Genes Cloning Of Terminase For SGAs Biosynthesis Metabolismic Pathway And Relevant MiRNA Screening In Potato

Potato steroidal glycoalkaloids (SGAs) was an important secondary metabolites existingin solanaceae and liliaceae plants, which had a close relation to plant biotic response andproduct quality as well as a wide pharmacological activity in pharmacology. Solanidine:UDP-glucosyltransferase (SGT) was a key terminal enzyme of the SGAs anabolic pathway inSolanum tuberosum. It will be of great importance in studying the characteristic of SGT genestructure and encoded enzyme protein which involved in regulating SGAs synthesis andproducing SGTs by microbial fermentation in plant. The cDNA sequence of sgt1, sgt2and sgt3were cloned by RT-PCR from potato tubers and the encoded protein structure, property andfunction were predicted by bioinformatics method. Differential expression microRNA wereselected by smallRNA high-throughput sequencing technology. The function of thesemicroRNA target gene were analysed by bioinformatics methods. MicroRNAs related tointermediate products and enzymes related to Glycoalkaloid biosynthesis metabolismicpathway were selected finally. These results may provide a theoretical basis for furtherseparation of the enzyme protein and studying its activity, it also provid a reference aboutSGAs expression and spatial distribution in other genetic system. The key results are asfollows:1. Total RNA was extracted from Solanum tuberosum and three cDNAs of GlycoalkaloidSynthase gene were obtained by reverse transcription polymerase chin reaction (RT-PCR). Thesequencing results show that Three SGTs enzyme gene fragments were obtained from Solanumtuberosum each of which contains1467-1518bp and encodes a peptide of488～505aminoacids. Similarity comparisons show that they share over99.12%similarity in nucleotidesequence and over99%similarity in amino acid sequence with those of other plant SGTasegene (U82367.2, DQ218276.1and DQ266437) in GenBank. The enzyme protein structureand properties of three glycosyltransferase genes were predicted by bioinformation method, theresult showed that three genes contian UDPG glycosyltransferase conserved domain and manyimportant functional sites the PI=5.52-5.62. The3D structure of protein was predicted byhomology comparative modeling in Swiss-Model, the results showed that the3D structure ofSGTs was highly similar to that of the glycosyltransferase, and which have the ability ofsynthesizing steroid glycoside alkaloids, and its sequence was submitted with GenBank: sgt1,No: JN695005; sgt2, No: JN695006; sgt3, No: HM188447.2. SmallRNA were extracted from tubers and leaves of five different genotype potatos which were induced by the red light. The miRNA library was constructed by small molecularRNA high-throughput Illumina/Solexa sequence technology. The small molecules RNA whichregulate potato glycoside biosynthesis metabolic pathways were found by bioinformationanalysis. The results indicated that153known miRNAs were detected in four potato miRNAslibraries, which belong to115miRNA families and its abundance ranged from1to123803reads. The target genes forecast analysis showed that21miRNAs involved in regulating56target genes directly or indirectly related to glycosidic alkaloids synthesis, where30targetgenes directly involved in regulating terminase gene synthesis related to SGAs synthesispathway (sgt1, sgt2and sgt3).