Transgenic expression vector for regulating seed color of brassica napus L., and construction method and application of transgenic expression vector

A technology of Brassica napus and expression vectors, applied in the field of genetic engineering, can solve the problems of slow progress in genetic and molecular mechanism research, complex and lack of genetic mechanism of grain color, etc.

Active Publication Date: 2020-05-29
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the lack of natural yellow seed gene resources, and the complex genetic mechanism of grain color and unstable traits lead...

Method used

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  • Transgenic expression vector for regulating seed color of brassica napus L., and construction method and application of transgenic expression vector
  • Transgenic expression vector for regulating seed color of brassica napus L., and construction method and application of transgenic expression vector
  • Transgenic expression vector for regulating seed color of brassica napus L., and construction method and application of transgenic expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Cloning of BnMYB47 Gene RNAi Fragment

[0037] RNAi primers were designed according to the multiple alignment results obtained from the BnMYB47 gene sequence (the accession number of ganbank is BnaAnng36570D) for the grain color traits of Brassica napus. The forward primer contains restriction enzyme cutting sites BamHI+AatII, and the nucleotide sequence is as SEQ ID NO Shown in: 2, reverse primer contains restriction enzyme cutting site XbaI+NcoI, nucleotide sequence is shown in SEQ ID NO: 3, with the cDNA of Brassica napus black seed line Zhongyou 821 as template, for from cabbage Amplification of BnMYB47 gene interference fragment in B. napus.

[0038] The amplification reaction system is shown in Table 1. The PCR reaction amplification program is: 94°C pre-denaturation for 2 min → 35 amplification cycles (94°C denaturation for 45 s → 58°C annealing for 45 s → 72°C extension for 1 min) → 72°C extension for 10 min. The reaction amplification product was det...

Embodiment 2

[0041] Example 2 Construction and identification of RNAi-BnMYB47 transgene expression vector

[0042] Use restriction endonucleases NcoI+AatII to completely double-enzyme digest the positive clone extraction plasmid and pFGC5941M plasmid of the BnMYB47 gene pMD19-T connected with the interference fragment, and recover the antisense fragment and the open-circle fragment of the BnMYB47 gene after electrophoresis detection. pFGC5941M vector backbone, and the recovered fragments were ligated overnight at 16°C with T4 DNA ligase. The obtained recombinant plasmids were transformed into Escherichia coli JM109 competent cells again, and the bacterial liquid plasmids of PCR-positive clones were extracted.

[0043]Use the restriction enzymes BamHI+XbaI to completely double-enzyme digest the plasmid extracted from the positive clone of the BnMYB47 gene pMD19-T connected to the interference fragment and the plasmid after the recombination of the antisense fragment and pFGC5941, and recove...

Embodiment 3

[0044] Example 3 Application of RNAi Fragment of BnMYB47 Gene

[0045] The application of the RNAi fragment of the BnMYB47 gene in the regulation of the grain color of Brassica napus and the genetic improvement and breeding of rapeseed yellow seed breeding and genetic engineering.

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Abstract

The invention belongs to the field of gene engineering, and particularly relates to a transgenic expression vector for regulating seed color of brassica napus L., and a construction method and application of the transgenic expression vector. The transgenic expression vector is RNAi-BnMYB47 transgenic expression vector, a nucleotide sequence of the RNAi segment of the RNAi-BnMYB47 transgenic expression vector is shown as SEQ ID NO:1, and the RNAi-BnMYB47 transgenic expression vector is obtained by the steps of cloning a RNAi segment of a BnMYB47 gene, connecting the RNAi segment with a pFGC5941M plasmid, performing conversion and plasmid extraction. The RNAi segment of the BnMYB47 gene and the expression vector can be applied to regulate the seed color of the brassica napus L., the difficulty of obtaining stable yellow-seeded brassica napus breeding due to instable seed color property of the brassica napus L. is overcome, conditions for obtaining a stably inherited new seed material oftransgenic yellow seeds of the brassica napus L. are created, and it is of great guiding significance for the brassica napus L. yellow seed breeding and genetic engineering for performing genetic improvement of brassica napus L. quality and breeding practice.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a transgene expression vector for regulating the grain color of Brassica napus and its construction method and application. Background technique [0002] Rapeseed is one of the five largest oil crops in the world and occupies an important position in the world oil crops. Seeds are the main harvesting organs of rapeseed, and the seed coat accounts for about 10-20% of the dry weight of the seed and 20-30% of the dry weight of the cake. The seed coat is rich in pigments and polyphenols, which will seriously affect the oil quality and Feed value of cake. Therefore, improving the composition of Brassica napus seeds and increasing the oil content per unit area is one of the main goals of Brassica napus breeding. Brassica napus has the advantages of thin seed coat, high protein and oil content, high cake protein content, low content of cellulose and pigment and other tox...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/66A01H5/00A01H6/20
CPCC12N15/8261C12N15/66
Inventor 曲存民李加纳卢坤徐新福王瑞唐章林梁颖刘列钊
Owner SOUTHWEST UNIVERSITY
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