Cloning And Functional Analysis Of MsABCG1,an ABC Transporter Gene From Medicago Sativa

Alfalfa,as a legume forage,is known as the "king of forage" because of its high nutritional value,excellent quality,and good palatability.In recent years,due to climate change,alfalfa is often exposed to drought,cold damage and other abiotic stresses,which has a greatly effect on its quality and yield.Using transgenic technology to improve the resistance of alfalfa has become one of the common and effective methods.ATP binding cassette(ABC)transporter is one of the largest and oldest protein superfamily.It is widely present in various organisms.It mainly transports substrates with the energy produced by hydrolysis,which plays an important role not only in plant growth and development but also in resistance to external biotic and abiotic stresses.In this study,the full length of MsABCG1 gene was cloned by homologous cloning method,and its sequence was analyzed by bioinformatics method;the expression level of MsABCG1 in different tissues and different treatments was analyzed by q RT-PCR technology;The vector p CAMBIA1300-MsABCG1 was constructed for subcellular localization.In addition,the heterologous expression of MsABCG1 was also performed in tobacco for functional analysis by Agrobacterium-mediated method.The main findings are as follows:1.The full length of alfalfa MsABCG1 gene was successfully cloned.Sequence analysis showed that,its ORF region was 2061 bp,and,it encoded 686 amino acids with 6transmembrane domains.2.The expression level of MsABCG1 gene under drought,Na Cl,ABA and other treatments was analyzed by q RT-PCR.The results showed that the MsABCG1 gene was mainly expressed in the roots of alfalfa,with relatively lower expression level in the stems and leaves,and it was response to each treatment in a certain degree.Especially under drought treatment,the expression level of MsABCG1 gene was increased significantly in the stems and leaves.3.The vectors p CAMBIA1300-MsABCG1 and p CAMBIA1302-MsABCG1 were successfully constructed for molecular function of MsABCG1.The result of subcellular localization showed that MsABCG1 protein was mainly expressed on the plasma membrane.4.The MsABCG1 was transformed into tobacco by Agrobacterium tumefaciens strain GV3101.We found that MsABCG1 could improve the resistance to drought stress in tobacco.Further study showed that MsABCG1 may improve the drought resistance of tobacco by regulating its relative water content,stress responsive gene expression and stomata state.In summary,the function of MsABCG1 gene was initially verified by cloning the full length of MsABCG1 gene,bioinformatics analysis,expression pattern analysis,and functional analysis of the transgenic tobacco.