The zmglk44 gene regulating water use efficiency in maize under drought and its application
A corn and gene technology, applied in application, genetic engineering, plant genetic improvement and other directions, to achieve the effects of improving drought resistance, drought resistance phenotype, and drought resistance physiological indicators
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Embodiment 1
[0035] The acquisition of the ZMGLK44 gene that regulates corn drought is efficient
[0036] According to the CDNA sequence of ZMGLK44, the PCR amplification is designed to design a pair of homologous recombinant primers. The primer sequence is as follows:
[0037] Zmglk44-OE-F: 5’-GCACTAGTAGTCCCCCCCGGGGCGCGCCCCATGGGGGGGACGTCGG-3 '; Zmglk44-OE-R: 5’-cGTCGTATGGGTACATGCCCACGTTTTCCCTGTGTAGCC-3 '.
[0038] The above PCR amplification uses Vazyme Phanta Max Super-Fidelity DNA Polymerase for genes amplification. The reaction system is as follows:
[0039] 2X Phanta Buffer 10ul dntp 0.4ul Zmglk44-OE-F 0.8ul Zmglk44-OE-R 0.8ul PHANTA MAX 0.4ul CDNA 100ng DDH 2 O
7.1ul
[0040] The reaction program is: 95 ° C 3min; 95 ° C 15s; 60 ° C 15s; 72 ° C 60s; 72 ° C 5min; 35 cycle, cycle,
[0041] That is, the PCR product is obtained. After testing, the PCR product sequence is shown in SEQ ID No.1, which is shown in SEQ IDNO.1, and it...
Embodiment 2
[0043] 1.zz0153-RD101P-3HA carrier Construction
[0044] 1) The corn B73 self-intersection drought-induced expression gene ZMRD101 promoter sequence such as SEQ IDNO.1 is shown as a template design primer pair, as follows: RD101-F: 5’-CCTGTCAACACTGata GTTT TTCACTTTTTTTTTTTTCTGGCAAT-3 ', RD101-R: 5’-CCGGGATACTAGC Gtttaaac Gctcacgttgccttg-3 (the lower line is PMEI enzyme cutting point);
[0045] 2) Interpretation of genes ZMRD101 sequence with corn drought is used as templates for PCR, gene PCR products, sequencing its nucleotide sequence is shown in SEQ ID No.3; its PCR amplification conditions are as follows:
[0046] reaction system:
[0047] 2X Phanta Buffer 10ul dntp 0.4ul Positive primer RD101-F 0.8ul Reverse primer RD101-R 0.8ul PHANTA MAX 0.4ul CDNA 1.5ul DDH 2 O
6.9ul
[0048] PCR reaction conditions: 95 ℃ prestigious 3min; 95 ℃ degeneration 30SEC; 60 ° C annealing 30SEC; 72 ° C extension of 75SEC; after 35 times; 72 ° C extensi...
Embodiment 3
[0068] Example 3 GM corn plant system obtained
[0069] 1. Example 2 income carriers are sent to Jiangsu Weimi Biotechnology Co., Ltd. for transformation.
[0070] 2. Screen T1 -positive plants by applying herbicides and harvest seeds, and cultivate T2 generations;
[0071] 3. Continue to detect the PCR test of the T2-generation plant to determine that the destination gene has not occurred without genetic separation and loss, and finally obtained two positive pure convergence genetic corn homes and corresponding negative separation materials containing a stable and genetic inheritance of ZMGLK44; / Zmglk44-OE-R).
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