Analysis Of Overexpression Of BetA-ZmPIS-TsVP To Improve Cotton Salt Tolerance

High salt stress could affect the growth,development,and yield of crops,and improving the salt tolerance of crops could play a significant role in the sustainable development of agriculture.The cotton(Gossypinum hirsutum)material used in this study was transfected with transgenic Escherichia coli choline dehydrogenase gene(betA),Zea mays phosphatidylinositol synthase gene(ZmPIS),Thellungiella salsuginea tonoplast H+-PPase gene(TsVP).The physiological and biochemical indicators were used to investigate the possible mechanism responsible for the improved salt tolerance of transgenic cotton,which could provide a theoretical basis for cultivating excellent salt-tolerant cotton lines.In this experiment,seed germination experiments were carried out on three lines(HN28,HN29,HN30)transfected with betA-ZmPIS-TsVP and wild-type cotton(Lu21).The germination rate of transgenic cotton under salt stress increased by 18.75%-21.38%,and the length of hypocotyl was increased by 39.89%-59.97%compared to wild-type cotton Lu21.Following the treatment with 200 mM NaCl for 4h,the water absorption rate of transgenic cotton seeds increased by 6.03%-10.91%,and the ion leakage rate decreased by 19.60%-23.90%compared to Lu21.Moreover,the seeding rate of transgenic cotton in saline-alkali soil was increased by 69.22%-76.88%,and the yield of seed cotton was increased by 9.42%-16.81%compared to Lu21.These findings indicate that transgenic cotton has better salt tolerance than wild-type cotton.In this study,transgenic cotton was treated with salt stress for 21 d,and after treatment,some physiological and biochemical parameters were evaluated.Transgenic cotton exhibited a higher net photosynthetic rate than Lu21,indicating that transgenic cotton could show enhanced photosynthetic performance even under salt stress.Furthermore,it was observed that transgenic cotton leaves had lower hydrogen peroxide contents and superoxide anion production rate compared to Lu21.Moreover,the activities of CAT,POD,APX,and SOD in transgenic cotton leaf cells were higher than in Lu21 leaf cells,indicating that transgenic cotton has better antioxidant capacity under salt stressed.The ABA content in leaves of transgenic cotton plants under potted salt stress was significantly higher than in Lu21.The expression of ABA synthesis-related genes GhNCED3(XM＿016864700.1),GhABA2(Ghir＿A05G007800),and GhAAO3(Ghir＿D05G007960)was observed to be high,while the expression of ABA degrading genes GhCYP707A(Ghir＿A05G007800)was observed low in the leaves of transgenic cotton line HN30 under salt stress than Lu21.These expression patterns show that the ABA synthesis was increased,and its degradation was decreased in the leaves of HN30 compared to wild-type cotton.Therefore,the cotton transfection with betA-ZmPIS-TsVP may improve the salt tolerance of cotton by regulating ABA synthesis under salt stress.In our study,we performed transcriptome sequencing analysis on 12 cotton samples using the DNBSEQ platform,and each sample obtained an average of about 6.39 Gb of data.Under salt stress conditions,the number of down-regulated genes in the betA-ZmPIS-TsVP transgenic cotton line HN30 was 1040,and the number of up-regulated genes was 1211 compared to wild-type cotton.Furthermore,the differential expression of genes involved in starch and sucrose metabolism,glyceride metabolism,MAPK signaling pathway-plant,ABC transporter,fatty acid elongation,and other pathways in KEGG Pathway enrichment was observed under salt stress conditions.These pathways are responsible for the metabolism of carbohydrates,lipids,amino acids,and MAPK signal transduction in cotton cells.The expression levels of GhPYR/PRL(Gh＿D01G1269),GhPP2C(Gh＿D09G1743),GhMAPK6(BGI＿novel＿G001403),and other genes play an essential role in MAPK signal transduction pathway was observed to be changed in HN30 transgenic cotton line under salt stress conditions.Particularly the expression of GhPP2C(Gh＿D09G1743),and GhMAPK6(BGI＿novel＿G001403)was observed to be increased.Given these findings,it can be speculated that the MAPK signal pathway may be related to the improvement of salt tolerance of transgenic cotton lines.In conclusion,the findings of our study showed that transgenic cotton lines exhibited a high seedling rate,better cotton seed yield,enhanced photosynthetic performance,and increased ability to scavenge reactive oxygen species under salt stress conditions.The heterologous expression of betA-ZmPIS-TsVP contributed to improving the salt tolerance of transgenic cotton.betA,ZmPIS,and TsVP facilitated increased ABA synthesis,and the MAPK signaling pathway’s reregulation might contribute to transgenic cotton’s enhanced ability to response salt stress conditions.