Identification And Preliminary Functional Study Of BeSWEETs Gene In Bambusa Emeiensis

As the main form of the existence and transportation of photochemical products in plants,sucrose plays an important role in the growth and development of plants and the response of plants to stress.SWEET protein is a new type of sugar transporter,which cooperates with SUT sucrose transporter to participate in the transportation of sucrose from source to sink.Bambusa emeiensis is an important economical bamboo species in Southwest China.There are no reports about the SWEET gene of Bambusa emeiensis.In this study,based on the SWEET gene sequence of other species and transcriptome data of Bambusa emeiensis,the bioinformatics and subcellular localization analysis of the selected SWEET genes were performed on the selected SWEET genes;and through bimolecular fluorescence complementation experiments and genetic transformation of wheat,it was found that the Be SWEET4-2,Be SWEET11-3,and Be SWEET1A-2proteins of Bambusa emeiensis can interact in the form of self-multimerization or pairwise multimerization;the heterologous overexpression of Be SWEET11-3 can delay the growth period of wheat,increase biomass and improve the filling degree of wheat,and improve the photosynthetic capacity of wheat.The main results are as following:（1）Nine Be SWEET genes sequences were screened from Bambusa emeiensis transcriptome database.Bioinformatics analysis shows that these Be SWEET genes contain Mt N3 conserved domains unique to the SWEET family,which are clustered into four branches,which may be involved in the transport of hexose,fructose and sucrose,respectively.（2）Three of the SWEET genes were cloned and named Be SWEET4-2,Be SWEET11-3,and Be SWEET1A-2,encoding 256,294,and 222 amino acids,respectively.The tissue expression and induced expression analysis were carried out by q RT-PCR.The results showed that Be SWEET4-2,Be SWEET11-3,and Be SWEET1A-2 were highly expressed in roots,stems,and leaf veins,respectively;under the conditions of exogenous addition of 2% fructose,glucose,and sucrose,Be SWEET4-2 and Be SWEET1A-2 responded to hexose significantly,while Be SWEET11-3 has a significant response to sucrose,indicating that the three genes may have functional differences.（3）Subcellular localization analysis showed that the three Be SWEET proteins were all located on the plasma membrane.Bimolecular fluorescence complementation experiments found that there is protein interaction between Be SWEET proteins.Among them,Be SWEET4-2 and Be SWEET1A-2 proteins form a protein complex through self-polymerization,and Be SWEET11-3 and Be SWEET1A-2 proteins form a protein complex through mutual binding.Using the Saccharomyces cerevisiae EBY.VW4000 mutant,a yeast complementary growth experiment of Be SWEET1A-2 protein was carried out,and it was found that Be SWEET1A-2protein has the ability to transport glucose,fructose,and galactose,and the ability to transport fructose is weaker than that of glucose and galactose.（4）An overexpression vector of Be SWEET11-3 was constructed,and wheat callus was transformed by gene gun,and 11 PCR-positive transgenic wheat plants were obtained.The phenotypic analysis of three T1 overexpression lines（OE11-3,OE12-1 and OE12-2）found that the overexpression of Be SWEET11-3 significantly increased the net photosynthetic rate and grain size of the leaves,which consistent with the results that rice grain size was promoted by the overexpression of Os SWEET4,suggesting that Be SWEET11-3 promotes the transport of nutrients to the endosperm during wheat filling.