Radix notoginseng mitogen-activated protein kinase kinase gene PnMAPKK1 and application thereof

A technology of mitogen activation and protein kinase, which is applied in the field of molecular biology and genetic engineering, can solve the problems of less research and achieve the effects of simple operation, shortened breeding cycle, and reduced use

Active Publication Date: 2016-07-13
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the research on the MAPK cascade system is mostly focused on the last MAPK, and a large number of MAPK genes have been isolated and identified, but there are few studies on the upstream of the MAPK cascade pathway, namely MAPKK and MAPKKK

Method used

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  • Radix notoginseng mitogen-activated protein kinase kinase gene PnMAPKK1 and application thereof
  • Radix notoginseng mitogen-activated protein kinase kinase gene PnMAPKK1 and application thereof
  • Radix notoginseng mitogen-activated protein kinase kinase gene PnMAPKK1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: PnMAPKK1 full-length cDNA cloning and sequence analysis

[0023] The root of Panax notoginseng was inoculated with Fusarium solani rot, total RNA was extracted from the root 8 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. 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 of total RNA, add 50 ngoligo (dT) and 2 μL dNTPMix (2.5 mMeach) in sequence, and mix with DEPC water. Make up the reaction volume to 14.5 μL; after mixing, heat denaturation at 70°C for 5 minutes, then quickly cool on ice for 5 minutes, then add 4 μL 5×First-standbuffer, 0.5 μL RNasin (200 U), 1 μL M-MLV (200 U), mix well and Centrifuge briefly, incubate at 42°C for 1.5h, take it out and heat at 70°C for...

Embodiment 2

[0026] Embodiment 2: plant overexpression vector construction

[0027] The E. coli plasmid pGEM-T-PnMAPKK1 inserted into PnMAPKK1 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 plasmids integrity and concentration. Restriction enzymes BamHI (TaKaRa) and Pst (TaKaRa) carried out double digestion of plasmids pGEM-T-PnMAPKK1 and pCAMBIA2300S respectively (100 μL system). The reaction system and operation process were as follows: take 20 μL pGEM-T-PnMAPKK1 and pCAMBIA2300S plasmids respectively, add 10 μL 10×Kbuffer, 4.5 μL BamHI, 5.5 μLPst , 60μLddH 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 PnMAPKK1 fragment and the large fragment of the pCAMBIA2300s vector were gel-recovered us...

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 (Nicotianatabacum 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 5-8d, transfer to light incubator (25°C, 16h / d light) after germination, and then monthly Subculture once with MS medium.

[0032] Take out the preserved Agrobacterium LBA4404 strain containing the pCAMBIA2300S-PnMAPKK1 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, and culture it to the culture medium at 28°C turbid. Pipette 1mL of turbid bacterial solution onto LB solid medium containing 50mg / LKm, and incubate at 28°C for 48h. Then scrape off an appropriate amount of Agrob...

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Abstract

The invention discloses a radix notoginseng mitogen-activated protein kinase kinase gene PnMAPKK1.A nucleotide sequence of the gene PnMAPKK1 is as shown in SEQ ID NO:1, and the gene PnMAPKK1 codes mitogen-activated protein kinase kinase.According to functional genomics related technical researches, the gene PnMAPKK1 has a function of improving pathogenic fungus resistance of plants.After the antifungal gene PnMAPKK1 is constructed to a plant expression vector and transferred into tobaccos to realize overexpression, transgenic tobacco plants have extremely high in-vitro antifungal activity.The transgenic tobacco plants with PnMAPKK1 overexpressed have an evident inhibition effect on growth of fusarium solani, colletotrichum gloeosporioides, verticillium fusarium and botryosphaeria.

Description

technical field [0001] The invention relates to related technologies in the fields of molecular biology and genetic engineering, in particular to a notoginseng mitogen-activated protein kinase kinase gene PnMAPKK1 with antifungal activity and its application. Background technique [0002] Globally, reduced food production caused by pathogens such as bacteria, viruses and fungi has been an important issue. Due to the increasing global population, the demand for food will continue to increase in the next 40 years, and by 2050, food production will need to increase by another 70% to meet people's needs. Due to the infection of pathogenic bacteria, the average yield reduction of corn, barley, rice and soybean is about 12%, the average yield reduction of peanut and potato is about 24%, and the yield reduction of wheat and cotton is about 50% and 80% respectively (OerkeEC.Croplossestopests.JAgricSci, 2006, 144(1):31–43). In the United States alone, the economic loss caused by pa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/84A01H5/00
CPCC12N9/1205C12N15/8205C12N15/8282
Inventor 刘迪秋陈瑞崔秀明曲媛杨野白智伟关瑞攀
Owner KUNMING UNIV OF SCI & TECH
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