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Application and method of brassica napus BnMAPK2 gene

A Brassica napus, gene technology, applied in the directions of application, genetic engineering, plant genetic improvement, etc., can solve the problem that the effect has not yet been reported, and achieve the effect of improving tolerance

Active Publication Date: 2021-01-29
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, the role of MAPK2 gene in Brassica napus in plant growth and development and heavy metal stress environment has not been reported yet.

Method used

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  • Application and method of brassica napus BnMAPK2 gene
  • Application and method of brassica napus BnMAPK2 gene
  • Application and method of brassica napus BnMAPK2 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1, BnMAPK2 overexpression vector construction

[0029] Using the known Arabidopsis AtMAPK2 gene sequence to search the Chinese cabbage genome database, predict the possible BnMAPK2 gene, and search the GenBank EST and GSS databases at the same time. The total RNA of mixed tissues and organs of Brassica napus Zhongyou 821 strain was extracted and cDNA was reverse transcribed, and the 5'RACE sequence and 3'RACE sequence of the target gene were synthesized by RECA method. RACE primers were designed according to the cDNA and EST sequences in the database, respectively using FMPK2-51 (SEQ ID NO.5)+RACE 5P (SEQ ID NO.7), FMPK2-52 (SEQ ID NO.6)+RACE 5P (SEQ ID NO.7), FMPK2-31 (SEQ ID NO.4)+RACE 3P (SEQ ID NO.9) (Table 1) primers for the first amplification, respectively to obtain FMPK2-51'R, FMPK2-52'R and FMPK2-31'R products. The PCR amplification conditions were: pre-denaturation at 94°C for 5 min → 35 amplification cycles (denaturation at 94°C for 30 s → anneal...

Embodiment 2

[0034] Example 2, Transformation of Arabidopsis wild-type plants and acquisition of Arabidopsis transgenic lines overexpressing BnMAPK2

[0035] After the recombinant vector was transformed into Agrobacterium, the wild-type Arabidopsis was transformed by the Agrobacterium carrying the recombinant vector pCAMBIA1300-BnMAPK2 by flower dipping method, cultured in the dark at 22°C for 24 hours, and Arabidopsis seedlings were taken out for routine culture. Single plant received T 0 generation seeds. T 1 Substituting seeds, extracting T 1 Genomic DNA of leaves of the first generation seedlings was used as a template, and a total of 16 plants ( figure 1 ). Plants detected as positive, harvested seeds T 2 After generations, the homozygous strains were screened using hygromycin-containing medium.

[0036] 13 of them were selected to extract young leaf RNA for PCR identification, and the qualified cDNA was obtained by reverse transcription method. With AtACT2 as the reference gene...

Embodiment 3

[0037] Example 3. Investigation of the agronomic traits of Arabidopsis transgenic lines overexpressing BnMAPK2

[0038] Referring to the seeding method of the Arabidopsis TAIR10 database operation manual, five Arabidopsis lines (OEMAPK2-25, OEMAPK2-5, OEMAPK2-9, OEMAPK2-12, OEMAPK2-13) and wild-type Arabidopsis overexpressed BnMAPK2 Mustard (WT) seeds were sterilized and sown on sterilized culture soil after vernalization. During the growth period, observe and record the growth and development of different transgenic Arabidopsis lines and wild-type Arabidopsis lines, including: emergence stage, four-leaf stage, eight-leaf stage, ten-leaf stage, bolting stage and stem length 5cm , 10cm, and 15cm dates, and take pictures of the bolting stage and mature plants for comparative analysis. When the Arabidopsis seeds are mature and harvested, count and record their agronomic traits, including: plant height, effective length of main inflorescence, number of pods of main inflorescence,...

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Abstract

The invention discloses an application and method of a brassica napus BnMAPK2 gene, and successfully constructs an overexpression vector driven by a 35S promoter by using a cloned full-length coding sequence of the brassica napus BnMAPK2 gene. Genetic transformation is carried out on plants through an agrobacterium infection method, and positive transgenic plants are obtained. The agronomic traitinvestigation on transgenic plants shows that the agronomic traits of the plants can be improved and the tolerance to heavy metal cadmium can be improved by overexpression of the brassica napus BnMAPK2 gene.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of the Brassica napus BnMAPK2 gene, and also to a method for improving plant agronomic traits and / or increasing cadmium tolerance. Background technique [0002] Rapeseed, together with soybeans, sunflowers and peanuts, is known as the world's four major oil crops, and it is also the oil crop with the largest sowing area and the widest regional distribution in my country. my country's rapeseed oil production accounts for about 40% of the total domestic vegetable oil production. Rapeseed oil has distinctive nutritional characteristics, reasonable fatty acid composition, rich in sterols, vitamin E and other functional substances, and is the oil with the most comprehensive nutritional structure among the bulk edible vegetable oils. , which is of great benefit to improving people's living standards and physical health. As a widely planted cultivar in my country, Brassicana...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82A01H5/00A01H6/20
CPCC07K14/415C12N15/8261C12N15/8271
Inventor 梁颖王珍李咸杨陆俊杏彭茜雷友梅袁大双
Owner SOUTHWEST UNIVERSITY
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