Brassica napus Bna.A08IDD7 gene promoter and application thereof

A technology of bna.a08idd7, Brassica napus, applied in the direction of application, genetic engineering, recombinant DNA technology, etc., to achieve the effect of good application potential

Active Publication Date: 2020-12-01
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But after all, it is not derived from plants, and the application of this

Method used

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  • Brassica napus Bna.A08IDD7 gene promoter and application thereof
  • Brassica napus Bna.A08IDD7 gene promoter and application thereof
  • Brassica napus Bna.A08IDD7 gene promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0028] Example 1 clone of promoter of Bna.A08IDD7 gene in Brassica napus

[0029] The 2000bp promoter sequence upstream of translation initiation site (ATG) of Brassica napus Bna.A08IDD7 was searched on NCBI, and the PCR amplification primer Pro-Bna.A08IDD7-F / R and vector general primer pM13F / R were designed and cloned by Primer Premier 5.0. BamHI restriction site was added to the 5' end of the primer before promoter amplification, and NcoI restriction site was added to the 5' end of the primer after promoter amplification. Table 1 shows the designed primer sequences.

[0030] 1. general primer sequence of Bna.A08IDD7 gene promoter amplification primer and vector

[0031]

[0032]

[0033] Using genomic DNA of Brassica napus ZS11 as template, high fidelity enzyme provided by TAKARA Company was used. Amplification. Amplification system (50μL): PCR reaction procedure: pre-denaturation at 98℃ for 30s；; Denaturing at 90℃ for 10s, annealing at 56℃ for 15s, stretching at 72℃ for...

Example Embodiment

[0035] Example 2 Construction of Plant Promoter Vector

[0036] The recovered PCR product was connected to the flat-ended T-vector pEASY-Blunt simplecloning vector provided by Quanquanjin Company, and transformed into Escherichia coli DH5α competent; Colony PCR was used to select the bacterial solution with correct sequencing results for propagation, and the plasmid T-BnaIDD7-pro containing the promoter sequence of Bna.A08IDD7 gene was extracted. The detection results are as follows Figure 3 . The T-BnaIDD7-pro vector and the target vector pCAMIBIA1305.1 were double-digested by restriction enzymes BamHⅠ and NcoⅠ ⅰ of Thermo scientific Company. Then, T4 DNA Ligase provided by Promega Company was used to connect the promoter fragment of Bna.A08IDD7 gene with the completely digested pCAMIBIA1305.1 vector skeleton, and the promoter of Bna.A08IDD7 gene was used to replace pCAMIBIA1305.1(Ping Li, Yan Pei, Xianchun Sang, Yinghua Ling，ZhenglinYang，Guanghua He， Transgenic indica rice expre...

Example Embodiment

[0043] Example 3 Transformation of Agrobacterium tumefaciens GV3101 with Plant Recombinant Expression Vector

[0044] The competent Agrobacterium tumefaciens were purchased in the laboratory, and the constructed expression vector plasmid pCAMBIA1305.1-Bna.A08IDD7::GUS was transformed into the competent cells of Agrobacterium tumefaciens GV3101 by liquid nitrogen cold shock method.

[0045]Operation: take a 100μL competent tube of Agrobacterium GV3101 stored at -80℃, thaw it in ice-water bath, then add the expression vector plasmid pCAMBIA1305.1-Bna.A08IDD7::GUS, mix it gently with a gun head, first ice-water bath for 5min, then quickly heat-shock it in a 37℃ water bath for 5min after cold shock with liquid nitrogen for 8min, and then add 800μL in clean bench. Suck 600μL of supernatant, resuspend the remaining bacterial solution, and spread it evenly on YEB solid culture medium with antibiotic KAN 50mg / L+GEN 25mg / L+RIF 20mg / L+STR 25mg / L, and put it in an incubator at 28℃ for invert...

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Abstract

The invention discloses a brassica napus Bna.A08IDD7 gene promoter and application thereof, the nucleotide sequence of the promoter is shown as SEQ ID NO.5, and the promoter can be strongly expressedin roots, leaves, stems, flowers, siliques, seeds and other tissues of arabidopsis thaliana; therefore, it is indicated that the sequence has very strong expression activity and characteristics of a constitutive promoter, and can be used as a strong constitutive promoter to be applied to plant genetic engineering research. Therefore, the promoter creates excellent germplasm resources and other genetic engineering research aspects; the promoter has good application potential, can replace a cauliflower mosaic virus (CaMV) 35S promoter commonly used in current plant genetic engineering research,constructs a plant transgenic expression vector, is applied to plant genetic engineering, and can widely cultivate transgenic plants with high biosafety.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the Bna.A08IDD7 gene promoter of Brassica napus, and also relates to the application of the promoter. Background technique [0002] my country is the world's largest producer of rapeseed (Brassica napus) and consumer of rapeseed oil. It can provide about 5.2 million tons of high-quality edible oil every year, accounting for 47% of domestic vegetable oil. However, at present, the supply situation of edible vegetable oil in my country is still relatively severe, and the self-production is insufficient, which seriously threatens the safety of edible vegetable oil in my country. Rapeseed production mainly utilizes fallow fields in winter, and does not compete with food crops for land. Currently, there are still 66hm of fallow fields in the Yangtze River Basin 2 All of the above have great potential for developing the rapeseed industry. [0003] The plant gene promoter plays a key role ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82A01H5/00A01H6/20
CPCC07K14/415C12N15/8222
Inventor 卢坤刘绪梅段回春曲存民李加纳
Owner SOUTHWEST UNIVERSITY
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