Agrobacterium-Mediated Genetic Transformation Of Salt Tolerance Related CmP5Cs Gene In Vegetable Soybean

Vegetable soybean [Glycine max (L.) Merr.] is popular with consumers for its rich nutrition. With the expansion of market demand, vegetable soybean cultivation area is increasing rapidly. But the yield and quality of vegetable soybean have been seriously affected by the constant exacerbation of soil salinization and secondary salinization. In this study, we have cloned a △1-pyrroline-5-carboxylate synthetase gene (P5CS) from salt tolerate pumpkin rootstock, and have transferred the gene into vegetable soybean. The main results were as follows:1. The open reading frame sequences of CmP5CS gene was obtained by homologous cloning and RT-PCR from’Shintosa Supreme’ pumpkin (Cucurbita maxima x C. moschata). Sequence analysis showed that the coding sequences of this gene is 2,154bp, encoding 717 amino acids. CmP5CS shares high nucleotide sequences identity with the P5CS from Cucumis melo (92.34%) Its GenBank accession number is JN606859. The deduced protein shared conserved y-glutamyl kinase (y-GK) and y-glutamate semialdehyde (GSA) domains, putative ATP and NADPH-binding sites and two Leu-rich regions with other plant P5CS proteins.2. Site-directed mutagenesis by overlap extension PCR was used to remove the SacI and XbaI restriction sites in CmP5CS sequence. The target fragment was inserted between CaMV35S promoter and NOS terminator of the plant expression vector pCMBIA3301 after confirmed by restriction enzyme and sequencing. Then the herbicide resistant vector pCMBIA3301-CmP5CSm was transformed into Agrobacterium LBA4404.3. Effects of genotype, basal medium, combinations of plant growth regulators and other factors on shoot regeneration from cotyledonary nodes of vegetable soybean were studied. The result showed that, the shoot regeneration frequency of the six genotypes ranged from 53.5% to 88.9% and three of them reached 88.9%,87.5% and 83.3% respectively on B5 medium supplemented with 1 mg·L-1 TDZ,0.05 mg·L-1 NAA, and 5 mg·L-1 AgNO3. This study established highly efficient shoot regeneration from cotyledonary nodes of vegetable soybean.4. The effect of genotype, co-cultivation conditions, selective pressure and other factors on genetic transformation efficiency of vegetable soybean were studied. The result showed that the rate of transient expression of GUS gene of ’NY-1001’was Significantly higher than other 15 cultivars. Orthogonal test showed the best co-cultivation condition for Agrobacterium-medidited transformation of ’NY-1001’was:pH5.4,3 mM L-Cysteine (L-Cys) and 200 μM Acetosyringone (AS) in medium of co-cultivation. The influencing extent of the three factors in co-cultivation period was in the following order:L-Cys, pH-value, AS. The concentration of bialaphos for selection was 4 mg-L"1, and the selection began after one week after co-cultivation. The resistant shoots were transferred to shoot elongation medium containing 0.5 mg·L-1 GA3 for shoot elongation, and then rooted in rooting medium containing 1 mg·L-1 IBA. Finally, we obtained 8 transgenic plants which have shown resistance to Basta. The average transformation rate was 1.06%. PCR and southern blot have confirmed that CmP5CS gene has been integrated into the vegetable soybean genome.