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Cleistogamy character regulation gene BnaC03. FBA, floral organ specific expression promoter PFBA and application thereof

A technology for closed flower pollination and gene regulation, applied in the field of genetic engineering, can solve the problem of no breakthrough in breeding work, and achieve the effect of reducing genetic drift

Active Publication Date: 2022-06-03
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although botanists have actively explored the inheritance of cleistopollination traits and the excavation and breeding of germplasm, no breakthroughs have been made in related breeding work, and new genes that control cleistopollination traits need to be continuously excavated to provide a basis for breeding work

Method used

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  • Cleistogamy character regulation gene BnaC03. FBA, floral organ specific expression promoter PFBA and application thereof
  • Cleistogamy character regulation gene BnaC03. FBA, floral organ specific expression promoter PFBA and application thereof
  • Cleistogamy character regulation gene BnaC03. FBA, floral organ specific expression promoter PFBA and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Cloning of closed flower pollination trait control gene BnaC03.FBA and its promoter PFBA

[0037] 1. Discovery and cloning of BnaC03.FBA, the control gene for closed-flower pollination traits

[0038]Cloning the closed flower pollination control gene, the gene number is BnaC03G0156800ZS, the gene encodes an F-box protein comprising the FBA (F-box associated domain) domain, and the gene is named as BnaC03.FBA, and the full-length genome sequence is shown in SEQ ID NO. 3. According to the published double 11 reference genome in Brassica napus (http: / / cbi.hzau.edu.cn / bnapus / ), the primers used to amplify the closed-flower pollination trait control gene were designed, the upstream primer P1: 5'-ATGGAGAACTCGGACTTTGC -3' and downstream primer P2: 5'-TTAAATGTAGATGCTTGGTT-3'; total RNA was extracted from fresh leaves of Brassica napus, and then cDNA was reversely synthesized by Reverse Transcription kit (Takara), and the synthesized cDNA was used as Template, PCR a...

Embodiment 2

[0042] Example 2: Construction of plant overexpression vector containing BnaC03.FBA gene

[0043] 1. Construction of 35S promoter-driven gene BnaC03.FBA overexpression vector

[0044] The overexpression vector pBI121 digested with Xba I was used. By introducing the homologous sequence of the vector insertion site at the 5' end of the primer, the amplified product of the BnaC03.FBA gene and the linearized cloning vector have completely identical sequences (15bp) capable of mutual homologous recombination. The gene cloning primers are: upstream primer P5 (5'-AGAACACGGGGGACTCTAGAATGGAGAACTCGGACTTTGC-3'), downstream primer P6 (5'-CCACCCGGGGATCCTCTAGATTAAATGTAGATGCTTGGTTTGTG-3'). The nucleotide sequence of SEQ ID NO. 2 in the sequence table cloned in Example 1 was subjected to PCR amplification with primers P5 and P6. After the reaction, the PCR amplification product was detected by 1% agarose gel electrophoresis. AXYGEN spin column gel recovery kit, follow the instructions to re...

Embodiment 3

[0047] Example 3: Obtainment of BnaC03.FBA overexpressing transgenic rape

[0048] The plant vectors PFBA::BnaC03.FBA and 35S::BnaC03.FBA constructed in Example 2 were transformed into Agrobacterium EHA105 competent cells by heat shock method, and were coated on LB antibodies containing kanamycin and rifampicin. cultured at 28°C for 12h, pick out the single colony of Agrobacterium that grows, screen positive clones and sequence them, and then inoculate the positive clones with correct sequencing in 20ml YEB liquid culture containing kanamycin and rifampicin culture medium, cultured at 28°C and 150rpm for 2 days, and then inoculated the bacterial liquid in 300ml YEB liquid medium containing kanamycin and rifampicin at a volume ratio of 2%, at 28°C. , Cultivated at 150rpm for 18 hours to OD600=0.8-1.2. After the cultivation, the cells were collected by centrifugation at 5000 rpm for 20 minutes, and then suspended in 250 ml of a solution containing 5% sucrose and 0.1% Silwetl-77...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a cleistogamy character control gene BnaC03. FBA, a key promoter PFBA and application of the cleistogamy character control gene BnaC03. FBA and the key promoter PFBA in cleistogamy character genetic regulation. The invention provides the application of the gene BnaC03. FBA in the formation of the cleistogamy character of the rape for the first time. The promoter PFBA, the cleistogamy character control gene BnaC03. FBA, the encoding product BnaC03. FBA protein and the expression vector containing the gene BnaC03. FBA fragment can be used for producing transgenic plants with cleistogamy characters so as to obtain new germplasm resources, and the application prospect is wide.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a closed-flower pollination trait regulation gene BnaC03.FBA, a flower organ-specific expression promoter PFBA and applications thereof. Background technique [0002] Closed-flowered pollination: During flowering, the petals are always closed, wrapping the pistil and stamen, so that pollination is completed in a closed space. The closed-flower pollination trait is helpful for the preservation of germplasm in the process of crop breeding and the cultivation of new ornamental horticultural flower forms. [0003] At present, there are few studies on the control genes and molecular mechanisms of closed-flower pollination traits. AtMYB21 and AtMYB24 are two R2R3-MYB transcription factors in Arabidopsis thaliana, and the double mutant myb21myb24 failed to open flowers and exhibited a phenotype of closed-flower pollination (Mandaokar et al., 2006). By RNAi techn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/113C12N15/84A01H5/02A01H6/20
CPCC07K14/415C12N15/8231C12N15/827C12N15/8205Y02A40/146
Inventor 管荣展万书贝杨茂倪飞
Owner NANJING AGRICULTURAL UNIVERSITY
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