Application of panax japonicus transcription factor gene PjWRKY1

A technology of transcription factor and ginseng beads, applied in the fields of molecular biology and genetic engineering, can solve problems such as habitat destruction and depletion of ginseng beads, and achieve the effects of increasing yield, overcoming long artificial cultivation cycles and improving expression levels.

Active Publication Date: 2015-11-25
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, most of the medicinal materials of pearl ginseng are wild products, and disorder

Method used

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  • Application of panax japonicus transcription factor gene PjWRKY1
  • Application of panax japonicus transcription factor gene PjWRKY1
  • Application of panax japonicus transcription factor gene PjWRKY1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: PjWRKY1 Gene cloning and bioinformatics analysis

[0022] Because Ginseng contains more secondary metabolites, a two-step method is required for the extraction of total RNA. First, the improved guanidine isothiocyanate method was used for crude extraction, followed by digestion with DNase and extraction with chloroform to obtain relatively pure total RNA of Ginseng ginseng ( figure 1 ). Use reverse transcriptase M-MLV (promega) to synthesize the first strand of cDNA using the total RNA of Ginseng ginseng as a template. The reaction system and operation process are as follows: Take 5 μg Total RNA, add 50 ngoligo (dT) in sequence 15 , 2μL dNTP (2.5mMeach), DEPC water to a reaction volume of 13.5μL; after mixing, heating and denaturation at 70°C for 5min, cooling on ice for 5min, and then adding 4μL 5×First-standbuffer, 0.5μL RNasin (200U), 1μL M- MLV (200U), mix well and centrifuge briefly, incubate at 42°C for 1.5h, take it out and heat at 70°C for 10min t...

Embodiment 2

[0025] Embodiment 2: plant expression vector construction

[0026] Use the SanPrep column type plasmid DNA mini-extraction kit (Shanghai Sangong) to extract the insert PjWRKY1 coli plasmid pGEM-T- PjWRKY1 As well as the plasmid of the plant expression vector pCAMBIA2300S, 1 μL was used for agarose gel electrophoresis to detect the integrity and concentration of the extracted plasmid. use Bam H I(TaKaRa) and Xba I(TaKaRa) for plasmid pGEM-T- PjWRKY1 Carry out double digestion with pCAMBIA2300S (100μL system), the reaction system and operation process are as follows: take 20μL pGEM-T- PjWRKY1 or pCAMBIA2300S plasmid, add 10μL 10×Kbuffer, 5μL Bam H I, 5μL Xba I, 60 μL ddH 2 O, after mixing, centrifuge for a short time, and place at 37°C for overnight reaction. Spot all digested products on agarose gel for electrophoresis, and then PjWRKY1 The fragment and the pCAMBIA2300S large fragment were gel-recovered separately, and the SanPrep column DNA gel-recovery kit (Shang...

Embodiment 3

[0029] Embodiment 3: Agrobacterium-mediated genetic transformation of Ginseng

[0030] Take out the stored pCAMBIA2300S containing pCAMBIA2300S from the -80℃ refrigerator -PjWRKY1 The Agrobacterium EHA105 strain of the plasmid was inoculated in 5 mL of LB liquid medium containing 50 mg / L Km and 25 mg / L rifampicin, and cultured at 28°C until cloudy. Pipette 1mL of turbid bacterial solution onto LB solid medium containing 50mg / LKm, and incubate at 28°C for 48h. Scrape off an appropriate amount of Agrobacterium on LB solid medium and inoculate it in MGL liquid medium, add 40 mg / L acetosyringone, and shake at 28°C until OD 600 When it is 0.6, stop shaking the bacteria, and the obtained bacterial solution is used for infection.

[0031] The callus of Panax ginseng in good growth state was transferred to MS pre-medium (containing 35 mg / L acetosyringone) and pre-cultured for 3 days. After the pre-cultivation is completed, the callus is completely immersed in the above-mentioned A...

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Abstract

The invention discloses application of panax japonicus transcription factor gene PjWRKY1, namely, application in improving the expression quantity of key enzyme genes in the biological synthesis of panax japonicus saponins, and increasing the content of saponins in panax japonicus callus. The nucleotide sequence of the PjWRKY1 gene is shown as SEQ ID NO: 1, and WRKY transcription factors are coded. Through the adoption of functional genomics and techniques related to metabolic engineering, a panax japonicus PjWRKY1 transcription factor is proved to have the effect of positively regulating the biological synthesis of panax japonicus saponins; when the panax japonicus PjWRKY1 transcription factor gene disclosed by the invention is established on a plant expression vector and transferred into the panax japonicus callus for over expression, the expression quantity of the key enzyme genes in the synthetic route of panax japonicus saponins is increased, and the yield of panax japonicus saponins is increased.

Description

technical field [0001] The invention relates to the fields of molecular biology and genetic engineering, in particular to a transcription factor gene for ginseng saponin biosynthesis PjWRKY1 Applications. Background technique [0002] Ginseng Panaxjaponicus C.A. Mey.var. major (Burk.) C.Y.WuetK.M.Feng is Araliaceae Panax genus Panax The plant is named after the slender internodes of the rhizome, and the nodes expand into a spherical bead shape, and its rhizome is used as medicine. Pearl ginseng, as a medicinal material, was first recorded in "Southern Yunnan Materia Medica", and it is a variety recorded in the previous editions of "Chinese Pharmacopoeia". Medicinal pearl ginseng is mainly produced in Yunnan. It is a rare and commonly used traditional Chinese medicinal material. It is a traditional medicine of Yi, Naxi, Bai, Tibetan, Lisu and other ethnic minorities. At present, wild ginseng is mainly distributed in northwestern and northeastern Yunnan, and is common ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/84C12N5/10
Inventor 葛锋黄壮嘉刘迪秋
Owner KUNMING UNIV OF SCI & TECH
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