Cloning And Genetic Transformation Of Key Genes Related To Triterpenoicl Saponin Biosynthetic Pathway In Aralia Elata

Aralia elata (Miq.)Seem.,an important medical plant of the family Araliacea, canbe used for the treatment of hepatitis, inhibiting cancer, enhancing the body immunity,and so on.Triterpenoid saponinisthe main medicial composition inAralia elatawhosesapogenin is oleanane. The biosynthetic pathway of triterpenoid saponin is known,Squalene epoxidase is a key enzyme of this pathway. Itcatalyses Squalene into2,3-Oxidosqual Squalene,the content and activity of itinfluences the yield of endproducts.Therefore, ithas an important biological significance that we clone the geneand transfer it intomodel plant bybiotechnology.In this study, we cloned the Squalene epoxidase gene fromAralia elata usingRT-PCR.The sequencing results showed that the nucleotide sequence contained acomplete open reading frame,its full length was1644bp,encoding a polypeptide of547amino acids. Bioinformatics analysis of this Amino acid sequence showed it sharedabove90%identities to Panax gingeng, Panax notoginseng. It confirmed that we hadcloned the Squalene epoxidase gene in Aralia elata. The gene was firstly cloned fromAralia elata,therefore we submitted it into NCBI GenBank database,the number isGU354314.1. And then we predicted thestructureand biological properties of theproteinusing bioinformatics software.A partial sequence of gene GAPDH (627bp) was firstly cloned from Araliaelata,the GenBank number is JQ183068.1.And thenit was used as internal control genein Real-time qRT-PCR. In this study,expression differences in transcription level ofAeSE from lateral roots, stems, leaves and flowers of Aralia elate were analyzed byReal-time qRT-PCR. The results demonstrated that it was expressed in lateral roots,stems, leaves and flowers, the relative expression level of stems and leaves werehigher than lateral roots and flowers.In this study, the plant expression vector pCAMBIA3301-AeSE was successfullyconstructed, and wastransferred into tabacco by Agrobacterium tumefaciens-mediatedleaf disc transformation method. In the study, we obtained36positive tobacco seedlingsby molecular detection of506resistantplants,the conversion efficiencywas7.1%. The results of this study lay the foundation for further studying thefunctions of AeSEgene.Squalene synthase gene (SS) is another key gene in the biosynthetic pathway oftriterpenoid saponins.In this study, the T0generation seeds of transgenic tobacco(target gene is AeSS) were selected with kanamycin (50mg/L), the results showed atotal of267tobacco resistant seedlings wereselectedfrom830T0generation seeds oftransgenic tobacco,the germination rate was32.2%with kanamycin treatment(50mg/L).According to the PCR and PCR-Southern blotting analysis, a total of31positive T1seedlings were obtained.This study laid the foundation for further study on AeSS gene.