CN-121975860-A - Gene for regulating oil content of brassica napus seeds and application thereof

CN121975860ACN 121975860 ACN121975860 ACN 121975860ACN-121975860-A

Abstract

The invention discloses a gene for regulating and controlling oil content of brassica napus seeds and application thereof. The Bnadir1.C2 gene codes lipid transport protein, the nucleotide sequence of the Bnadir is shown as SEQ ID NO.1, and the amino acid sequence of the Bnadir is shown as SEQ ID NO. 2. The invention constructs knockout vector bnadir1.c2, the oil content of the obtained mutant seed is obviously reduced by 3.75% -4.28% compared with the wild type, and simultaneously constructs overexpression vector pBWA (V) HS-OE-Bnadir1.C2, and the oil content of the obtained transgenic overexpression plant seed is obviously improved by 5.05% -6.26% compared with the wild type by introducing the transgenic overexpression vector into agrobacterium GV3101 and transforming Westar. The invention verifies the biological function of Bnadir1.C2 gene for regulating and controlling the oil content of the cabbage type rape seeds from the positive and negative aspects, and provides important gene resources for molecular breeding of rape high-oil varieties.

Inventors

ZOU JUN
WANG HAO
WANG MENG
WANG JING

Assignees

华中农业大学

Dates

Publication Date: 20260505
Application Date: 20260409

Claims (4)

1. The application of one of the following in regulating the oil content of the brassica napus seeds is characterized in that: (1) The nucleotide sequence of the Bnadir1.C2 gene is shown as SEQ ID NO. 1; (2) The overexpression vector pBWA (V) HS-OE-Bnadir1.C2, and the overexpression vector pBWA (V) HS-OE-Bnadir1.C2 is based on the expression vector pBWA (V) HS, the Bnadir1.C2 gene is placed at the downstream of the 35S promoter; (3) Engineering strain containing the above overexpression vector pBWA (V) HS-OE-Bnadir1.C2, wherein the engineering strain is Escherichia coli; (4) Host bacteria containing the above-mentioned over-expression vector pBWA (V) HS-OE-Bnadir1.C2, wherein the host bacteria is Agrobacterium GV3101; (5) The knockout vector of the Bnadir1.C2 gene for regulating and controlling the oil content of the brassica napus seeds is the Bnadir1.c2, a CRISPR-Cas9 system is utilized, the sgRNA sequence of a targeted knockout site is GAGAGATAAACTGCTCGG, the targeted knockout site is positioned in the first exon region of the Bnadir1.C2 gene, and the nucleotide sequence of the Bnadir1.C2 gene is shown as SEQ ID NO. 1.
2. The use of one of the following to increase the oil content of a brassica napus seed, characterized in that: (1) The nucleotide sequence of the Bnadir1.C2 gene is shown as SEQ ID NO. 1; (2) The overexpression vector pBWA (V) HS-OE-Bnadir1.C2, and the overexpression vector pBWA (V) HS-OE-Bnadir1.C2 is based on the expression vector pBWA (V) HS, the Bnadir1.C2 gene is placed at the downstream of the 35S promoter; (3) Engineering strain containing the above overexpression vector pBWA (V) HS-OE-Bnadir1.C2, wherein the engineering strain is Escherichia coli; (4) Host bacteria containing the above-mentioned over-expression vector pBWA (V) HS-OE-Bnadir1.C2, wherein the host bacteria is Agrobacterium GV3101.
3. A method for increasing the oil content of cabbage type rape seeds is characterized in that an over-expression vector pBWA (V) HS-OE-Bnadir1.C2 is introduced into agrobacterium GV3101, and a genetic transformation method mediated by agrobacterium is utilized to transform a cabbage type rape variety Westar, so that a transgenic rape plant with remarkably increased oil content is obtained, wherein the over-expression vector pBWA (V) HS-OE-Bnadir1.C2 is based on an expression vector pBWA (V) HS, and the Bnadir1.C2 gene is placed at the downstream of a 35S promoter.
4. The application of one of the following in cultivating rape varieties with high oil quantity is characterized in that: (1) The nucleotide sequence of the Bnadir1.C2 gene is shown as SEQ ID NO. 1; (2) The overexpression vector pBWA (V) HS-OE-Bnadir1.C2, and the overexpression vector pBWA (V) HS-OE-Bnadir1.C2 is based on the expression vector pBWA (V) HS, the Bnadir1.C2 gene is placed at the downstream of the 35S promoter; (3) Engineering strain containing the above overexpression vector pBWA (V) HS-OE-Bnadir1.C2, wherein the engineering strain is Escherichia coli; (4) Host bacteria containing the above-mentioned over-expression vector pBWA (V) HS-OE-Bnadir1.C2, wherein the host bacteria is Agrobacterium GV3101.

Description

Gene for regulating oil content of brassica napus seeds and application thereof Technical Field The invention relates to the technical field of cabbage type rape genetic engineering and molecular breeding, in particular to a gene for regulating and controlling oil content of cabbage type rape seeds and application thereof. Background Rape is an important source of edible oil, next to palm and soybean, is one of three world oil crops. The edible oil consumption in China is mainly vegetable oil, and soybean oil, rapeseed oil, palm oil and peanut oil are mainly used in vegetable oil consumption. With the improvement of economic level, the requirements of people on the quality of edible oil are gradually improved, and high-quality rapeseed oil with low saturated fatty acid and high oleic acid becomes a main choice. The oil content is increased by 1%, which is equivalent to 2.5% of yield, the oil content of domestic rape is 40% -45%, and the oil content of the rape variety in China is greatly increased compared with the oil content of foreign rape which is 45% -48%. Seed oil content is a complex agronomic trait that is closely related to a variety of biological processes such as photosynthesis, embryo and seed coat development, material transport, fatty acid synthesis, accumulation, and degradation. Currently, more than 700 genes associated with oil accumulation have been reported in arabidopsis thaliana. Up to hundreds of oil content QTLs were located in canola (Yan et al 2022), but only orf188, bnaPMT6, bnaSFAR4, bnaSFAR45, bnaCIPK9, bnaTTG1, bnaTT1, bnaTT2, bnaTT8, bnaFAX6, bnaUK, etc. were cloned (Hu et al 2017,Hu et al 2021,Tang et al 2021,Li et al 2023,Wang et al 2023). Further identification of rape oil content related genes, creation of novel high oil material germplasm would be one of the long-term objectives of rape breeding. Therefore, there is a need to make intensive studies on the functions of the genes for regulating and controlling the oil content of the brassica napus seeds, and to clarify the roles of the genes in regulating and controlling the oil content of the brassica napus seeds so as to provide theoretical basis and gene resources for molecular breeding of high-oil varieties of the brassica napus. Disclosure of Invention The invention aims to overcome the defects of the prior art and provides a gene for regulating and controlling the oil content of brassica napus seeds and application thereof. The Bnadir1.C2 gene of the rape oil content is identified and cloned through QTL fine positioning, a CRISPR-Cas9 technology is utilized to create a mutant of the Bnadir1.C2, the oil content of the obtained rape mutant seed is obviously reduced, and the transgenic rape with the seed oil content obviously improved is obtained through over-expression of the Bnadir1.C2 gene. The invention verifies the important role of Bnadir1.C2 gene in regulating and controlling the oil content of the brassica napus seeds, and provides valuable gene resources for cultivating the high oil seeds of the brassica napus by a molecular breeding mode. In order to achieve the above purpose, the technical scheme of the invention is as follows: The invention provides a gene for regulating and controlling oil content of brassica napus seeds, which is a Bnadir1.C2 gene, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1. The invention also provides a lipid transport protein Bnadir1.C2 coded by the Bnadir1.C2 gene, the total 105 amino acids of the protein coded by the gene, and the amino acid sequence of the lipid transport protein Bnadir1.C2 is shown as SEQ ID No. 2. It should be clear that a person skilled in the art can, based on the amino acid sequences disclosed in the present invention, substitute, delete and/or add one or more amino acids thereto, and the protein sequence is aligned to have a homology of more than 90% without affecting the biological activity of the protein, resulting in a mutant sequence of said protein. Thus, the invention also includes derived proteins with high homology and biological activity obtained by substituting, deleting and/or adding one or more amino acids to the amino acid sequence shown in SEQ ID No. 2. Furthermore, it will be appreciated that in view of the codon degeneracy and the nature of the codon bias, those skilled in the art will be able to use codons appropriate for expression by a particular species, as desired. The gene and the protein can be cloned or separated from a rape variety Westar or can be obtained by a sequence chemical synthesis method. A primer pair for obtaining the sequence of the Bnadir1.C2 gene, wherein the primer pair is as follows: 5'-CATAAGTAGAAAATGGGGAAGAA-3', a primer F is shown as SEQ ID No. 3; primer R5'-TCAAGCTCCATTCTTCAAACT-3' is shown as SEQ ID No. 4. The Bnadir1.C2 gene codes for lipid transport proteins, and can regulate the oil content of seeds by regulating lipid synthesis and transport. Blast alignment analysis was performed on the brass