Application of gene BnaCYP705a12 in brassinolide biosynthesis and transgenic plant production

A technology of brassinosteroids and transgenic plants, applied in the fields of application, plant products, genetic engineering, etc., to achieve the effects of increasing yield, increasing planting density and harvest index, and plant dwarfing

Pending Publication Date: 2021-11-26
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report about brassinosteroid-related mutants in rapeseed. Therefore, brassinosteroid-deficient transgenic rapeseed can be obtained by transgenic engineering.

Method used

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  • Application of gene BnaCYP705a12 in brassinolide biosynthesis and transgenic plant production
  • Application of gene BnaCYP705a12 in brassinolide biosynthesis and transgenic plant production
  • Application of gene BnaCYP705a12 in brassinolide biosynthesis and transgenic plant production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Cloning of BnaCYP705a12 gene involved in brassinosteroid biosynthesis

[0039] The published French Brassica napus B.napus cv.'Darmor-bzh' genome (http: / / www.genoscope.cns.fr / brassicanapus / ) designed primers for amplifying genes involved in brassinosteroid biosynthesis , upstream primer P1: 5'-ATGGCAGCAATGATAGTTGA-3' and downstream primer P2: 5'-TTAAAGGCTGAGTCGAGAAA-3'; extract total RNA from fresh leaves of Brassica napus, and then use Reverse Transcription kit (Takara) to reverse Synthesize cDNA, and use the synthesized cDNA as a template to amplify the genes involved in the biosynthesis of brassinosteroids in rapeseed. After the reaction, perform 1% agarose gel electrophoresis detection on the PCR amplification products, and use AXYGEN spin column Type gel recovery kit, recover and purify, connect the recovered product into the vector pEasyBlunt Simple, and transform Escherichia coli (E.coli) DH5α competent cells by heat shock method. Positive clones were screened b...

Embodiment 2

[0043] Construction of Plant PFGC5941 Vector Containing BnaCYP705a12 Gene (RNAi-BnaCYP705a12)

[0044] Select interference fragment 170bp (SEQ ID NO.3) according to the conservative sequence of BnaCYP705a12 gene, add Asc I restriction endonuclease site at the upstream of SEQ ID NO.3 sequence, according to Asc I restriction endonuclease site and conservative Sequence design upstream primer, upstream primer P3:5'-TTACAATTACCATGGGGCGCGCCATCCCTCATCACACCATA-3', add Swa I restriction endonuclease site downstream of SEQID NO.3 sequence, according to Swa I restriction endonuclease site and conserved sequence Design downstream primers, downstream primer P4: 5'-TTAAATCATCGATTGGGCGCGCATCTACCAAACCTCCCTT-3'. The nucleotide sequence of SEQ ID NO.1 in the sequence listing cloned in Example 1 was amplified by PCR with primers P3 and P4, and the amplified product was the positive-sense strand of the BnaCYP705a12 gene. Add the Sma I restriction endonuclease site at the upstream of the SEQ ID N...

Embodiment 3

[0046] Obtaining RNAi-BnaCYP705a12 Transgenic Rapeseed

[0047] The plant RNAi-BnaCYP705a12 vector constructed in Example 2 was transformed into Agrobacterium EHA105 (Boerkin, NGC201-1) competent cells by the heat shock method, and coated on LB-resistant cells containing kanamycin and rifampicin. On the plate, culture at 28°C and 150rpm for 16 hours, pick out the single colony of Agrobacterium, screen the positive clones and sequence them, and re-inoculate the positive clones with correct sequencing in 20ml YEB liquid containing kanamycin and rifampicin In culture medium, cultivate 2 days under 28 ℃, 150rpm, then, inoculate bacterial liquid in the 300ml YEB liquid culture medium that contains kanamycin and rifampicin by volume ratio 2% inoculum again, at 28 Cultivate at 150 rpm for 18 hours until OD600=0.8-1.2. After the cultivation, the cells were collected by centrifugation at 5000 rpm for 20 minutes, and then the cells were suspended in 250 ml of a solution containing 5% s...

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Abstract

The invention discloses an application of a gene BnaCYP705a12 in brassinolide biosynthesis and transgenic plant production. The application of the gene BnaCYP705a12 in brassinolide biosynthesis is provided for the first time, and the nucleotide sequence of the gene BnaCYP705a12 is as shown in SEQ ID NO. 1. According to the application, the brassinolide biosynthetic gene BnaCYP705a12, the encoding product BnaCYP705a12 protein of the brassinolide biosynthetic gene BnaCYP705a12 and an expression vector containing the brassinolide biosynthetic gene BnaCYP705a12 gene segment can be used for producing brassinolide biosynthetic defect transgenic crops so as to obtain new germplasm resources, and a wide application prospect is achieved.

Description

technical field [0001] The invention belongs to the field of crop genetic breeding, and specifically relates to the application of gene BnaCYP705a12 in biosynthesis of brassinosteroid and production of transgenic plants. Background technique [0002] Rapeseed is one of the most important oil crops in the world, providing high-quality edible oil for humans, which can be used as high-protein animal feed and industrial raw materials. Plant type breeding plays an important role in improving crop yield. The research on rapeseed plant type is still relatively weak, and it is necessary to further excavate the ideal plant type germplasm resources and related genes, and deepen the research on rapeseed plant type. [0003] Brassinosteroids (BR) are a class of steroid hormones that widely exist in plants. They mainly promote cell elongation, division and cell wall relaxation, thereby further regulating plant elongation and affecting plant height. Blockage of BR biosynthetic pathways ...

Claims

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Application Information

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IPC IPC(8): C12N15/84C12N15/70C12N15/66A01H5/00A01H6/20
CPCC12N15/8218C12N15/8298C12N15/70C12N15/66C12N9/0081C12Y114/15006
Inventor 管荣展杨茂管乙键樊浩王杨铭
Owner NANJING AGRICULTURAL UNIVERSITY
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