| Cotton is an important cash crop in China,and it is also a pioneer crop of saline-alkali tolerance.Cotton planting in saline-alkali land is an important direction of China’s cotton industrial structure adjustment.Studies have shown that seedling stage is one of the key periods in response to salt stress,but the research on the mechanism of salt tolerance in cotton seedling stage is relatively slow.In this study,iTRAQ was used to sequence the root tissues of three salt-tolerant cultivars(The relative survival rates of the three salt-tolerant varieties,ND 13,Emian 9 and Ekangmian 3,treated with 150 mmol/L NaCl solution were 56.7%,73.3%and 50.9%,respectively)and three salt-sensitive cultivars(The relative survival rates of three salt-sensitive varieties,Jinmian 12,Lumian 14 and Zhong 078,treated with 150 mmol/L NaCl solution were 34.04%,32.95%and 33.30%,respectively)treated with 150 mmol/L NaCl solution for 0 h,0.5 h and 5 h at the three-leaf stage.The related candidate salt tolerance genes were screened out,and their functions were preliminarily verified.The main findings were as follows:1.A total of 3004 proteins were identified by proteome sequencing of samples from the two cultivar groups subjected to salt stress for 0 h,0.5 h and 5 h,including 101 differentially expressed proteins.The difference analysis between the salt-tolerant group and the salt-sensitive group showed that at 0h of salt stress,5 differentially up-regulated proteins and 6 differentially down-regulated proteins were identified;In the rapid response stage,21 differentially up-regulated proteins and 13 differentially down-regulated proteins were identified;In the specific response stage,29 differentially up-regulated proteins and 35 differentially down-regulated proteins were identified.2.Six differentially expressed proteins were identified at both the rapid response stage and specific response stage,among which GhISR3,GhISR22,GhISR25 and GhISR27 were differentially up-regulated at both stages,GhISR34 and GhISR43 were differentially down-regulated in these two stages.There were ten differentially expressed proteins without functional annotations,among which GhISR46 and GhISR66 were differentially up-regulated,the eight proteins of GhISR9,GhISR10,GhISR36,GhISR43,GhISR78,GhISR85,GhISR86 and GhISR90 were differentially down-regulated.3.GO analysis of the differentially expressed proteins revealed that the main biological processes involved in the rapid response stage included metabolic process,cellular process and biological regulation;Cell components mainly include cell,cell part and organelle;Molecular functions mainly include catalytic activity and binding.The main biological processes involved in the specific response stage include metabolic process and cellular process;Cell components mainly include cell,cell part and protein-containing complex;Molecular functions mainly include binding and catalytic activities.4.KEGG analysis of differentially expressed proteins revealed that the differentially expressed proteins were mainly involved in Flavonoid biosynthesis,biosynthesis of amino acids,amino sugar and nucleotide sugar metabolism,Fructose and mannose metabolism and other pathways in the rapid response stage.In the specific response stage,the differentially expressed proteins were significantly enriched in Viral carcinogenesis,Ribosome,and Biosynthesis of amino acids.5.Two related candidate salt tolerance genes,GhISR3 and GhISR50,were selected and cloned.Among them,GhISR3 was identified in the rapid response phase and the specific response phase simultaneously,while GhISR50 was differentially expressed only in the specific response phase.VIGS(virus-induced gene silencing)was applied to GhISR3 and GhISR50 in ND 13,and the salt tolerance of the interfered plants was significantly lower than that of the control plants after salt treatment.It was preliminarily proved that GhISR3 and GhISR50 played an important role in response to salt stress at seedling stage.In conclusion,21 differentially up-regulated proteins and 13 differentially down-regulated proteins were identified in the rapid response stage in this study.In the specific response stage,29 differentially up-regulated proteins and 35 differentially down-regulated proteins were identified.The important roles of GhISR3 and GhISR50 in salt tolerance of upland cotton seedlings were preliminarily verified by VIGS technique. |
