oligo (dT), 2 μL dNTP Mix (2.5mM each), make up the reaction volume to 14.5 μL with DEPC water; after mixing, heat denaturation at 70°C for 5 min, then rapidly cool on ice for 5 min, then add 4 μL 5×First-stand buffer, 0.5 μL RNasin (200 U ), 1 μL M-MLV (200U), mix well and centrifuge briefly, incubate at 42°C for 1.5 h, take it out and heat at 70°C for 10 min to terminate the reactio...

Embodiment 2

[0025] Embodiment 2: plant overexpression vector construction

[0026] Use the SanPrep column type plasmid DNA mini-extraction kit (Shanghai Sangong) to extract the insert JsRNase coli plasmid pMD18-T- JsRNase As well as the plant expression vector pCAMBIA2300S plasmid, 1 μL was used for agarose gel electrophoresis to detect the integrity and concentration of the extracted plasmid. restriction endonuclease Eco RI (TaKaRa) and Bam HI(TaKaRa) against plasmid pMD18-T- JsRNase and pCAMBIA2300S for double enzyme digestion (100 μL system), the reaction system and operation process are as follows: take 20 μL pMD18-T- JsRNase and pCAMBIA2300S plasmid, add 10 μL 10×K buffer, 5 μL EcoRI , 5 μL Bam HI, 60 μL ddH 2 O, after mixing, centrifuge for a short time, and place at 37°C for overnight reaction. All digested products were subjected to agarose gel electrophoresis, and then SanPrep Column DNA Gel Extraction Kit (Shanghai Shenggong) was used for JsRNase The fragments and t...

Embodiment 3

[0029] Example 3: Plant genetic transformation mediated by Agrobacterium and screening of transgenic plants

[0030] The transgenic recipient in this experiment was tobacco ( N. tabacum L.). Tobacco seeds were soaked in 75% alcohol for 30s, washed with sterile water and washed with 0.1% HgCl 2 Soak for 8 minutes, then wash several times with sterile water, sow on 1 / 2 MS medium, culture in dark at 28°C for 5-8 days, transfer to light incubator after germination (25°C, 16 h / d light) , and then subculture once a month with MS medium.

[0031] Take out the stored pCAMBIA2300S-containing pCAMBIA2300S- JsRNase Agrobacterium LBA4404 strain of the plasmid was inoculated in 5 mL of LB liquid medium containing 50 mg / L Km and 20 mg / L rifampin in 20 μL, and cultured at 28°C until the medium was turbid. Pipette 1 mL of turbid bacterial solution onto LB solid medium containing 50 mg / L Km, and incubate at 28°C for 48 h. Then scrape off an appropriate amount of Agrobacterium on LB soli...

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Abstract

The invention discloses a Juglans sigillata Dode ribonuclease gene JsRNase and application thereof. The nucleotide sequence of the JsRNase gene is disclosed as SEQ ID NO:1. According to the coding ribonuclease, the research by functional genomics related technology confirms that the JsRNase gene has the function of enhancing fungus infection resistance of the plant. After the antifungal JsRNase gene is constructed into a plant expression vector and transformed into tobacco for overexpression, the transgenic tobacco plant has very high in-vitro antifungal activity. The JsRNase-overexpressed transgenic tobacco has obvious inhibiting actions on growth of Colletotrichum gloeosporioides, Sclerotinia sclerotiorum, Fusarium oxysporum and Gibberella moniliformis.

Description

technical field [0001] The invention relates to the field of research on molecular biology and genetic engineering related technologies, in particular to the gene of walnut ribonuclease with antifungal activity JsRNase and applications. Background technique [0002] Plant diseases are a very difficult problem in agricultural production, and the struggle between human beings and plant diseases runs through the development and progress of agriculture. The rise of classical genetics in the early 20th century has enabled people to successfully breed new disease-resistant varieties through cross-breeding and greatly increase food production. In recent years, with the continuous development and improvement of molecular biology theory and technology, people can directly, quickly and efficiently cultivate new disease-resistant varieties and new materials through genetic engineering, a modern biotechnology, which is a way to improve plant disease resistance. new method. [0003] R...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N15/84A01H5/00
Inventor 刘迪秋韩青陈朝银陈瑞葛锋
Owner KUNMING UNIV OF SCI & TECH
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