Relevant protein TaAOC for regulating and controlling cracking of plant anther as well as gene and application thereof
A related protein and plant flower technology, applied in the field of regulation and control of plant anther dehiscence-related protein TaAOC and its gene and application, can solve the problems of wheat and other problems
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Embodiment 1
[0067] Example 1: cDNA cloning and semi-quantitative analysis of TaAOC, a gene related to the regulation of anther dehiscence in the jasmonic acid metabolic pathway of wheat
[0068]The anthers of the hybrid wheat variety Jingmai 7 (Jingshenmai 2009003) were taken, and the total RNA of the anthers was extracted by Trizol method. 5'RACE kit (5'RACE System for Rapid Amplification of cDNA Ends Kit) (GIBCOBRL, CAT.NO.18374-058) and 3'RACE kit (3'RACE System for Rapid Amplification of cDNA Ends Kit) ( GIBCOBRL, CAT. NO. 18373-019) obtained the TaAOC gene, the full length of which is 845kb.
[0069] Total RNA from anthers of hybrid wheat variety Jingmai 7 (Jingshenmai 2009003) was extracted by Trizol method, and cDNA was obtained by reverse transcription with superscript II (purchased from Invitrogen) reverse transcriptase. Primers P1 and P2 were designed according to the sequence of TaAOC gene coding region. Using the cDNA obtained by reverse transcription as a template, PCR ampl...
Embodiment 2
[0075] Example 2: Construction of TaAOC monocot transgenic expression vector
[0076] 1. Construction of recombinant expression vector
[0077] Construction of pAHC-TaAOC monocotyledonous recombinant expression vector: The cDNA obtained by reverse transcription of the total RNA of the hybrid wheat variety Jingmai 7 (Jingshenmai 2009003) was used as a template, and PCR was carried out with specific primers containing BamHI and SacI linker sequences Amplification; then BamHI and SacI double digestion PCR product, recovery, the digestion product is inserted into the vector pAHC25 CaMV 29A promoter between the BamHI and SacI digestion sites, to obtain the recombinant vector pAHC-TaAOC.
[0078] The primer sequences are as follows:
[0079] AOC[BamHI]: 5′-CGGGATCTATCATCCGTTAGC-3′
[0080] AOC[SacI]: 5′-CGAGCTCACGCACGCAAGTTC-3′
[0081] 2. Acquisition and identification of transgenic wheat
[0082] 1) The recombinant plasmid pAHC-TaAOC obtained above is mediated by the gene gun ...
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