Cloning And Expression Analysis Of A Alliinase Gene (AsALy1) Full-length CDNA From Allium Senescens L.

Mountain Garlic(Allium senescens L.)is a perennial wild Allium species of Liliaceae,which is a traditional medicine in China with effects of anti-bacterial biological,anti-oxidantion,anti-tumor,reducing blood pressure,reducing blood fat,lowering blood sugar,anti platelet aggregation,protect hepar and other physiological functions.Allicin is the main medicinal component,which is produced by the reaction of alliinase with alliin.Consequently,Cloning and analysis of alliinase gene,not only provide a theoretical basis of the further study on alliinase,but also laid the foundation for the industrial application of allicin in the field of medicine.Mountain Garlic as the experimental material in this study.The Allium senescens L.alliinase(tentatively named As ALy1)gene c DNA was cloned and analysised of expression and bioinformatics.Concrete study method as follows: the sequence of conserved region of As ALy1 gene and actin gene were cloned by RT-PCR.The 3'/5' end unknown sequence and ORF sequence of As Aly1 gene were cloned by RACE technique,and the full-length c DNA sequence of As ALy1 gene was obtained by electronic splicing.The expression of As ALy1 gene in different tissues was analyzed by Real Time PCR;Amino acid sequence of As Aly1 gene was analyzed by bioinformatics.2The results showed that the length of conserved region sequence of As ALy1 gene was 862 bp;The full-length c DNA sequence of As ALy1 gene with a length of 1655 bp was obtained by RACE technique,its open reading frame(ORF)length of 1440 bp and encoding a protein of 479 amino-acid residues,the protein relative molecular mass was 54950.82 Da,and a theoretical isoelectric point was 8.70;The predicted As ALy1 protein had a transmembrane region that was between 13 and 32 amino acids sites;There was a secondary structure that mainly consisted alpha helix,random coil and beta strand;The predicted As ALy1 protein had an aspartate aminotransferase superfamily domain,a C-terminal catalytic domain,a EGF like domain and an aspartate/ methionine/ tyrosine aminotransferase domain;It had serine,threonine and tyrosine phosphorylation sites and N-glycosylation sites.The conserved region sequence length of actin gene was 386 bp;After real-time fluorescence quantitative PCR analysis suggested that the expresstion level of As ALy1 gene in stems was 0.7 times,36 times and 157 times high as expresstion level in flowers,leaves and seeds,respectively,while As ALy1 gene hardly expression in the roots,indicating that the enzyme was a bulb alliinase.