Cloning And Functional Analysis Of Homocysteine S- Methyltransferases In Wild Triticum Monococcum L.

Selenium is one of the 14 trace elements necessary for the human body with many physiological functions.Selenium deficiency can lead to a variety of diseases.Proper selenium supplementation by daily diet is an effective method.Wheat is an ideal selenium-enriched food.Triticum monococcum L,one of the A genome donor species of common wheat?Triticum aestivum AABBDD?has a large amount of useful genes for wheat yield,resistance improvement which is not available in modern common wheat.In the present stydy,a Homocysteine s-methyltransferases genes?HMT?was isolated and named TmHMT1 from the accession of Triticum monococcumL,10-1 was used to.The bioinformatics and biochemical characteristics of the TmHMT1 were systematically analyzed.The primary physiological functions of TmHMT1 was verified by prokaryotic expression system using transgenic Escherichia coli.The present study provided new beeding elements for selenium-enriched wheat breeding programme.The conclusions are as follows:1.The HMT1 gene was cloned from 10-1 and designated as TmHMT1.The TmHMT1 gene ORF was 975 bp in length.It is speculated that the TmHMT1 gene encodes 324 amino acids and the predicted molecular mass is about 35.16kDa.Bioinformatics analysis TmHMT1 contains a GGCCR conserved zinc chain structure and three conserved cysteine residues:Cys236,Cys303,Cys304,which is a necessary site for HMT enzyme catalytic activity.2.The phylogenetic tree found that the amino acid sequence of TmHMT1 was98.14%similar to that of AetHMT1,the similarity with ZmHMT1 was 85.97%,and the similarity with AtHMT1 was 66.15.%,the similarity with BoHMT1 is 52.18%.3.After incubation for 3 h at 200 rpm and 37°C,the expression level of the target protein with IPTG of 0.10 mM was the highest and the obtained TmHMT1 protein product was consistent with the predicted molecular size.4.The Escherichia coli?BL21?carrying the and TmHMT1 gene and an ordinary E.coli strain?BL21?carrying an empty vector were cultured in M9 medium containing100?M sodium selenite and 100?M disodium selenate respectively,and It was found that the OD₆₀₀ value of the transgenic TmHMT1 gene strain was higher than that of the control under the stress of 100?M sodium selenite and 100?M disodium selenate,indicating that the TmHMT1 gene can improve the selenium tolerance of E.coli.5.The Escherichia coli?BL21?carrying the and TmHMT1 gene and an ordinary E.coli strains?BL21?were cultured in M9 medium containing 100?M sodium selenite for24 h,The results showed that Se concentration of transgenic Ecoli strain was higher than the control,indicating that the TmHMT1 gene can enhance the selenium-enriching ability of E.coli.6.The temporal and spatial expression of TmHMT1 gene in the different organs of10-1 was eleucidated by qPCR.The results demonstrated that Se treatment could stimulate the expression level of TmHMT1 gene in all organs of 10-1 and the expression level was highest in roots and low in stems and leaves,indicating that TmHMT1 gene work mainly in Triticum monococcum L.root.