Functional Analyses Of Gene MfWAK And MsLRPK

Alfalfa is known as "the king of forage",which is an important source of crude protein for the development of animal husbandry in C hina.But the alfalfa is vulnerable to chilling injury.Medicago falcata Lis a kind of important legume forage,which exhibits great tolerance to cold and drought.As a result,it is an important material for the reasearch of plants' resistance to stresses like low-temperture.In this paper,the function of low temperature response gene Mf WAK and MsLRPK was analyzed,Followed are the the main results of this study.TransgenicMedicago truncatula(Medicago truncatula G.)plants were analyzed by PCR,and real-time quantitative RT-PCR.Regenerated plants from 5 resistant callis were obtained,The expression of geneMf WAK was determined by real-time quantitative PCR.The results showed that the geneMf WAK was highly expressed in transgenic plant A35-8,A14,A41,A1-4 and A17,and the highest expression level of gene Mf WAK was in plant A1-4.The cold resistance of transgenic plants was tested,t he results of semi-lethal temperature showed that the semi-lethal temperature of wild type was about-2?,and the semi-lethal temperature of transgenic plants was between-3? and-6?,and the difference was significant.The lowest semi-lethal temperature of transgenic plant was A1-4(-6?),the highest semi-lethal temperature of transgenic plant was A17(-3 ?.).The result showed that the expression of geneMf WAK can significantly improve the cold resistance of Medicago truncatula.In the preliminary study of our laboratory,it was found that the expression of Mf LRPK inMedicago falcata was inhibited by low temperature,while the expression of the gene Mt LRPK in Medicago truncatula was induced by low temperature.In this paper,the full length of c DNA of homologous gene MsLRPK was cloned from alfalfa.The length ofthe ORF was 2173 bp,and the GC content was 41.79%.The encoded protein consisted of 723 amino acids,belongs to the transmembrane protein,which contains extracellular leucine-rich repeat(LRR)domains and intracellular protein kinase domain.The relative expression of geneMsLRPK of alfalfa in response to low temperature stress was detected by fluorescence quantitative PC R.The results showed that the induction was started at 2h after low temperature treatment,the highest expression of MsLRPK gene was at 24 h after low temperaturetreatment.The CRISPR/Cas9 system was used to genetically edit the gene MsLRPK,and the transgenic alfalfa plants were obtained.After PC R detection,42 positive transgenic plants were obtained.PC R products were directly sequenced to detect target mutations,and three mutant transgenic plants 68-1,76-2 and 19-2 were obtained.The relative electrical conductivity of wild type and mutant alfalfa was determined.The results showed that the conductivity of target mutant plants was significantly lower than that of wild type after 1 h treatment at-8 ?,which indicated that the deletion of MsLRPK gene increased the cold resistance of alfalfa.