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Panax notoginseng antimicrobial peptide gene PnSN1 and application thereof

An antimicrobial peptide and gene technology, applied in Panax notoginseng antimicrobial peptide gene PnSN1 and its application fields, can solve the problems of easily inducing diseases and insect pests, affecting the yield and quality of Panax notoginseng raw medicinal materials, and achieve reduction of environmental pollution, broad market application prospects, The effect of shortening the breeding cycle

Active Publication Date: 2016-08-17
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because Panax notoginseng is warm and humid, and is sensitive to light, it is required to be cultivated under a sunshade net. Its unique growth environment is easy to induce the occurrence of diseases and insect pests, especially fungal diseases such as root rot and black spot, which have seriously affected the origin of Panax notoginseng. Yield and quality of medicinal materials

Method used

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  • Panax notoginseng antimicrobial peptide gene PnSN1 and application thereof
  • Panax notoginseng antimicrobial peptide gene PnSN1 and application thereof
  • Panax notoginseng antimicrobial peptide gene PnSN1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: PnSN1 full-length cDNA cloning and sequence analysis

[0023] The root of Panax notoginseng was inoculated with Fusarium solani rot, the total RNA was extracted from the root 12 hours after inoculation, the treated root of Panax notoginseng was ground into powder with liquid nitrogen, then transferred into a centrifuge tube, and extracted by guanidine isothiocyanate method total RNA. The 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 ...

Embodiment 2

[0026] Embodiment 2: plant overexpression vector construction

[0027] The Escherichia coli plasmid pGEM-T-PnSN1 inserted into PnSN1 and the plant expression vector pCAMBIA2300S plasmid were extracted using the SanPrep column plasmid DNA mini-extraction kit (Shanghai Sangong), and 1 μL was used for agarose gel electrophoresis to detect the extracted Plasmid integrity and concentration. Restriction enzymes BamHI (TaKaRa) and Pst (TaKaRa) carried out double digestion of plasmids pGEM-T-PnSN1 and pCAMBIA2300S respectively (100 μL system). The reaction system and operation process were as follows: take 20 μL pGEM-T-Pnsnakin and pCAMBIA2300S plasmids respectively, and add 10 μL 10×K buffer, 4.5 μL BamHI, 5.5 μL Pst , 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 the PnSN1 fragment and the large fragment of the pCAMBIA2300s vector were gel-recover...

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 preserved Agrobacterium LBA4404 strain containing the pCAMBIA2300S-PnSN1 plasmid from the -80°C refrigerator, take 20 μL and inoculate it into 5 mL of LB liquid medium containing 50 mg / L Km and 20 mg / L rifampicin, 28 Cultivate until the medium becomes 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 Agr...

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Abstract

The invention discloses a panax notoginseng antimicrobial peptide gene PnSN1. The nucleotide sequence of the PnSN1 gene is shown as SEQ ID NO:1, and the PnSN1 gene is used for coding snakin antimicrobial peptide. The functional genomics correlation technique study proves that the PnSN1 gene has the function of improving the plant fungal infection resistance capability; the antifungal PnSN1 gene is built onto a plant expression vector and is transferred into tobaccos for overexpression; the transgenosis tobacco has high in vitro antifungal activity. The transgenosis tobacco subjected to PnSN1 overexpression has the strong inhibiting effect on the growth of fusarium oxysporum, fusarium solani, verticillium fusarium, botryosphaeria dothidea and beaded gibberella.

Description

technical field [0001] The invention relates to the research fields of molecular biology and genetic engineering related technologies, in particular to a notoginseng snakin antibacterial peptide gene PnSN1 with antifungal activity and its application. Background technique [0002] Globally, plant diseases caused by pathogenic bacteria are an important factor limiting crop yield, which can reduce crop yield by 20%-40%. The traditional methods of controlling plant diseases are mainly the use of chemical pesticides, improved cultivation management measures and the cultivation of new resistant varieties. Although these methods have achieved certain results, they have also brought some problems, such as environmental safety, food safety and new virulent pathogens appearance etc. With the rapid development of biotechnology, the use of genetic engineering to breed new disease-resistant varieties can not only overcome the many drawbacks of the above-mentioned control methods, but a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/84A01H5/00
CPCC12N15/8205C12N15/8282C07K14/415
Inventor 刘迪秋陈瑞崔秀明杨野曲媛关瑞攀白智伟
Owner KUNMING UNIV OF SCI & TECH
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