Cloning Chorispora Bungeana Sucrose Transporter Gene CB7399 And Exploring Its Physiological Function

Chorispora bungeana is a perennial cruciferae species living in high altitude periglacial area.The unique growth environment endows them distinct characters of growth and developme-nt,such as:unique morphology feature and diffrent antifreeze mechanism and so on. This study focuses on these special physiological phenomenons.Cloned sucrose transporter gene CB7399 from Chorispora bungeana which homologous AtSUT4 from Arabidopsis thaliana.and confirmed expression of CB7399 rises obviously under the cold condition.To further reveal some physiologic function for the encoding proteins of CB7399,The CB7399 is overexpressed in Arabidopsis thaliana.This paper mainly explored in following aspects:(1) acquired the full-length CB7399 cDNA 1557bp in total which is Homologous to AtSUT4 from Arabidopsis thaliana based on blast in NCBI. Using SigalIP 3.0 forecast found that the encoding protein have no signal peptide sequence; Both TMHMM2.0 and Phyre2 online predicted tools showed that the gene encoding protein with 12 transmembrane structure domains.(2) Detected gene expression level of AtSUT4 and CB7399 respond to chling by RT-PCR.which show that CB7399 is strongly upregulated and AtSUT4 keeps unchange respond to cold.(3) using the Gateway system with TOPO, LR reaction obtained three CB7399 gene overex-pression vectors, respectively named pEarlygate101-CB7399, pEarlygate104-CB7399, pEarlygate100-CB7399. And transformed into agrobacterium strain GV3101, inflores-cence infect Col,a kind of Arabidopsis thaliana ecotype, and respectively obtained 5 pEarlygate101-CB7399 Lines,7 pEarlygate104-CB7399 Lines,4 pEarlygate-100 CB7399 Lines of T3 generation of seeds.(4) Observed subcellular localization of transgenic plants of overexpression CB7399 by confocal fluorescence microscope. Which showed that either N-terminal or C-terminal labeled YFP, the encoding protein is both located on the plasma membrane and tonoplast membrane.(5) RT-PCR tests expression level of CB7399 in pEarlygate104-CB7399 different Lines, and choose three different expression level lines(Line3、Line4 and Line 10). sugar content and cold stress conductivity of blades are tested, the results show that the three Lines sucrose and fructose content in the transgenic plants leaf was lower than those of Col, On the other hand, under cold stress both cold acclimation (CA) and without cold acclimation (NCA) pEarlygate104-CB7399 transgenic plants’ LT50 of leaves are slightly higher than the Col, especially L10.Regardless of 1/2 ms culture medium containing 1% sugar or without sugar, moreover the three lines under the condition of 150 mm,200 mm NaCl show that more excuberant,and higher seed germination rate than Col, slightly longer than Col in root length.(6) To analyze differences between over-expression CB7399 and Arabidopsis thaliana AtSUT4 physiological effect, we built AtSUT4 two overexpression vectors:pEarlygate101-AtSUT4, pEarlygate100-AtSUT4, five and three Lines respectively over-expression of genetical modified seeds are harvested. RT-PCR to detect expression level of pEarlygate 100-CB7399, pEarlygate-100 AtSUT4 transgenic plants of different Line. Transgenic plants leaf sugar level is tested,which shows sucrose content has decreased, but the difference is pEarlygate 100-CB7399 transgenic plants leaf fructose has also dropped.LT50 determination showed that the transgenic plants are slightly higher than Col.(7) Dark culture show that both pEarlygate100-CB7399,and pEarlygate-100 AtSUT4 transgenic plants’hypocotyl and root have a bit shorter than Col, and soil culture observed show that blades of pEarlygate100-CB7399 transgenic L1, L10 and pEarlygate 100-AtSUT4 L3 have rollover phenomenon.We cloned the gene CB7399 belongs to typeⅣ sucrose transpoter, which is localted on plasma membrane and tonoplast membrane.and particpart transport of sucrose.On the other hand, Ectopic expression in this study is applied to research some species that can’t grow in the normal condition.