Gene related to drought resistance of oilseed rape, overexpression vector, cell line, host bacterium and application of gene, overexpression vector, cell line and host bacterium

A drought resistance, genetic technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as yield reduction or no harvest, economic loss, etc.

Active Publication Date: 2021-06-25
HENAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Rapeseed (Brassica napus L.) is one of the most important oil crops in the world. During its growth, it is often affected by various environmental factors, resulting in reduced or no harvest, resulting in serious economic losses.

Method used

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  • Gene related to drought resistance of oilseed rape, overexpression vector, cell line, host bacterium and application of gene, overexpression vector, cell line and host bacterium
  • Gene related to drought resistance of oilseed rape, overexpression vector, cell line, host bacterium and application of gene, overexpression vector, cell line and host bacterium
  • Gene related to drought resistance of oilseed rape, overexpression vector, cell line, host bacterium and application of gene, overexpression vector, cell line and host bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1, the cloning of Brassica napus BnWRKY255 gene

[0029] Use RNA extraction and separation reagent (Trizol, Invitrogen) to extract the total RNA of Brassica napus seedlings. The specific method is: collect about 80-150 mg of Brassica napus seedlings, put them into the correspondingly labeled EP tube, then add 1ml Trizol reagent, and mix quickly Place on ice for 8-10min; add 0.2ml chloroform, gently invert up and down for 15-30s at a uniform speed, and let stand on ice for 5-8min; centrifuge at 12000rpm at 4°C for 15-20min; transfer the supernatant to a new RNase free EP tube Add 0.5ml of pre-cooled isopropanol and mix gently, let stand for 3-5min, and centrifuge at 12,000g for 20min at 4°C to precipitate RNA; wash the RNA pellet with 1ml of 75% ethanol, let it dry for 5min, dissolve in an appropriate amount of DEPC-treated Store in water at -80°C for later use.

[0030] The gene overexpression vector was constructed according to Gateway recombination technol...

Embodiment 2

[0039] Embodiment 2, the acquisition of transgenic Arabidopsis plants of BnWRKY255

[0040] In order to study the role of BnWRKY255, transgenic plants overexpressing BnWRKY255 were constructed in Arabidopsis, and the transgenic lines were identified, according to the semi-quantitative results as follows: figure 2 As shown, three independent homozygous T3 transgenic lines highly expressing BnWRKY255 were randomly selected for further phenotypic analysis.

[0041] 1. Construction of an overexpression vector for Brassica napus BnWRKY255 transgenic plants: the sequence-verified fragment obtained in Example 1 was recombined into the pDONR207 vector through the BP reaction (BP Clonase II EnzymeMix, Invitrogen) using the Gateway technology of Invitrogen Company, and transformed into Escherichia coli DH5α In the competent cells, the entry clone was obtained by screening with 50mg / L Qingda, and then the plasmid was extracted and recombined the BnWRKY255 gene into the pEarleyGate103-RF...

Embodiment 3

[0044] Example 3, Determination of Drought Sensitivity of Transgenic BnWRKY255 Arabidopsis

[0045] To study the effect of mannitol on the root growth of transgenic BnWRKY255 Arabidopsis thaliana, the method is as follows: Transgenic and wild-type Arabidopsis seeds harvested at the same time after disinfection and cleaning were sown in 1 / 2MS solid medium, and vernalized at 4°C for 2 days Place them in a long-day culture room (22°C, light for 16 hours, and darkness for 8 hours). After 3 days of germination, move the seedlings in the same growth state to 1 / 2 MS solid medium containing 200 mM and 300 mM mannitol in the ultra-clean workbench. , put into the long-day cultivation room, place vertically, count the root length and take pictures after cultivating for 7 days. The experimental results showed that: under the influence of mannitol, the root growth of the BnWRKY255 overexpression line was significantly inhibited. The root length of the overexpression line was statistically...

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Abstract

The invention belongs to the technical field of plant molecular biology, and particularly relates to a gene related to drought resistance of rape, an overexpression vector, a cell line, host bacteria and application thereof. The gene is BnWRKY255, and the nucleotide sequence of the gene is as shown in SEQ ID NO: 1. The BnWRKY255 gene is applied to transformation of dicotyledons to produce drought-sensitive dicotyledons, the BnWRKY255 gene plays a negative regulation role in drought resistance, the sensitivity of an overexpressed plant to ABA is reduced, and a reference basis and related genes are provided for deep understanding of a stress resistance signal network in which rape WRKY participates.

Description

technical field [0001] The invention belongs to the technical field of plant molecular biology, and specifically relates to a gene related to drought resistance of rapeseed, an overexpression vector, a cell line, a host bacterium and applications thereof. Background technique [0002] Rapeseed (Brassica napus L.) is one of the most important oil crops in the world. During its growth, it is often affected by various environmental factors, which leads to yield reduction or failure, resulting in serious economic losses. As one of the largest transcription factor families in plants, the WRKY transcription factor family is widely involved in the regulation of plants to biotic and abiotic stresses, growth and development, and metabolic processes. More than 70% of the WRKY transcription factors in Arabidopsis have effects on pathogens and salicylic acid treatment, and WRKY transcription factors also play roles in responses to low and high temperature, water stress, high CO2 levels,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H6/82A01H6/20A01H5/00
CPCC07K14/415C12N15/8273C12N15/8293
Inventor 王道杰杨翠玲王勇锋赵恬丁群英剧凌岳
Owner HENAN UNIVERSITY
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