Cloning Of Wheat S-adenosylmethionine Synthase Gene TaSAMS10 And Analysis Of Its Function In Response To Hypoxia

Wheat(Triticum aestivum L.)is one of the main food crops,and its high and stable yield is of great strategic significance for ensuring food security in my country.The winter wheat area in the Huaihe River Basin and the middle and lower reaches of the Yangtze River is the main wheat producing area in China.Affected by the monsoon circulation and other factors,the precipitation is abundant,and waterlogging disasters occur frequently,which seriously affects the growth and development of wheat.The earliest and most direct damage of waterlogging to plants is hypoxia stress.So far,there are few reports on the molecular regulation mechanism of hypoxia stress in wheat.Therefore,mining the key genes of wheat in response to hypoxia stress,and exploring and analyzing the molecular mechanism of hypoxia stress regulation in wheat are of great significance for breeding moisture-tolerant wheat varieties.In this study,two spring wheat varieties with significantly different waterlogging tolerance were used as materials,and the results of the proteome under hypoxia stress in the previous wheat showed that the expression of TaSAMS family genes was significantly up-regulated under hypoxia.Further bioinformatics analysis and expression pattern analysis of the family found that the family member S-adenosylmethionine synthase gene TaSAMS10 was significantly up-regulated in response to hypoxia stress.The gene was further cloned and an overexpression vector was constructed to obtain homozygous Positive overexpressing Arabidopsis lines.Hypoxic treatment of overexpressing Arabidopsis thaliana to analyze its function in response to hypoxia.The main results are as follows:1.Analysis of the wheat TaSAMS gene family,a total of 13 wheat SAMS members were identified,which were named TaSAMS1-TaSAMS13 according to their chromosomal locations.All members are unevenly distributed on the seven chromosomes.The conserved domains and conserved motifs among family members are conserved in position and structure.Phylogenetic analysis showed that the protein encoded by TaSAMS10 was most closely related to Hv SAMS2 in barley,and the sequence similarity was as high as 99.2%.2.Bioinformatics analysis showed that the ORF of TaSAMS10 gene was 1185 bp in length and consisted of 394 amino acid residues.The molecular weight of the encoded protein is 42.83 k Da,and the theoretical PI is 5.58,which is a relatively stable hydrophilic protein.Sequencing of the promoter sequences of two wheat varieties with different tolerance revealed that there were 4 single-base differences.The analysis of homeopathic elements found that the hypoxia-sensitive variety contained 23 CAAT-boxes,which was 1 more than that of the hypoxia-tolerant variety.indivual.The online site predicts that TaSAMS10 is subcellularly localized in the cytoplasm.Family expression pattern analysis showed that TaSAMS10 was significantly expressed under waterlogging stress.3.The TaSAMS10 gene was cloned from waterlogging-tolerant wheat roots by homologous cloning.The expression pattern of TaSAMS10 in wheat under waterlogging stress showed that in waterlogging tolerant cultivars,TaSAMS10 was tissue-specific,specifically expressed in roots,and was induced to express by waterlogging,and the highest relative expression level could reach 26 times.In the waterlogging-sensitive cultivars,the expression was induced by waterlogging in both roots and stems,the highest being 24-fold and 13-fold,respectively,and the average expression level in roots was higher than that in stems.However,in leaves that were not induced by waterlogging stress,the expression level was maintained at the same level as before treatment 3 d before treatment,and the expression level after 4 d treatment4.Construct the wheat TaSAMS10 gene overexpression vector,transform wild-type Col Arabidopsis thaliana through Agrobacterium,screen for hygromycin resistance medium and identify by PCR to obtain a positive homozygous Arabidopsis thaliana overexpression line,which is verified by q RT-PCR.It was verified by q RT-PCR that the TaSAMS10 gene was successfully overexpressed in Arabidopsis and strongly responded to hypoxia stress.The phenotypic observation of Arabidopsis thaliana treated with hypoxia showed that the overexpression line withered earlier than the wild type,but it had a longer survival time than the wild type.The results showed that Arabidopsis overexpressing TaSAMS10 could prolong the survival time of Arabidopsis under hypoxia in response to hypoxia stress.Analysis of subcellular localization results showed that TaSAMS10 was localized in cell membrane,nucleus and cytoplasm.