Soybean folic acid synthesis key enzyme ADCS and gene and application thereof
A key enzyme gene, key enzyme technology, applied in soybean folic acid synthesis key enzyme ADCS and its gene and application field
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Embodiment 1
[0166] Cloning and sequence analysis of embodiment 1, GmADCS gene
[0167] 1. Obtain the EST sequence of soybean ADCS gene
[0168] The CDS sequence (AtADCS) of the Arabidopsis ADCS gene (AtADCS), with a size of 2760bp, was compared with the EST sequence of soybean through NCBI. There were 8 soybean EST sequences, which were highly consistent with the 2050-2714bp of AtADCS, and were named 3 respectively. 'EST1-8, an EST sequence with a size of 281bp, which is highly consistent with AtADCS's 1345-1592bp, named mEST. And the 8 sequences of 3'EST1-8 were compared and found that there were only differences in individual bases and sequence lengths between them. After analysis, the downstream primer 3 was designed using the 3'EST5 terminal sequence with high homology as a template 'EST5-R1 / R2, using the mEST sequence as a template to design the upstream primer mESTF1 (see Table 1 for the primer sequence).
[0169] The sequences of 3'EST1, 3'EST2, 3'EST3, 3'EST4, 3'EST5, 3'EST6, 3'...
Embodiment 2
[0181] Embodiment 2, the expression pattern of GmADCS gene in soybean
[0182] The transcription level of GmADCS gene in different tissues and organs of soybean was analyzed by RT-PCR. Soybean roots, stems, and leaves grown for 8 weeks were taken, and total RNA was extracted by the Trizol method, reverse-transcribed into cDNA as a template, and GmActin-specific primers GmActin11F, GmActin11R (primer See Table 2 for the sequence), design gene-specific primers GmADCS-FSP and GmADCS-RSP (see Table 2 for the primer sequences) according to the CDS sequence of the GmADCS gene, and the results show that the gene can be transcribed in roots, stems and leaves, wherein, Expression levels are highest in leaves and weaker in roots and stems (e.g. figure 2 shown).
[0183] Table 2. Gene-specific primers for GmACTIN and GmADCS
[0184] Primer name
Embodiment 3
[0185] Example 3, the acquisition of transgenic GmADCS gene Arabidopsis and the determination of its folic acid synthesis precursor pABA content
[0186] The CDS sequence of GmADCS was constructed between the restriction sites NcoI and Pml I of the plant overexpression vector pCAMBIA1302, and the recombinant expression vector pCAMBIA1302-GmADCS was obtained driven by the cauliflower mosaic virus (CaMV) 35S promoter.
[0187] The above-mentioned recombinant vector was transformed into Agrobacterium LBA4404 by electroporation, and the correct clones identified by enzyme digestion were picked, and the wild-type Arabidopsis was infected by the Agrobacterium-mediated flower dipping method, and the harvested seeds were recorded as the T0 generation. The seeds of the T0 generation were first screened on the resistance medium of 1 / 2MS+hygromycin B (25mg / L), and 13 positive green seedlings (referred to as the T1 generation) were obtained, and moved into the greenhouse for 20 days, and t...
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