Stem mustard stress resistance gene bjefh1 and its plant expression vector and application

A technology of plant expression vector and stem mustard, which is applied in the field of stress-resistant gene BjEFh1 and its plant expression vector, can solve the problems of few reports on molecular mechanism research, less research on calmodulin-like and calcium-dependent protein kinase functions, and achieve Variety improvement, the effect of improving stress tolerance characteristics

Active Publication Date: 2017-02-08
CHONGQING UNIV OF POSTS & TELECOMM
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  • Application Information

AI Technical Summary

Problems solved by technology

It has been reported that calcium-binding proteins include calmodulin, calmodulin-like protein, and calcium-dependent protein kinase, among which the report on the function of calmodulin is the most; but there are few studies on the function of calmodulin-like protein and calcium-dependent protein kinase. There are few reports on its molecular mechanism

Method used

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  • Stem mustard stress resistance gene bjefh1 and its plant expression vector and application
  • Stem mustard stress resistance gene bjefh1 and its plant expression vector and application
  • Stem mustard stress resistance gene bjefh1 and its plant expression vector and application

Examples

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experiment example 1

[0023] Experimental Example 1: Cloning of Stress Resistance Gene BjEFh1 in Stem Mustard

[0024] 1 Main reagents: column type small amount plant total RNA extraction kit (W7021) was purchased from Shanghai Huashun Biotechnology Co., Ltd.; DNA purification and recovery kit was purchased from Tiangen Biochemical Technology Co., Ltd.; 5'-Full RACE Kit kit , M-MLV Reverse Transcriptase, Premix Ex Taq, DL2000DNA Marker, and pMD19-T vectors were all purchased from Dalian Bao Biological Engineering Co., Ltd.

[0025] 2 Experimental methods and steps

[0026] Acquisition of BjEFh1 gene sequence fragment SEQ ID NO.4: use column type small amount plant total RNA extraction kit to extract the total RNA of the tuber mustard stem, and take a small part of the total RNA to detect OD260 / OD280 by ultraviolet spectrophotometer The ratio of 1.93, the brightness of 28S by agarose gel electrophoresis is about 2 times that of 18S, indicating that the RNA has high purity and no degradation, which ...

experiment example 2

[0058] Experimental Example 2: Construction of the recombinant plant expression vector pCAMBIA1302-BjEFh1 of the stem mustard stress-resistant gene BjEFh1

[0059] 1 Main reagents: common plasmid extraction kit was purchased from Tiangen Biochemical Technology Co., Ltd.; T4DNA ligase, restriction endonuclease Bgl II and BstE II were purchased from Dalian Bao Biological Engineering Co., Ltd.; strains containing plasmid pCAMBIA1302 were purchased from our laboratory save.

[0060] 2 Construction of recombinant plant expression vector pCAMBIA1302-BjEFh1

[0061] 2.1 Primer design: Design upstream and downstream primers according to the sequence of the coding region of BjEFh1 gene SEQ ID NO.1, and respectively introduce restriction site Bgl II (AGATCT) and BstE II (GGTAACC) sequences, the primers are:

[0062] Upstream primer BjEFh1-Bgl: 5'-AGATCTATGGAGAAGAGCTTATTG-3'

[0063] Downstream primer BjEFh1-Bst: 5'-GGTTACCTTAGCAGAAGCTACTC-3'

[0064] 2.2 PCR amplification: use the fi...

experiment example 3

[0070] Experimental Example 3: Genetic Transformation of Arabidopsis

[0071] 1 Main reagents: Fluorescence quantitative PCR kit SYBR Premix Ex Taq TM Kit was purchased from Dalian Bao Biological Engineering Co., Ltd.; W7021 Column Small Plant Total RNA Extraction Kit was purchased from Shanghai Huashun Biotechnology Co., Ltd.; DNase was purchased from Promega.

[0072] 2 Cultivation of Arabidopsis thaliana and Agrobacterium infection experiment

[0073] ① Take wild-type Arabidopsis seeds, sterilize with 75% ethanol for 1 min, and then wash with sterile water; then sterilize with 5% NaClO for 10 min, wash with sterile water for 5 times, and remove the residual NaClO. Add a certain amount of sterile water, and vernalize at 4°C for 3-4 days;

[0074] ②Cultivate Arabidopsis thaliana seeds that have been vernalized for 3-4 days on the MS blank medium for 7 days under light, and after the seedlings grow, transplant them to the vermiculite medium (vermiculite: nutrient soil: perl...

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Abstract

The invention discloses a tumorous stem mustard stress resistance gene BjEFh1 as well as a plant expression vector and application thereof. The base sequence of the stress resistance gene BjEFh1 is as shown in SEQ ID NO. 1; the recombinant plant expression vector pCAMBIA1302-BjEFh1 constructed by the invention is formed by connecting the SEQ ID NO. 1 sequence in a plant expression vector pCAMBIA1302; the plant expression vector is used for plant genetic transformation, and the BjEFh1 gene can realize over-expression under promotion of a CaMV35S promoter to synthesize a large quantity of BjEFh1 protein, thus enhancing stress resistance of plant.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a stress-resistant gene BjEFh1 derived from stem mustard mustard and its plant expression vector and application. Background technique [0002] Abiotic stresses such as high salinity, drought and low temperature have a great impact on the yield and growth of crops. Therefore, the research on plant stress resistance has always been one of the hotspots in the field of botany research. [0003] Stem tumor mustard (Brassica juncea var.tumida Tsen et Lee), also known as cabbage head, is a Brassicaceae plant, the main raw material of mustard mustard, and one of the main economic crops in Chongqing, Sichuan, Zhejiang and other regions of my country. For many years, the related research on the stem mustard has been mainly concentrated in the fields of genetic breeding, breeding of improved varieties, cultivation technology, quality and safety, etc. In recent years, there have been repo...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82C07K14/415A01H5/00
Inventor 向浏欣汪露刘吉军王小艳郑慧邓朝伟高翔
Owner CHONGQING UNIV OF POSTS & TELECOMM
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