Verification And Analysis Of Interacting Protein Of Sugarcane Sucrose Transporter ShSUT4

The vacuoles in cane stem parenchyma cells are the main storage places for sucrose.The sucrose transporters on the vacuole membrane are the carriers of sucrose entering and leaving the vacuoles,and play an important role in regulating the accumulation of sucrose in the vacuoles.However,the effect of sucrose transporters on sucrose accumulation in sugarcane vacuolar membrane is still a problem to be solved.Studying the molecular regulation mechanism of sucrose transporters in sugarcane vacuolar membrane is an effective way to solve this problem.This study focused on screening and validation of the interacting protein of the vacuolar sucrose transporter Sh SUT4.The ubiquitin yeast two-hybridization technique was used to screen the interacting proteins.The interacting proteins were further verified by Bi FC.Gene expression analysis and transgenic studies were conducted on the coding genes of their validation interacting proteins.The main research results are as follows:1.85 positive clones were screened by Split Ubiquitin System,and 35 proteins were obtained.It contains zinc finger A20 and AN1 domain stress related proteins,ATPase family AAA domain related proteins,thylakoid related proteins,cytochrome related proteins,60 S ribosomal protein-related proteins,ribosomal proteins of S5 P family 40 S ribosomal proteins,histones,abscisic acid stress maturation related proteins.2.The decoy protein ShSUT4 and interacting protein were fused with the C-terminal fragment and N-terminal fragment of green fluorescent protein(GFP),respectively,to construct BiFC vector,and the tobacco BiFC experiment was carried out.The results showed that Sh SUT4 could interact with Cyb6-f protein and ShASR2 protein,and the interaction sites were both in the cell membrane.Further mapping of rice protoplasts showed that ShASR2 localized the nucleus and cytoplasm,indicating that Sh SUT4 and ShASR2 interacted to transfer the expression of ShASR2 from the cytoplasm to the cell membrane.3.qRT-PCR was applied to the expression of ShASR2 under drought and salt stress.After 20%PEG6000 treatment,the expression of ShASR2 decreased at the initial stage,increased at 12 h,and was 5 times higher than that of the control at 24 h.Under 250mmol/L Na Cl salt stress,the expression of ShASR2 decreased,increased and decreased,indicating that ShASR2 responds to drought and salt stress,and it is presumed that Shas R2 regulates cell sugar content and thus affects cell osmosis.4.Further analysis of ShASR2 gene expression in leaves and stem nodes of RNAi plants with high glycemic overexpression and low glycemic RNAi plants showed that ShASR2 gene expression was up-regulated in OE plants(leaves and stem nodes)and down-regulated in RNAi plants,indicating a positive regulatory relationship between Sh SUT4 and ShASR2.It can control sucrose content in sugarcane stalk cooperatively.5.The ShASR2 gene editing vector ASR2-ShCAS9 was constructed for the genetic transformation of sugarcane,and 30 resistant seedlings were obtained.Finally,10 positive transgenic plants were tested for gene editing sites.