Corn drought-resistant transcription factor gene Zmhdz9 and application thereof
A technology of transcription factor and corn, applied in application, genetic engineering, plant gene improvement, etc., can solve the problem that HD-Zip gene transcription factor has not been reported, and achieve the effect of improving drought tolerance
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Embodiment 1
[0029] Cloning of Example 1 Zmhdz9 Gene
[0030] The maize inbred line H8186 was selected as the experimental material, and the three-leaf stage maize leaf tissue was extracted and reverse transcribed for future use.
[0031] The biological information of Zmhdz9 was retrieved from the corn database, and the transcript ID was GRMZM2G041462_P01. Design specific amplification primers using the gene sequence as a template, combine the multiple cloning restriction sites of the cloning vector at the same time, select the BglII and BstEII restriction sites to introduce at both ends of the Zmhdz9 sequence, and the specific amplification primer sequences are as follows:
[0032] Zmhdz9-F: 5'-CACTTTATTGTGAAGATAGTGGAAA-3'
[0033] Zmhdz9-R: 5'-CCATCTAATTCAACAAGAATTGGGACAAC-3'
[0034] Using cDNA as a template, Zmhdz9-F and Zmhdz9-R as primers, the high-fidelity enzyme Primer STAR Max Premix (2×) produced by TAKARA Company was used to perform the amplification reaction. The amplificatio...
Embodiment 2
[0040] Example 2 Bioinformatics analysis of Zmhdz9 gene
[0041] After the sequencing result was compared with the sequence in the database by MEGA6.0 software, it was consistent with the nucleotide sequence shown in SEQ ID NO.1, indicating that the Zmhdz9 gene was successfully cloned. After sequence analysis, it was found that the coding region of Zmhdz9 gene was 1408bp in length, encoding a total of 239 amino acids, with a PI of 5.39 and a MW of 26.24kD. Analysis of its protein sequence revealed that the protein encoded by the Zmhdz9 gene contained two prominent conserved domains, the domain and the domain connected to it, and the domain contained multiple repeated leucine residues. It was predicted that the Zmhdz9 gene Belongs to the HD-Zip I family of transcription factors. The members of the maize HD-ZipI family whose functions have been reported and verified were selected, and the gene members with high homology were screened, and the protein sequences of these gene mem...
Embodiment 3
[0042] Example 3 Construction of Zmhdz9 gene overexpression vector
[0043] According to the multiple cloning site of pCAMBIA3301 and the sequence characteristics of the target gene, BglII and BstEII restriction sites were added to both ends of the target gene Zmhdz9, and the positive plasmid obtained by cloning was used as a template for amplification to obtain PCR amplification fragments. The amplification primer sequences are as follows:
[0044] Zmhdz9-F: 5'-actcttgaccatggtagatctCACTTTATTGTGAAGATAGTGGAAA-3'
[0045] Zmhdz9-R: 5'-ggggaaattcgagctggtcaccCCATCTAATTCAACAAGAATTGGGACAAC-3'
[0046] Transform the recombinant plasmid into Escherichia coli competent cells, shake the bacteria for sequencing and save, extract the plasmid, and use BglII and BstEII to double-enzyme digest the plasmid to obtain a small target fragment. The connection system is as follows:
[0047] Drug Name Dosage (μl) Target gene fragment 6 Pcambia3301 large fragment 2 10...
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