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Rapeseed transcription factor bnwrky184, cloning method, vector, host cell and application

A technology of transcription factor and cloning method, applied in the field of plant molecular biology, can solve the problems of prolonging the degradation cycle, affecting the effect of soil fertility, affecting the fermentation and utilization of biomass energy, and reducing the degree of lignification and inhibiting the development of sclerenchyma. Effect

Active Publication Date: 2020-09-25
HENAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the existence of lignin also brings many negative effects to human production practices. For example, too much lignin in vegetables will affect the appetite and digestibility, and high lignin content in forage grass will affect the digestion and absorption of livestock, reducing the The nutritional value of forage grass is reduced; too high lignin content also affects the fermentation and utilization of biomass energy by humans. Too much cellulose in green manure will prolong the degradation cycle and affect the fertility of the soil

Method used

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  • Rapeseed transcription factor bnwrky184, cloning method, vector, host cell and application
  • Rapeseed transcription factor bnwrky184, cloning method, vector, host cell and application
  • Rapeseed transcription factor bnwrky184, cloning method, vector, host cell and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Cloning of BnWRKY184 Sequence of Brassica napus Drought Tolerance Protein Coding Gene

[0026]Use RNA extraction and separation reagent (Trizol, Invitrogen) to extract total RNA of Brassica napus seedlings. The specific method is: collect 150 mg of Brassica napus seedlings, put them into correspondingly labeled EP tubes, then add 1 mL of Trizol reagent, mix quickly and place on ice 10min; add 0.2mL chloroform, gently invert up and down for 30s at a uniform speed, and let stand on ice for 8min; centrifuge at 12000rpm at 4°C for 20min; transfer the supernatant to a new RNase free EP tube, add 0.5mL pre-cooled isopropyl Gently mix with alcohol, let stand for 3-5min, 4°C, centrifuge at 12000g for 20min to precipitate RNA; wash RNA pellet with 1mL 75% (v / v) ethanol, let it dry for 5min, dissolve in appropriate amount of DEPC-treated water, -80°C Save for later.

[0027] Obtain the cDNA sequence of BnWRKY184 by RT-PCR amplification, the specific method is:

[0028...

Embodiment 2

[0036] The acquisition of embodiment 2 transfection BnWRKY184 gene plant

[0037] 1. Construction of the BnWRKY184 gene plant overexpression vector of Brassica napus: the RFP gene (red fluorescent protein gene) was recombined into the pEarleyGate103 plasmid to obtain the pEarleyGate103-RFP vector, which was used for future use; the reverse transcription product prepared in Example 1 was used as a template The PCR reaction and the PCR program are the same as in Example 1, and the primers used for PCR are BnWRKY184-F2 and BnWRKY184-R2; the BnWRKY184 gene obtained by the amplification of the sequencing verification is passed through the BP reaction (BP Clonase II Enzyme Mix, Invitrogen) using the Gateway technology of Invitrogen Company Recombined into the pDONR207 plasmid to obtain the pDONR207-BnWRKY184 recombinant vector, transformed into Escherichia coli DH5α competent cells, and obtained entry clones by screening with 50 mg / L gentamicin, and then extracted the plasmids and pa...

Embodiment 3

[0045] Example 3 Staining of Transgenic Arabidopsis Inflorescence Stem Transverse Section and Observation of Cell Wall Thickness

[0046] Taking the Arabidopsis thaliana T3 generation transgenic line obtained in Example 2 and the wild type Arabidopsis thaliana as the experimental objects, the stem segments of the overexpressed T3 generation Arabidopsis thaliana and the wild type Arabidopsis thaliana that had grown for 35 days at the same position were taken respectively, and were free-handed. Sections were subjected to Wiesner staining. Wiesner staining: the sections were stained with 2% (v / v) phloroglucinol (dissolved in 95% ethanol) for 5 minutes, then soaked in 15% (v / v) HCl for 3 minutes, and then mounted and observed directly. Due to the transgene, the diameter of the transgenic plants is small, and the development of the secondary wall is affected, so we use the ratio of the width of the fluorescent beam to the width of the sclerenchyma to reflect the difference between ...

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Abstract

The invention belongs to the technical field of plant molecular biology, and particularly relates to a rape transcription factor BnWRKY184, a cloning method, a carrier, a host cell and an applicationto preparation of lignin-related transgenic plants. The nucleotide sequence of the rape transcription factor BnWRKY184 is represented as SEQ ID NO.1, the amino acid sequence is represented as SEQ ID NO.2, the length of the nucleotide sequence is 645 bp, and the length of the amino acid sequence is 215 amino acids. The provided rape transcription factor BnWRKY184 has a negative control effect, andcan directly overexpress to inhibit expression of plant lignin and cellulose synthetic genes, so that biosynthesis of lignin and cellulose is blocked, development of sclerenchyma is inhibited, the degree of lignification is reduced, and theoretical basis and related genes are provided for follow-up modification of lignin and cellulose content of rape and seed breeding of rape for vegetables, rapefor feed and rape for green manure.

Description

technical field [0001] The invention belongs to the technical field of plant molecular biology, and specifically relates to a rapeseed transcription factor BnWRKY184, a cloning method, a carrier, a host cell and an application. Background technique [0002] Rapeseed is the largest oil crop in my country, and domestic rapeseed oil accounts for more than 55% of the oil production of domestic oilseed crops. The development of rapeseed production has important strategic significance for maintaining the security of national edible oil supply. In recent years, a large number of studies of Chinese rapeseed scientific and technological workers have fully proved the multifunctional utilization value of rapeseed. Focusing on oil use, expanding the functions of feed, fertilizer, vegetable, flower, and honey according to local conditions can greatly improve the efficiency of rapeseed planting. With the large-scale promotion of "double low" rapeseed, the stems and leaves of rapeseed can...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/10C12N15/84C12N1/21A01H5/00A01H6/20
CPCC07K14/415C12N15/1096C12N15/8246C12N15/8255C12Q2531/113
Inventor 王道杰杨俊楠陈浩丁群英杨翠玲
Owner HENAN UNIVERSITY
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