Germplasm Creation And Stress Resistance Analysis Of LjHsp70-15 Transgenic L. Japonicus

Stress seriously affects the growth,yield and quality of pasture.Heat shock protein(Hsp),induced by high temperature,low temperature,drought and salinity,can repair damaged cells and reduce damage.Lotus japonicus is a perennial high-quality leguminous forage mainly distributed in warm and humid areas,with high nutritional value and good palatability.In this paper,the transformation of model plant Arabidopsis thaliana and transient expression of Lotus japonicus were used to explore the stressresistant regulation function of heat shock protein gene LjHsp70-15 of Lotus japonicus,and further use this gene to create a new germplasm of Lotus japonicus forage with stress tolerance,so as to provide basis for the cultivation and application of stressresistant forage in karst areas with bare rocks,barren soil layers and high temperature and drought.The main research results are as follows:Analysis of the genome(version number V3.0)showed that LjHsp70 family of Lotus japonicus was composed of 18 members,with the number of exons and introns ranging from 1 to 8,belonging to acidic hydrophilic protein,which contained response and regulation elements such as low temperature,drought,cell cycle,defense and stress response,auxin and light response.Among them,LjHsp70-15 gene has the largest number of exons and introns,which are located in endoplasmic reticulum and contain typical stress response elements such as low temperature and drought induction.The expression vector p SH-35S-LjHsp70-15 was constructed to carry out the plant genetic transformation of LjHsp70-15 gene.Eight transgenic(TP)Arabidopsis thaliana plants were obtained by the method of stained flowers.It was found that the expression of LjHsp70-15 did not affect the development of leaf,flower and rosette branches of Arabidopsis thaliana,but promoted the plant height to some extent.Treatment with Na Cl at different temperatures and concentrations showed that the expression of LjHsp70-15 gene improved the germination and growth ability and salt stress tolerance of Arabidopsis thaliana seeds.Under the stress of 42? and 4?,the expression of LjHsp70-15 gene can maintain the cell membrane integrity of Arabidopsis thaliana,and realize its stress protection by increasing the activity of protective enzymes and reducing the content of MDA.Optimizing transient genetic transformation parameters to obtain TP Lotus japonicus which transiently expresses LjHsp70-15 gene.Under high temperature and Na Cl stress,LjHsp70-15 may be involved in the regulation of cell membrane structural stability under stress.Four strains of TP Lotus japonicus which constitutively expressed LjHsp70-15 gene were obtained by Agrobacterium-mediated genetic transformation.Under the stress of 42? high temperature,4? low temperature,250 mmol/L mannitol and 150mmol/L Na Cl,TP has good tolerance to high temperature,low temperature,drought and salt stress,which is related to the increase of protective enzyme activity and the decrease of MDA content.The research shows that LjHsp70-15 gene has a good function of stress resistance regulation,which can effectively improve the effects of stress factors such as high temperature,low temperature,drought and high salt on swallowwort grass.The research results can provide a certain basis for molecular breeding of stress-resistant grass.