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Application of brassica napus Bna.A05DAD1 gene and method

A technology of Brassica napus and genes, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve problems such as undeveloped research

Active Publication Date: 2020-08-11
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that DAD1 responds to wound induction and participates in the jasmonic acid synthesis pathway, but most of the current research focuses on the molecular mechanism of DAD1 regulating anther dehiscence, and the mechanism of DAD1 participating in silique development and increasing seed weight has not yet been studied

Method used

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  • Application of brassica napus Bna.A05DAD1 gene and method
  • Application of brassica napus Bna.A05DAD1 gene and method
  • Application of brassica napus Bna.A05DAD1 gene and method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, Bna.A05DAD1 overexpression vector construction

[0028] According to the whole genome DNA sequence and CDS sequence (SEQ ID NO.1) of Brassica napus published on NCBI (http: / / www.ncbi.nlm.nih.gov / ), overexpression primers were designed using Geneious 4.8.5 software Primers for amplifying the Bna.A05DAD1 CDS sequence and general primers for related vectors. The primer sequences are shown in Table 1 below, and all primers were sent to BGI for synthesis.

[0029] Table 1. Overexpression primers for amplification of Bna.A05DAD1 CDS sequence primers and related vector general primers

[0030]

[0031]

[0032] The sequences shown in SEQ ID NO.2 and SEQ ID NO.3 were used as primers, and Brassica napus cDNA was used as a template for PCR amplification, and the amplified products were recovered and ligated into the pENtry-D / TOPO entry vector. After sequencing and verifying that the Bna.A05DAD1 CDS sequence was correct, the Gateway method was used to recombine...

Embodiment 2

[0033] Embodiment 2, transformation Arabidopsis wild-type plant

[0034] 1. Transformation of Agrobacterium and identification

[0035] The Agrobacterium competent cell GV3101 was transformed by liquid nitrogen cold shock method to obtain the engineering bacteria GV3101-pEarlyGate101-DAD1 containing the pEarlyGate101-DAD1 vector. Then, the engineering bacteria were transformed into Arabidopsis thaliana by soaking the flowers, and the specific steps were as follows:

[0036] (1) Arabidopsis thaliana seedlings were germinated on 1 / 2 MS medium, and transferred to nutrient soil for about 10 days. Grow in an incubator at 20±2°C, 16 hours of light and 8 hours of darkness; bolting and topping after one month, used for experiments about 50 days, and the fertilized flower buds and siliques were removed the day before dipping;

[0037] (2) Agrobacterium culture: Shake the bacteria in 200mL YEB liquid medium of YEB+Kan 50mg / L+Gen25 mg / L+Rif 20mg / L+str 25mg / L added with antibiotics in 2...

Embodiment 3

[0044] Example 3. Obtaining and phenotypic identification of Arabidopsis transgenic lines overexpressing Bna.A05DAD1

[0045] Three homozygous transgenic lines that heterologously overexpressed BnaA05DAD1 in Arabidopsis thaliana ecotype Colombia were obtained by the above-mentioned Arabidopsis transformation and inbred screening methods, which were named ovAtDAD1-1, ovAtDAD1-2 and ovAtDAD1-3, respectively. Compared with wild-type Arabidopsis, heterologous overexpression of Bna.A05DAD1 plants grew more vigorously ( figure 2 , A). qRT-PCR was used to detect the expression level of Bna.A05DAD1 in the homozygous individual plants of the heterologous overexpression transgenic line and the leaves of wild-type Arabidopsis (WT). The results showed that compared with the control plants, the transgene overexpression homozygous The transcription level of the plants was significantly increased ( figure 2 , B). Through the investigation of the agronomic traits of Arabidopsis thaliana,...

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Abstract

The invention discloses an application of a brassica napus DAD1 gene and a method. By using a cloned full-length coding sequence of the brassica napus DAD1 gene, an overexpression vector driven by a 35S promoter is successfully constructed. Arabidopsis thaliana and brassica napus are respectively subjected to genetic transformation by an agrobacterium infection method, T3-generation positive transgenic plants are obtained, and agronomic trait investigation of the transgenic plants shows that: overexpression of the brassica napus Bna.A05DAD1 can significantly increase the silique length, the number of seeds per silique and the thousand seed weight of seeds, and the brassica napus Bna.A05DAD1 has important significance in increasing the yield of brassica napus.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of BNA.A05DAD1 gene of Brassica napus, and also relates to a method for increasing the yield of Brassica napus. Background technique [0002] The records of edible vegetable oils in our country have been archived since the Song Dynasty, mainly including sesame oil, soybean oil, vegetable oil and tea oil. More than a thousand years later, Brassica napus L. has been widely planted all over the world, and it is called the world's four major oil crops together with soybeans, sunflowers and peanuts. In addition to being an important source of edible vegetable oil for humans, rapeseed is also an important protein feed and raw material for industries such as biodiesel production. At present, the output of rapeseed oil accounts for about 55% of the total amount of edible vegetable oil in my country. However, as a big consumer of edible oil in China, the self-sufficiency rate...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82A01H5/00A01H6/20
CPCC07K14/415C12N15/8261
Inventor 卢坤刘淼曲存民张凯梁颖李加纳
Owner SOUTHWEST UNIVERSITY
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