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Rape BnaBPA03 gene as well as application and method of rape BnaBPA03 gene in regulation and control of rape plant type

A technology of pk7fwg2.0-bnabpa03 and rapeseed, which is applied in the field of plant genetic engineering and plant breeding, can solve the problems that rapeseed is less researched and is in the positioning stage

Pending Publication Date: 2022-08-02
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, there have been many studies on the branch angle in Arabidopsis (branch angle), rice (tiller angle), and maize (stem-leaf angle), and related genes have been cloned and identified, but There are few studies on rapeseed and it is still in the stage of positioning. There are few reports on the cloning and functional analysis of related genes, which still need to be further explored

Method used

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  • Rape BnaBPA03 gene as well as application and method of rape BnaBPA03 gene in regulation and control of rape plant type
  • Rape BnaBPA03 gene as well as application and method of rape BnaBPA03 gene in regulation and control of rape plant type
  • Rape BnaBPA03 gene as well as application and method of rape BnaBPA03 gene in regulation and control of rape plant type

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Identification and acquisition of BnaBPA03 gene

[0046] In Brassica napus, the BP gene has two members. The bioinformatics method was used to analyze the two member genes to determine that the gene with the highest expression and the highest similarity was the target gene—BnaA03g23610D (https: / / www.genoscope. cns.fr / brassicanapus / ), which is designated as BnaBPA03 (abbreviated as BnaA03).

[0047] The primers were designed according to the BnaBPA03 gene sequence on "https: / / www.genoscope.cns.fr / brassicanapus / ", and the primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The primer sequences are:

[0048] BnaA03-F (SEQ.ID.NO.3): 5'-ATGGAAGAATATCAACATGAAAGCAGATCC-3'

[0049] BnaA03-R (SEQ.ID.NO.4): 5'-TTATGGTCCAAGACGAATAAGGACCATC-3'

[0050] Rape Y127 (from Huazhong Agricultural University) was used as the experimental material, and it was grown in the greenhouse for about 6 weeks (growth conditions: 22±2°C, 22h (light) + 2h (dark), 60% to...

Embodiment 2

[0070] Example 2: Expression pattern and subcellular localization of BnaBPA03 gene

[0071] In order to explore the differential expression of gene BnaBPA03 in various tissues of Brassica napus and its localization in cells, qRT-PCR technique was used to detect the expression of BnaBPA03 in various tissues of Brassica napus. The material is Y127, each tissue at the full bloom stage is selected, and after sampling and quick freezing, RNA from each tissue is extracted and cDNA is synthesized according to the method in Example 1.

[0072] Design real-time quantitative PCR primers according to the non-conserved region of the BnaBPA03 gene sequence, and the primer sequences are:

[0073] BnaA03-Q-F (SEQ.ID.NO.7): 5'-AGCCATCCACAATACTCAAGAA-3'

[0074] BnaA03-Q-R (SEQ.ID.NO.8): 5'-CGCCGTAATTTTATCAACCACT-3'

[0075] The Brassica napus Actin gene (GenBank: AF111812.1) was selected as the internal reference gene, and the internal reference primer sequences were:

[0076] Actin-QF (SE...

Embodiment 3

[0085] Example 3: Construction of BnaBPA03 gene overexpression vector

[0086] In this example, the method of Gateway was used, and the CACC sequence was added to the 5' of the upstream primer, and in order to express the green fluorescent protein, the terminator was removed when the BnaBPA03 gene sequence was amplified. The primer sequences were designed as follows:

[0087] 2.0-A03-F (SEQ.ID.NO.11): 5'-CACCATGGAAGAATATCAACATGAAAGCAGATCC-3'

[0088] 2.0-A03-R (SEQ.ID.NO.12): 5'-TGGTCCAAGACGATAAGGACCATCGCCC-3'

[0089] First, the bacterial liquid obtained in Example 1 was taken out from the -70°C ultra-low temperature refrigerator, activated, and the plasmid was extracted. Using the high-fidelity enzyme 2×Phanta MAX Master Mix (purchased from Nanjing Novizan Biotechnology Co., Ltd.), the extracted plasmid was used as a template to amplify the BnaBPA03 gene sequence. The difference from Example 1 is that this amplification The obtained fragment has four more bases of CACC be...

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Abstract

The invention belongs to the technical field of plant genetic engineering and plant breeding, and particularly relates to an oilseed rape BnaBPA03 gene and application and method of the oilseed rape BnaBPA03 gene to regulation and control of oilseed rape plant types. A BnaBPA03 gene is cloned, an overexpression vector pK7FWG2.0-BnaBPA03 of the gene is constructed, brassica napus is transformed, an overexpressed stable transformation strain is obtained in the brassica napus, a transgenic plant with a small branching angle and a compact plant type is obtained, the obtained transformation strain is smaller in branching angle and leaf angle, and the obtained plant type is more compact in structure; the method has important guidance reference significance for production and breeding of economic crops such as oilseed rape, and certain available germplasm resources are provided for improving oilseed rape plant types and realizing high-density planting of oilseed rape.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering and plant breeding, in particular to a rape BnaBPA03 gene and an application method and method for regulating rape plant type. Background technique [0002] Rape (Brassica napus L.) is an important oil crop, an important source of edible vegetable oil and biofuel, and can also be used as ornamental, with broad market prospects. With the growing population and increasing demand for renewable energy, the market demand for rapeseed oil continues to grow, and my country's rapeseed industry has a huge supply and demand gap and is heavily dependent on imports. Therefore, increasing the yield of rapeseed and improving the self-sufficiency rate of rapeseed oil have extremely important strategic significance for ensuring the security of vegetable oil supply in my country. High-yield breeding of rapeseed has become the primary goal of rapeseed breeding. [0003] The improvement of plant t...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/84C12N1/21C12N15/65A01H5/00A01H6/20C12R1/01
CPCC07K14/415C12N15/8205C12N15/8261C12N15/8212C12N15/65C12R2001/01
Inventor 谭小力薛怡萱李玉龙耿瑞李雷
Owner JIANGSU UNIV
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