Screening And Identification Of Interaction Partner Of Sucrose Transporter ShSUT4 In Sugarcane

Sucrose is the main product of sugarcane photosynthesis,and it is also the main form of photosynthate transport,distribution and storage in sugarcane.When sucrose enters the storage parenchyma vacuole for accumulation,sucrose transporter is needed to control the sucrose concentration in vacuole,while SUT4 sucrose transporter on vacuole membrane plays an important regulating role for sucrose accumulation and directly decides the economic value of sugarcane.Now,how does sucrose transporter in sucrose regulate the accumulation mechanism of sucrose remains unknown.On basis of cloning sucrose transporter gene ShSUT4,yeast two-hybrid technology and bimolecular fluorescence complementation(Bi FC)technology were used to study the interacting factors of ShSUT4 in this paper.The research results are described as follows:(1)Split Ubiquitin System(SUS)was used to filter the interacting proteins of ShSUT4.80 positive clones were filtered,and 18 interacting proteins were obtained after sequencing analysis,including 3 photosynthesis related proteins,3 transport proteins,4 transcription factors and other proteins.Photosystem II subunit R(Psb R),RF2 a transcription factor,copper transporter COPT5.1-like,phosphate transporter PHT and other proteins were selected for one-to-one verification.The results showed that ShSUT4 interacted withPsbR,RF2 a,COPT5.1-like and PHT,ShSUT4 itself didn’t form dimer,and high concentration sucrose was more conducive to interaction than low concentration sucrose.Bi FC was used to further prove that ShSUT4 interacted with ShPsbR,ShRF2 a,ShCOPT5.1-like and ShPHT on epidermis cell membrane of tobacco.(2)The gene editing carrier ShPsbR-Cas9 was built.Genetic transformation of sugarcane callus was done after transformation of agrobacterium strain EHA105.After recovery,filtering,differentiation,rooting and other steps,45 positive plants were obtained through Bar gene detection and gene editing carrier sg RNA expression cassette detection.(3)According to expression analysis for different parts of sugarcane,ShPsbR showed the highest expression quantity in-1 leaves of ROC22,the expression quantity in +1 leaves came second,and the expression quantity in stem and root was the lowest,which indicated that ShPsbR mainly existed in photosynthetic chlorenchyma of sugarcane;under conditions of both drought and salt stress,ShPsbR gene showed up-regulated expression and showed significant up-regulated expression at 12 h of stress treatment.It indicated that ShPsbR could affect synthesis and transport of sugar and regulate the osmotic potential of cells by regulating leaf photosynthesis,thus responding to drought and salt stress.(4)The expression quantities of ShPsbR in +1 leaves and-1 leaves of ShSUT4-RNAi plant(the sucrose content of sugarcane can be reduced by inhibiting ShSUT4 expression)significantly reduced compared with CK,and the expression quantities in +1 leaves and-1leaves of ShSUT4 overexpression plant(overexpression ShSUT4 could cause sucrose content rise of sugarcane)showed no significant variation,so we speculated that ShPsbR expression variation could change the rate of sucrose synthesis through leaf photosynthesis,thus affecting sucrose transport efficiency of ShSUT4 and maintaining the sucrose homeostasis in cells.