Corn (zea mays) derived growth period related protein ZmHUB2 and coding gene and application thereof
A growth period, corn technology, applied in the field of protein and its coding gene and application, can solve the problems of low temperature, affecting yield, short sunshine time, etc.
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Embodiment 1
[0079] Example 1, the acquisition of ZmHUB2 and its coding gene
[0080] ZmHUB2 is obtained from maize inbred phylogeny 31 by RACE technology (Yang Hui, Wang Guoying, Dai Jingrui. Research on transformation of maize excellent inbred phylogeny 3 and ensemble 31. Journal of Agricultural Biotechnology 2001, 9 (4) 334 -337, publicly available from China Agricultural University) to clone a gene associated with reproductive period. The full-length cDNA of this gene can be amplified by PCR using the following primers and using the cDNA obtained by reverse transcription of the total RNA of maize inbred 31 as a template: ZmHUB2__5': 5'-ATG GAT TCC ACA GCT CTT CAA TAT G- 3'; ZmHUB2 3': 5'-TCA TAT CTT CAC CTC CCT AAC GTC G-3'). The electrophoresis results of the PCR products were as follows: figure 1 shown. The amplified products were recovered and connected to pMD18-T simple Vector (Takara) respectively, and transformed into Escherichia coli DH5α. Positive clones were screened throug...
Embodiment 2
[0081] Example 2, C of ZmHUB2 3 HC 4 Zinc finger protein domain polypeptide ZmHUB2E has the function of ubiquitin ligase
[0082] The 2311-2538th position of sequence 1 was amplified by PCR with the primers F-5'-AAGAATTCGAGACTGAAGAGACTAC-3' and R-5'-TTGTCGACTCATATCTTCACCTCCCT-3' using the cDNA obtained by reverse transcription of the total RNA of maize inbred lineage 31 as a template (ZmHUB2E shown in the 754-828 positions of coding sequence 2), EcoRI and SalI restriction sites were added to the two ends of ZmHUB2E respectively, the PCR product was connected to the pMD18-Tsimple vector, and sent to the company for sequencing.
[0083] reaction system:
[0084]
[0085] The PCR reaction program was: first round: denaturation at 95°C for 5 min, second round: denaturation at 95°C for 50 sec, annealing at 56°C for 50 sec, extension at 72°C for 50 sec, 30 cycles, third round: extension at 72°C for 10 min. After the reaction, 1.0% agarose gel electrophoresis detection.
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Embodiment 3
[0090] Example 3. Inhibiting the expression of ZmHUB2 in maize and shortening the growth period of transgenic maize
[0091] 1. Construction of pTCK303-ZmHUB2 interference vector
[0092] The 2172-2538th nucleotides of sequence 1 were amplified using the cDNA obtained by reverse transcription of the total RNA of maize inbred 31 as a template, and 5'-GGGGTACCACTAGTATCCTCTGAGAAAGAATACG-3' and 5'-GGGGATCCGAGCTCTCATATCTTCACCTCCCTAAC-3' as primers, Two pairs of restriction sites, KpnI / SpeI and SacI / BamHI, were added to the two ends of the C-terminal domain of the ZmHUB2 gene by PCR amplification, and the PCR product was connected to the pMD18-Tsimple vector and sent to the company for sequencing. Use KpnI / BamHI and SpeI / SacI to recover the bands of about 395 bp in the clones with correct sequencing respectively. The interference fragment with the KpnI / BamHI restriction site was first combined with the interference vector pTCK303 (Zhen Wang, Changbin Chen, Yunyuan Xu, Rongxi Jiang,...
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