Cloning And Expression Analysis Of Tripterygium Wilfordii Transmembrane Transporter TwMATE2 And TwMATE3

Tripterygium wilfordii Hook.f.is an important natural medicine resource.The sesquiterpene alkaloids extracted and isolated from T.wilfordii plants have good medical and agricultural activities and great economic value.However,the content of secondary metabolites of T.wilfordii is low,extraction and separation are difficult,and the cost of chemical synthesis is high.The use of metabolic engineering to regulate plant metabolic pathways is one of the methods to increase the production of secondary metabolites.MATE(Multidrug and Toxic compound Extrusion)protein is an important type of transmembrane transporter protein,which is involved in secondary metabolite transport and other processes.Therefore,we clone,locate and function the MATE transporter gene related to secondary metabolite transport,To lay the foundation for understanding the principle of transmembrane transport of sesquiterpene alkaloids,increasing the content of active substances and simplifying the subsequent separation operation.The current research results are as follows:1.Two T.wilfordii MATE protein genes were cloned,TwMATE2 open reading frame is 1473 bp,encoding 490 amino acids;TwMATE3 open reading frame is 1518 bp,encoding 505 amino acids.Bioinformatics analysis showed that the molecular weight of TwMATE2 protein is about 53.9 k Da;the molecular weight of TwMATE3 protein is 54.5 k Da,which are all unstable hydrophobins and have 12 transmembrane helices in the secondary structure.Fluorescence quantitative analysis TwMATE2 was expressed at low levels in the roots and stems of Tripterygium wilfordii,and the expression level was high in the leaves;TwMATE3 gene was expressed in large amounts in the roots,but at low levels in the stems and leaves.2.The subcellular localization vectors p CAMBIA1302-TwMATE2,p CAMBIA1302-TwMATE3,and the red fluorescent localization label p CAMBIA1301-TIP-RFP expression vector were constructed.After transformation,Agrobacterium was co-injected into tobacco leaves for transient expression.The fluorescence observation of laser confocal microscopy revealed that TwMATE2 and TwMATE3 proteins were highly coincident with the fluorescence of TIP protein located in the vacuole membrane.The results showed that both MATE proteins were located on the tobacco leaf vacuole membrane.3.The eukaryotic expression vectors p YES2/CT-TwMATE2 and p YES2/CT-TwMATE3 were constructed,transformed into yeast cells INVSc1 and induced by galactose expression,protein identification resulted in TwMATE2 and TwMATE3 protein bands with molecular weights of approximately 53.9 k Da and 54.5 k Da,respectively.The results of substrate transport experiments TwMATE2 and TwMATE3 protein have the function of transporting wilforine.4.The p CAMBIA1301-TwMATE2,p CAMBIA1301-TwMATE3 overexpression vectors were constructed.transformed Agrobacterium tumefaciens ATCC15834 to infect T.wilfordii callus for induction,successfully obtained transgenic hairy roots,and laid the foundation for further study of T.wilfordii MATE gene function.