Preliminary Analysis On The Function Of 7sugar Transporter (SWEET) Genes In Jasmine

Jasmine[Jasminum sambac(L.)Ait] is a genus of the Oleaceae.Its flowers are open at night,and it is a typical nocturnal flower.Jasmine flowers are widely used as raw material for scented tea and perfume industry.During bloom,the synthesis of volatiles,opening of petals and pollination in the late flowering stage are all energy-consuming,so that the sugar supply is one of the important factors that determine the development of jasmine.SWEET belong to a group of sugar transporter involved in the transport and distribution of plant photosynthesis products.In addition,SWEET proteins are also involved in important physiological processes such as pollen development,nectar secretion and stress response.Previously seven unigenes from JsSWEET family specifically expressed in flowers were identified and cloned via transcriptome analysis.These transporters may play important roles in the flowers of jasmine.In this study,the full-length cDNA sequence of JsSWEET gene was obtained from T vector containing 7 JsSWEET family genes obtained in the early stage of the experiment.The expression level of 7 JsSWEET family gene members before and after the opening of jasmine was studied by fluorescence quantitative PCR.Construction of Evolutionary Tree for protein sequences.The plant expression vectors of 7 genes were constructed by Gateway fusion technique,and the vectors were transiently transformed into jasmine protoplasts to determine the localization of JsSWEET protein.The localization of JsSWEET protein was determined.PK-JsSWEET9,PK-JsSWEET5,PK-JsSWEET1 plant expression vector was selected and transformed into Petunia by leaf disk method.The transgenic plants were obtained successfully.The yeast expression vector was constructed and the sugar transport function complementation test was carried out in the yeast expression system to verify the sugars transported by the seven members.The main research contents and results are as follows:1.In order to explore the phylogenetic relationship between SWEET genes in jasmine flowers,the SWEET protein sequences of model plant Arabidopsis thaliana and monocotyledonous rice were obtained by NCBI search,and the evolutionary trees were analyzed and drawn by MUSCLE and MEGA software.Samples of jasmine flowers were collected at 14:00h(before bract),17:00 h(bract stage),23:00 h(semi-open)and 5:00 h(full blooming).The phylogenetic tree analysis and mapping were performed using MUSCLE and MEGA software.Samples of Jasmine flowers were taken at 14:00 h(before the flowerbed),17:00 h(flowering period),23:00 h(semi-open)and 5:00 h(full open).Total RNA of Jasmine flower was extracted from four time periods,and the first chain of DNA was synthesized by reverse transcription.Seven JsSWEET genes were detected by real-time fluorescence quantitative PCR.The results showed that JsSWEET1,JsSWEET2,JsSWEET5,JsSWEET9,JsSWEET10 genes expressed the highest at 23:00 h,JsSWEET16 gene expressed the highest at 17:00 h and JsSWEET17 gene expressed the highest at 14:00 h.2.The binary vector of plant expression was constructed.The cDNA sequences of the JsSWEET1,JsSWEET2,JsSWEET5,JsSWEET9,JsSWEET10,JsSWEET16 and JsSWEET17 genes were subcloned from the T vectors containing the JsSWEET family gene obtained in the previous stage.PK7FWG2 was selected as the backbone vector.Seven jasmine PK-JsSWEET plant expression vectors with C-terminal GFP fluorescent label were constructed by Gateway fusion technology,named PK-JsSWEET1,PK-JsSWEET2,PK-JsSWEET5,PK-JsSWEET9,PK-JsSWEET10,PK-JsSWEET16,PK-JsSWEET17.3.By means of protoplast transformation,seven JsSWEET transporters were expressed in the protoplasts of jasmine.The results showed that JsSWEET5 and JsSWEET9 transporters were localized at the plasma membrane of the protoplasts.JsSWEET10 and JsSWEET2 transporter also mainly distributed in plasma membrane,but also in some organelles of cytoplasm.The subcellular localization of JsSWEET16,JsSWEET1 and JsSWEET17 transporter was similar and the fluorescence distribution in the cytoplasmic intimal system was more obvious than thatin the plasma membrane,but it was different from the cytoplasmic fluorescence expressed by GFP alone.4.The PK-JsSWEET1,PK-JsSWEET5,PK-JsSWEET9 and PK-JsSWEET17 vectors were used to transform petunia(W115)using Agrobacterium tumefaciens-mediated leaf disc method.After screening kanamycine resistant transgenes(T0),6 transgenic JsSWEET1 plants,2transgenic JsSWEET5 plants,47 transgenic JsSWEET9 plants and 3transgenic JsSWEET17 plants were obtained.Preliminary phenotypic observation showed that the plants over-expressing JsSWEET9 showed that the plants were dwarf,the leaves turned yellow,and the Corolla of some plants showed extra whisker or petal shape,while those infected by JsSWEET5 Agrobacterium tumefaciens showed that the plants were short,some of them were narrow and long,and the flowers became smaller.The plants infected by Agrobacterium tumefaciens JsSWEET1 and JsSWEET17 were to be observed.5.The seven JsSWEET yeast expression vectors pRS-JsSWEET1,pRS-JsSWEET2,pRS-JsSWEET5,pRS-JsSWEET9,pRS-JsSWEET10,pRS-JsSWEET16,pRS-JsSWEET17 and pRS426 control plasmids were transformed into Saccharomyces cerevisiae EBYVW4000,respectively.After selection in SD(-ura)medium with maltose as substrate,the yeast solution of different genes was dripped into different sugar substrate SD(-ura)media respectively.The results showed that pRS-JsSWEET5 transgenic yeast could grow on SD(-ura)medium containing maltose,glucose,fructose,mannose,sucrose or galactose.However,it could not grow on SD(-ura)medium with deoxyribose and maltose as common substrates.pRS-JsSWEET1 grew well on SD(-ura)medium with maltose and galactose as substrates.pRS-JsSWEET1,pRS-JsSWEET2,pRS-JsSWEET9,pRS-JsSWEET10,pRS-JsSWEET16,pRS-JsSWEET17 and pRS426 transgenic yeast can only grow on maltose medium and maltose and deoxyribose common substrate medium.These studies suggest that JsSWEET5 transporter may transport glucose,fructose,mannose,sucrose,galactose and deoxyribose;JsSWEET1 transporter may transport galactose;The transport properties of JsSWEET2,JsSWEET9,JsSWEET10,JsSWEET16,JsSWEET17 transporter need tobe further confirmed,which may play a role in organelle.