Genetic Transformation Of StP5CS Gene Into Vegetable Soybeans And Obtaining The Salt-tolerant Germplasm

Vegetable soybean[Glycine max?L.?Merrill],also named as 'mao dou' in Chinese,is produced mainly in lower-middle reaches of the Yangtze River and southeast coastal areas of China and has been consumed mainly as a vegetable.As it is rich in protein,unsaturated fatty acid,vitamins,minerals,fibers and isoflavones,vegetable soybean has become highly popular in the domestic and overseas markets.Vegetable soybean is also one of the important exported vegetables and its production area is continued to expand,owing to the increasing market demand and relatively high profits.However,the yield and quality of vegetable soybean have been seriously affected by the soil salinization and secondary salinization which are aggravated by the poor agricultural practices such as improper irrigation and over-fertilization.To improve the salt tolerance of vegetable soybean,a novel P5CS gene was isolated from salt-tolerant Solanum torvum Swartz and then was introduced into vegetable soybean cultivar NY 1001 through an Agrobacterium-mediated.transformation method.Fertile transgenic plants were generated successfully and the T₂ and T₃ homozygous transgenic lines were tested for salt tolerance.The main results are listed as follows:1.A novel P5CS gene,designated StP5CS?GenBank accession number:JN606861?was isolated and characterized from leaves of Solanum torvum Swartz cv.Torvum Vigor.Sequence analysis showed that the StP5CS was 2232 bp in length,consisting of a 42 bp 5'untranslated region,a 2154 bp complete open reading frame encoding a polypeptide of 717 amino acids and a 36 bp 3' untranslated region.The native molecular weight of the deduced protein was estimated to be 77.87 kDa and the isoelectric point was estimated to be 6.26.StP5CS shared 94%,77%,75%,74%and 74%homology in nucleotide sequence with the P5CS of tomato?tomPR02?,soybean?GmP5CS?,moth bean?VaP5CS?and Arabidopsis thaliana?AtP5CSl,AtP5CS?,respectively.Six highly conserved characteristic domains including y-GK domain,GSA-DH domain,putative ATP and NAD?P?H-binding sites,and two Leu-rich regions were identified in StP5CS,indicating StP5CS may plays the similar functional roles as other P5CS proteins in proline biosynthesis.Moreover,molecular evolutionary genetics analysis revealed that StP5CS was most closely to tomPR02 and NtP5CS.2.A binary vector pCAMBIA3301-T800-StP5CS which carried a bar gene conferring bialaphos resistance,an intron-gus reporter gene encoding?-glucuronidase and a StP5CS gene for proline synthesis was successfully constructed.The intermediate vector pCAMBIA3301-T800 was first generated.To this intermediate vector,the CaMV35S promoter and the NOS terminator was added between the EcoRI and Hind?restriction enzyme sites of pCAMBIA3301.The StP5CS flanked with BamHl and SalI restriction enzyme sites was then inserted between the CaMV35S promoter and the NOS terminator region of the intermediate vector to generate a pCAMBIA3301-T800-StP5CS.The resulting recombinant construct which was confirmed by restriction enzyme digestion and sequencing was transformed into A.tumefaciens strain EHA105 through the freeze-thaw method.3.Two independent T₂ homogyzous StP5CS-transgenic vegetable soybean lines?coded Z1-3 and Z2-7?were successfully generated through the Agrobacterium-mediated transgenic process.The cotyledonary-node explants were infected with EHA105 harboring the recombinant StP5CS expression vector.After 4 days of co-cultivation and shoot induction with 4 mg·L^-1 bialaphos,a total of four GUS positive shoots were identified through a half-leaf GUS assay method in the shoot elongation stage.After rooting and acclimatization,two independent fertile transgenic T₀ plants which were confirmed by PCR and Southern blot analyses were eventually generated.Herbicide painting assay verified that these two transgenic plants highly tolerant to herbicide Basta.Chi-square??2?analysis revealed that the transgenic plants transmitted the transgenes into their progenies in a Mendelian segregation pattern with a 3:1 ratio.Two T₂ homogyzous transgenic lines?HTLs?were successfully generated by PCR analysis and used for further evaluation of salt-tolerance.4.The HTLs were subjected to salt tolerance ability investigation over two generations?T₂ and T₃?and results showed that overexpression of StP5CS led to enhanced salt tolerance in transgenic vegetable soybeans.The T₂ and T₃ HTLs were examined for salt tolerance in pot and hydroponic cultures,respectively.Under NaCl stress conditions,the leaf scorch scores of T₂ and T₃ HTLs were significantly lower than those of wild-type?WT?plants.The plant height,leaf area,relative chlorophyll content,and number of fresh pods of T₂ and T₃ HTLs were significantly higher than those of WT plants.Compared with WT plants,T₂ and T₃ HTLs had significantly higher levels of proline and significantly lower levels of membrane lipid peroxidation.Quantitative RT-PCR analysis showed that high StP5CS transcript levels were observed in the two HTLs under both NaCl stress and normal conditions,while no StP5CS transcript was detected in the WT plants,as specific primers were designed for the amplification of StP5CS.Two-way ANOVA analysis revealed that the expression levels of StP5CS were significantly influenced by NaCl treatment,line and their interaction.These results indicate that StP5CS overexpression in HTLs results in enhanced salt tolerance associated with higher levels of proline accumulation under salinity stress.The new salt-tolerant germplasm can be utilized to improve salinity tolerance in vegetable soybean breeding.