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Julans sigillata Dode proline-enriched protein gene JsPRP1 and applications thereof

A technology of Yangbi big bubble walnut and proline protein, which is applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve problems such as differences in expression characteristics, and achieve the effects of shortening the breeding cycle, reducing environmental pollution, and reducing the use of

Active Publication Date: 2015-07-15
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Wyatt et al. found 3 genes encoding PRPs in soybean: SbPRP1 , SbPRP2 and SbPRP3 , despite showing high homology in nucleotide and amino acid sequences, their expression properties are quite different

Method used

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  • Julans sigillata Dode proline-enriched protein gene JsPRP1 and applications thereof
  • Julans sigillata Dode proline-enriched protein gene JsPRP1 and applications thereof
  • Julans sigillata Dode proline-enriched protein gene JsPRP1 and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: JsPRP1 Full-length cDNA cloning and sequence analysis

[0023] 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. Reverse transcriptase M-MLV (promega) was used to synthesize the first strand of cDNA using total RNA as a template. The reaction system and operation process were as follows: take 5 μg total RNA, add 50 ng oligo (dT), 2 μL dNTP Mix (2.5 mM Each), the reaction volume was made up to 14.5 μL with DEPC water; after mixing, heated and denatured at 70°C for 5 min, then rapidly cooled on ice for 5 min, then added 4 μL 5×First-stand buffer, 0.5 μL RNasin ( 200U), 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 termi...

Embodiment 2

[0026] Embodiment 2: plant overexpression vector construction

[0027] Use the SanPrep column type plasmid DNA mini-extraction kit (Shanghai Sangong) to extract the insert JsPRP1 coli plasmid pMD18-T- JsPRP1 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- JsPRP1 and pCAMBIA2300S for double enzyme digestion (100 μL system), the reaction system and operation process are as follows: take 20 μL pMD18-T- JsPRP1 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 JsPRP1 The fragments and the ...

Embodiment 3

[0030] Example 3: Plant genetic transformation mediated by Agrobacterium and screening of transgenic plants

[0031] The transgenic recipient in this experiment was tobacco ( Nicotiana tabacum L.). Tobacco seeds were soaked in 75% alcohol for 30 s, 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.

[0032] Take out the stored pCAMBIA2300S-containing pCAMBIA2300S- JsPRP1 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 ...

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Abstract

The invention discloses Julans sigillata Dode proline-enriched protein gene JsPRP1 and applications thereof. The nucleotide sequence of the JsPRP1 is represented by the SEQ ID No.1, and the encoding is rich in proline protein. Through the related technologies and researches of functional genomics, the results show that JsPRP1 gene has a function that can improve the plants' performance on resisting fungal infection. The anti-fungus gene JsPRP1 can be built on an expression carrier and over-expressed in tobacco, and the transgene tobacco plants has a strong in-vitro activity on resisting fungi. The transgene tobacco, where the JsPRP1 gene is over-expressed, has a prominent inhibiting effect on the growth of colletotrichum gloesporioides, sclerotinia sclerotiorum, botryosphaeria, and catenulate gibberella.

Description

technical field [0001] The invention relates to the field of molecular biology and genetic engineering related technology research, in particular to the proline-rich proline protein gene of Yangbio walnut with antifungal activity JsPRP1 and applications. Background technique [0002] Plants may be infected by pathogenic bacteria throughout the growth period. Among the various plant diseases, fungal diseases account for about 70-80% of plant diseases. The rise of classical genetics in the early 20th century has enabled people to successfully breed new disease-resistant varieties through cross-breeding, thereby greatly increasing food production. However, conventional breeding has the disadvantages of long cycle, time-consuming and labor-intensive, and few beneficial mutations, which cannot fundamentally solve the problem of plant diseases. In recent years, with the continuous development of molecular biology theory and technology, people can not only deeply understand the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/84A01H5/00
Inventor 刘迪秋韩青陈朝银陈瑞葛锋
Owner KUNMING UNIV OF SCI & TECH
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