Nucleotide sequence capable of mediating plant male fertility and method of using nucleotide sequence
A nucleotide sequence, male sterility technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, plant peptides, etc.
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no. 1 example
[0123] The first embodiment, obtaining the sequence of ZmMYB103
[0124] From the known sequence of Arabidopsis AtMYB103 (as shown in SEQ ID NO:3 in the sequence listing), the nucleotide sequence of the transcription factor ZmMYB103 of maize variety B73 is shown in SEQ ID NO:1 in the sequence listing, and its amino acid sequence As shown in SEQ ID NO:2 in the sequence listing.
[0125] To regulate male sterility by disrupting ZmMYB103 function, a dominant ZmMYB103 repressor needs to be established. The 12 amino acid EAR sequence was shown to function as a dominant repressor motif when fused to other transcription factors, such as LLDDLELRLGFA. The short nucleotide sequence encoding the EAR sequence is fused to the 3' end of the ZmMYB103 open reading frame to generate a ZmMYB103+G+EAR fusion, the nucleotide sequence of which is shown in SEQ ID NO:4 in the sequence listing.
[0126] The ZmMYB103+G+EAR nucleotide sequence was synthesized by Nanjing KingScript Biotechnology Co.,...
no. 2 example
[0127] The second embodiment, construction of recombinant expression vector and transformation of recombinant expression vector into Agrobacterium
[0128] 1. Construction of recombinant cloning vector DBN01-T containing ZmMYB103+G+EAR nucleotide sequence
[0129] Ligate the ZmMYB103+G+EAR nucleotide sequence into the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), and follow the instructions of the pGEM-T vector produced by Promega to obtain the recombinant cloning vector DBN01-T , its construction process is as follows figure 1 Shown (wherein, Amp represents the ampicillin resistance gene; f1 represents the replication origin of phage f1; LacZ is the LacZ start codon; SP6 is the SP6 RNA polymerase promoter; T7 is the T7 RNA polymerase promoter; ZmMYB103+G+EAR It is the nucleotide sequence of ZmMYB103+G+EAR (SEQ ID NO: 4); MCS is the multiple cloning site).
[0130] Then, the recombinant cloning vector DBN01-T was transformed into Escherichia coli T1 competent ce...
no. 3 example
[0137] The third embodiment, the acquisition and verification of maize plants transferred to the ZmMYB103+G+EAR nucleotide sequence
[0138] 1. Obtaining corn plants transferred to the ZmMYB103+G+EAR nucleotide sequence
[0139] According to the commonly used Agrobacterium infection method, the immature embryos of the aseptically cultured maize variety Zong 31 (Z31) were co-cultured with the Agrobacterium described in 3 in the second example, so that the The T-DNA in the recombinant expression vector DBN100369 (including the ZmMAC2 promoter sequence, ZmMYB103+G+EAR nucleotide sequence, the promoter sequence of the maize Ubiquitin gene, the PMI gene and the Nos terminator sequence) was transferred into the maize genome, A maize plant transformed with the ZmMYB103+G+EAR nucleotide sequence was obtained; at the same time, a wild-type maize plant was used as a control.
[0140] For Agrobacterium-mediated transformation of maize, briefly, immature embryos were isolated from maize an...
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