Screening Of Lycium Salt-resistant Germplasm Resources And Identification Of Salt-resistant Genes

As pioneer plants and building plants for the management of desert and saline-alkaline lands in northwest China,the ecological benefits of windbreaks and sand-fixing,soil improvement and ecological environment improvement,as well as the value of food,nutrition,and medicinal use have received intensive attention in recent years.In order to solve the problem of rational selection of Lycium germplasm improved in saline-alkali land,based on the physiological responses of 20 Lycium planting resources to salt stress,combined with statistical software such as principal component analysis and cluster analysis,different germplasm resources were determined.Strong and weak salt resistance,a set of simple and reliable salt-resistant germplasm resources screening evaluation system was established.The RACE method was used to clone the HKT1,NHX1 and SOS1 genes of Lycium,and bioinformatics analysis was performed to verify the expression characteristics of the three salt-resistant genes in Amur.On this basis,the salt resistance genes were transferred to the South Mustard for subsequent salt identification.The results are as follows:(1)Thephysiologicalindexes such asK~+/Na~+ratio,chlorophyll,betaine,malondialdehyde,GSH content and relative permeability of cell membranes and the three salt-resistant genes HKT1,NHX1 and SOS1 in 20 Lycium germplasm under treatment with300 mM NaCl.The analysis of expression levels,using a combination of principal component analysis and cluster analysis,reduced the ten salt-related physiological indicators to a representative four new principal components.The principal component analysis results were used to rank the salt tolerance of 20 alfalfa germplasms.The order from strong to weak was:L.ruthenicum>L.barbarum No.2>L.Chimesevar.potaninii>L.barbarum No.1>baitiao>damaye>L.Chinens>ningqicai No.1>Mengqi No.1>L.barbarum No.5>baihua>M1>yuanguo>Huangguobian>zibing>NingxiaHuangguo>L.hongzhi>L.yunnanens>Xiaomaye>L.barbarum No.7.According to the results of cluster analysis,the anti-salt ratings of 20 alfalfa germplasms were divided into four categories:high-resistance,medium-resistance,more sensitive,and most sensitive.(2)The full lengths of the three salt-resistant genes of HKT1,NHX1,and SOS1 were cloned,and the sequences of the 5'UTR and 3'UTR regions and the AATAA tailing signal of the 3'UTR region were clarified.Through multiple sequence alignments,it was found that the three genes and their encoded protein sequences are highly homologous to tobacco,tomato,potato,and other solanaceous plants.They all locate in the membrane structure and are lipophilic proteins except for the SOS1 protein.The prediction of conserved domains and spatial structures revealed that the HKT1 protein has a conserved domain and spatial structure of the TrkH protein family,while the NHX1 and SOS1 proteins have a Na~+/H~+transporter domain and a spatial structure.(3)Quantitative expression analysis of three genes using the most resistant salt variety,‘L.ruthenicum',was performed.It was found that their expression in young Lycium is tissue-specific,and their response in leaves is higher than that in roots and stems.The changes were even greater,and the relative expression patterns of the three genes under different temporal dynamics were similar.They all reached the maximum at 24 hours of salt stress.(4)The Lycium's HKT1 and NHX1 genes have been constructed on the plant binary overexpression vector pCAMBIA1304 and transformed into Agrobacterium tumefaciens and transformed into Arabidopsis thaliana wild-type Col-0 by dipping method,and then screened by antibiotics and PCR T1 generation positive seedlings were selected for subsequent functional verification.