The Expression Of Highland Barley Melatonin Synthetase And Biological Characteristics Research

As a kind of grain crop with strong resistance to stress,highland barley has been paid more and more attention by agriculture and industry because of its rich nutritional value and unique biological activity.Melatonin is an indole substance,which can be used as a plant hormone to promote seed germination and plant growth and development,as well as an antioxidant to maintain the dynamic balance of reactive oxygen species in plant cells.At present,there is no research on endogenous melatonin synthesis pathway in highland barley at home and abroad.In this experiment,"Zangqing 2000"was used as the material to study the changes of the contents of endogenous melatonin and other metabolites in highland barley at different germination stages under salt stress,and the gene of melatonin synthetase was preliminarily screened through transcripomics data.q RT-PCR was used to analyze the expression level of melatonin synthesis gene under salt stress to further explore the correlation between endogenous melatonin content and melatonin synthesis gene in highland barley.In addition,the gene of5-hydroxytryptamine-n-acetyltransferase（SNAT）and caffeic acid-O-methyltransferase（COMT）of melatonin synthesis pathway in highland barleywas cloned.Moreover,the expression vectors of SNAT gene and COMT gene were constructed and introduced into E.coli,and the recombinant proteins were induced by IPTG,the enzymatic characteristics of the highland barley SNAT and COMT genes were preliminarily studied.The main research results are as follows:1.Under salt stress,the contents of total polyphenols,total flavonoids,lignin and melatonin in barley seeds under salt stress conditions were significantly higher than those under normal conditions.With the extension of germination time,the contents of total polyphenols,total flavonoids and melatonin in barley first increased and then decreased,and reached the maximum on the third day,which were 8.38 mg/g,1.982mg/g and 13.702 ng/g,respectively.The contents of anti-stress substances such as melatonin,total polyphenols,total flavonoids and lignin in barley increased significantly under salt stress,and the change trend was consistent with the extension of germination time.Melatonin could not only directly remove reactive oxygen species in the body as an antioxidant,but also improve the contents of other metabolites as a signal molecule to maintain the normal growth of plants.Improve the resistance of barley to salt stress2.Transcriptome data analysis showed that more than 20%of the genes had significant changes under salt stress.Gene Ontology（GO）analysis showed that DEGs were mainly involved in antioxidant stress,osmoregulation,protein-DNA complex,cofactor binding and other processes under salt stress.The Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analysis revealed that DEGs were mainly enriched in phenylpropane synthesis pathway,glutathione metabolism,tryptophan metabolism pathway,photosynthesis and other pathways.This suggested that highland barley enhances its resistance to the adverse effects of salt stress on plants by increasing the expression of stress-related genes.Besides,11 key genes of the melatonin synthesis pathway in highland barley were screened out according to transcriptomic data.The expression changes of two significantly different genes under different conditions were analyzed by fluorescence quantitative PCR technology.It was found that the change trend of key genes in the pathway of melatonin synthesis in highland barley was basically consistent with the change trend of endogenous melatonin content in highland barley.It was speculated that highland barley could resist the adverse effects of salt stress by increasing the expression of genes related to the synthesis pathway,thereby increasing the content of melatonin.COMT enzyme,as a kind of multisubstrate enzyme,can not only participate in the synthesis of melatonin,but also act as a key enzyme in the metabolic pathway of phenylpropane to generate total polyphenols,total flavonoids and lignin,indicating that melatonin,total polyphenols,total flavonoids and lignin can jointly improve the resistance of barley to salt stress.3.In this study,the full-length CDS sequence of SNAT gene of highland barley was screened and cloned.The total length of SNAT gene was 729 bp,encoding 243 amino acids.It was a hydrophilic unstable protein with the functional domain of acetyltransferase（GNAT）,which was highly similar to the amino acid sequence of SNAT in other plants.Construction of expression vector of E.coli to import,then express conditions optimization for barley SNAT protein.In vitro enzyme activity tests showed that highland barley SNAT protein had N-acetyltransferase activity,which could transfer acetyl group from acetyl-co A to5-hydroxytryptamine.The enzyme activity of SNAT was the highest at55℃,and the enzyme activity increased with the increase of substrate concentration.The reaction rate and substrate concentration showed a double reciprocal curve(K_m=371μm,V_max=4.5 nmol/min/mg protein).4.The full-length CDS sequence of COMT in highland barley was cloned.The full-length nucleotide sequence was 1074 bp,the start codon was ATG,encoding a total of 358 amino acids.The molecular weight of the protein was 38.7 k Da,and there was no signal peptide sequence at the N terminus.The 12～355 amino acids are typical of the type II oxymethyltransferase SAM domain,the S-adenosine-L-methionine binding site is cysteine at position 219,and the enzyme reaction site is aspartic acid at position 260.At 35℃,the enzyme activity of COMT was the highest.At 35℃,the enzyme activity of COMT was the highest.With the increase of substrate serotonin concentration,the content of melatonin produced by COMT also showed a gradient increase.The substrate concentration and enzymatic reaction rate showed a double reciprocal curve,K_m=230μm,V_max=2.1 nmol/min/mg protein.