Gene, overexpression vector, cell line, host bacterium and application thereof related to drought resistance of rapeseed

A technology of gene and gene coding, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve problems such as production reduction or crop failure, economic loss, etc.

Active Publication Date: 2022-07-05
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, overexpression vector, cell line, host bacterium and application thereof related to drought resistance of rapeseed
  • Gene, overexpression vector, cell line, host bacterium and application thereof related to drought resistance of rapeseed
  • Gene, overexpression vector, cell line, host bacterium and application thereof related to drought resistance of rapeseed

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1. Cloning of Brassica napus BnWRKY255 gene

[0029] Use RNA extraction and separation reagent (Trizol, Invitrogen) to extract total RNA from Brassica napus seedlings. The specific method is as follows: collect about 80-150 mg of Brassica napus seedlings. As for the correspondingly labeled EP tubes, add 1 ml of Trizol reagent and mix quickly. Place on ice for 8-10min; add 0.2ml of chloroform, invert up and down gently and evenly for 15-30s, 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, mix gently, let stand for 3-5min, centrifuge at 12000g for 20min at 4°C to precipitate RNA; wash the RNA precipitate with 1ml of 75% ethanol, air it for 5min, and dissolve it 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 technology, and prime...

Embodiment 2

[0039] Example 2. Acquisition of BnWRKY255 transgenic Arabidopsis plants

[0040] In order to study the role of BnWRKY255, transgenic plants overexpressing BnWRKY255 were constructed in Arabidopsis thaliana, and the transgenic lines were identified. According to the semi-quantitative results such as figure 2 As indicated, 3 independent homozygous T3 transgenic lines highly expressing BnWRKY255 were randomly selected for further phenotypic analysis.

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

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: the transgenic and wild-type Arabidopsis thaliana seeds harvested at the same period after disinfection and washing were sown in 1 / 2 MS solid medium, and vernalized at 4°C for 2 days. Cultured in a long-day culture room (22°C, light for 16 hours, dark for 8 hours), and after 3 days of germination, the seedlings with the same growth state were moved to 1 / 2 MS solid medium containing 200 mM and 300 mM mannitol respectively on the ultra-clean workbench. , placed in a long-day cultivation room, placed vertically, and the root length was counted and photographed after 7 days of cultivation. The experimental results showed that the root growth of BnWRKY255 overexpression lines was significantly inhibited under the influence of mannitol. The root length of the ove...

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Abstract

The invention belongs to the technical field of plant molecular biology, and in particular relates to a gene related to drought resistance of rapeseed, an overexpression vector, a cell line, a host bacteria and applications thereof. The gene is BnWRKY255, and the nucleotide sequence is shown in SEQ ID NO:1. The application is in transforming dicotyledonous plants to produce drought-sensitive dicotyledonous plants. The BnWRKY255 gene of the present invention plays a negative regulatory role in drought resistance, and reduces the sensitivity of overexpressed plants to ABA. The participating anti-stress signaling network provides reference and related genes.

Description

technical field [0001] The invention belongs to the technical field of plant molecular biology, and in particular relates to a gene related to drought resistance of rape, an overexpression vector, a cell line, a host bacteria 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, resulting in reduced yield or no harvest, 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 biotic and abiotic stresses, growth, development and metabolic processes in plants. More than 70% of WRKY transcription factors in Arabidopsis thaliana are responsible for pathogens and salicylic acid treatment, and WRKY transcription factors also play a role in response to low and high temperature, water stress, high CO2 level, ...

Claims

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Application Information

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Patent Type & Authority Patents(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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