Moleculuar Cloning And Functional Analysis Of Sterol Methyltransferase2of Spinacia Oleracea (SoSMT2) Under High Nitrate Stress

Soil secondary salinization is one of the main obstacles to the greenhouse vegetable growth, seriously restricting the sustainable development of vegetable production. The anion in the greenhouse soil is main NO3-, accounting for about67%-76%of the total anions. Soil salinization is related not only to the resources and ecological environment, but also to the sustainable agricultural development. Plant sterols play important roles in plant stress as signaling molecule, lipid raft fractions and membrane components. Sterol methyltransferases (SMTs) are key enzymes in sterol metabolism in higher plants, catalyzing the formation of C-24-and C-24-ethyl methyl sterols. SMT2catalyzes the second alkyl reaction using24-methylene-lophenol as substrate. This study compared nitrate stress resistance of two different spinach cultivars(Chaoji and Daye). The spinach SMT2gene (SoSMT2) was cloned, and transformed into the model plant Arabidopsis thaliana to investigate its function. The main results are as follows:(1) The salt tolerance difference during germination and seedling growth of two spinach cultivars (’Chaoji’and’Daye’) were compared. The results showed that10mM NO3-promoted the seed germination rate.60mM NO3-and160mM NO3-inhibited the germination of the two cultivars, while the germination rate of’Chaoji’ was higher than’Daye’.160mM NO3-inhibited the seedling growth of the two cultivars, and the growth of’Chaoji’was better than’Daye’. In addition, we also compared soluble sugar content, total protein content, antioxidant enzyme activities, nitrate reductase (NR) activity, MDA and H2O2content and other physiological indexes in the two cultivars under160mM NO3" stress, indicating that the salt(?)lerance of’Chaoji’was higher than’Daye’. Besides, the content of main plant sterol and total sterol content in two spinaches was determined by GC-MS. The result showed that the content of total sterol in’Chaoji’and’Daye’was increased and the increasement in’Chaoji’was higher than’Daye’. The increasement of the ratio of sitosterol to stigmasterol in’Chaoji’was less than’Daye’.(2) The sterol methyltransferase gene associated with nitrate stress was isolated from spinach root and designated SoSMT2. The full-length cDNA sequence of SoSMT2was1364bp, with the ORF of1086bp, which encodes361amino acids. SoSMT2expressed in root and leaves of two cultivars under the160mM NO3-treatment by RT-PCR, while the expression of SoSMT2in leaves of’Chaoji’was higher than in ’Daye’. In addition, the expression of SoSMT2in’Chaoji’and’Daye’ was induced by15%PEG,150mM NaCl,1mM salicylic acid and1mM H2O2stress treatment.(3) Transgenic expression vector was constructed and transformed into Arabidopsis thaliana using flower dip method. The germination rate of homozygote transgenic Arabidopsis seed was higher than the wild type (WT) Arabidopsis under60mM NO3-and160mMNO3-stress, indicating that SoSMT2improves the nitrate tolerance in transgenic Arabidopsis seeds. The seedling growth of WT and transgenic plants was inhibited by160mM NO3-stress treatment, and the inhibition in WT was more serious. The fresh weight, dry weight, soluble sugar content, total protein content, antioxidant enzyme activities, nitrate reductase (NR) activity, MDA and H2O2content and other physiological indexes after160mM NO3-stress treatements indicates that nitrate tolerance in transgenic Arabidopsis was higher than the WT plants. Besides, the plant sterol content was measured using GC-MS. The results showed that the increasement of total sterol content in transgenic Arabidopsis was higher than that of the WT plants. The ratio of sitosterol to stigmasterol remained relatively stable in the transgenics. The results suggested that SoSMT2impoved the nitrate tolerance might be due to the change of the plant sterols content.