Application of ZmPHR1 protein in regulating and controlling phosphorus content of corn
A technology of phosphorus content and protein, applied in the biological field, can solve the problems of lack of grain, lack of row, poor pollination and so on
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Embodiment 1
[0071] Example 1, the discovery of the ZmPHR1 protein and its coding gene (i.e., ZmPHR1 gene).
[0072] 1. The total RNA of maize inbred line B73 was extracted by TRizol method. The integrity of the total RNA of maize inbred line B73 was checked by formaldehyde-denaturing RNA agarose gel electrophoresis.
[0073] 2. Take the total RNA of the corn inbred line B73, according to SUPERSCRIPT II Instructions for use (Thermo Fisher Scientific products) synthetic corn inbred B73 single-stranded cDNA.
[0074] 3. Take the single-stranded cDNA of maize inbred B73, dilute it 10 times with water and use it as a template, and use Primer 1:5'-tataagcttATGAGGAACTTTAATCTGATGCAGT-3' and Primer 2:5'-gctctagaTTAACTTTTGCGCGC-3' primer pairs for PCR amplification, and recover pcR amplification products of about 1370bp.
[0075] The reaction system is 50 μL, consisting of 10 μL 5×Phusion HF Buffer, 4 μL 2.5 mM dNTP mix, 2.5 μL Primemer 1 aqueous solution (concentration 10 μM), 2.5 μL Primer 2 aqueous ...
Embodiment 2
[0080] Example 2, T 3 Acquisition and phenotypic identification of ZmPHR1 gene corn by generation homozygous
[0081] First, the construction of recombinant plasmid UBI: ZmPHR1
[0082] 1. Using the recombinant plasmid pMD18-ZmPHR1 as a template, the primer pair composed of primer 1:5'-ATGAGGAACTTTAATCTGATGCAGT-3' and primer 2:5'-TTAACTATCTTGCTTGCGC-3' was used to pcR amplify the PCR amplification product.
[0083] 2. Take the PCR amplification product obtained in step 1, use Taq enzyme to supplement A at the flat end, and obtain the PCR amplification product with A sticky end.
[0084] 3. Linearized vector pCXUN was obtained by using the restriction enzyme XcmI (NEB company) to digest the vector pCXUN (GenBank: FJ905215.1). The linearized carrier pCXUN has a T-stick end.
[0085] 4. By TA cloning method, the PCR amplification product with A sticky end and the linearized carrier pCXUN are connected to obtain the recombinant plasmid UBI:ZmPHR1.
[0086]Sequence the recombinant plas...
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