Clone Of OSC New Gene, Structure Of RNAi Carrier And Study Genetic Transformation Preliminarily In Betula Platyphylla Suk

Triterpenoid in Betula platyphylla Suk., such as betulin, betulinic acid and its derivatives, had significant effect on resisting HIV virus and restraining tumor. The triterpenoid components （betulin, oleanolic acid, dammarendiol, sterol, etc.） composed via the mevalonate pathway （MVA） with materials acetyl-CoA, synthetised in the cytoplasm. The common precursor,2,3- oxidation of squalene, under the regulation of different triterpenoid synthesis gene（LUS、CAS、β-AS、DS）, several products competed the same precursor, and inhibited synthetised in each other. The target product, betula and oleanolic acid’s synthesis were low, restricted the large-scale application in clinic. We used polymerase chain reaction （PCR） to clone birch new OSC gene homologous sequences-new gene of amyrin synthase and temporarily named BpDS.We completed the bioinformatics analysis and constructed phylogenetic tree. RNAi vectors of BpDS single and BpDS-CAS double gene were structured, we also did the genetic transformation,and looked forword to block triterpenoid biosynthetic branch process to promote the betulinic acid and betulin accumulation by gene engineering.The results of this study are as follows:1. The templets were the cDNA of Betula Platyphlla suk tissue culture seedling, cloned the OSC BPD by homologous sequence RT-PCR method. Analysed its molecular characteristics, physical and chemical properties, homology and phylogenetic evolution tree using bioinformatics software. The results showed that, we obtained the sequence and the fragment length was 2636bp, had high similarity with a surmised Oxidation squalene cyclase of Betula platyphylla var. japonica, the similarity was 92%. The similarity with amino acid sequence of Beta-amyrin synthase (（3-AS） in Panax ginseng,Aralia elata, Prunus mume and some other plants was 66%-71%,and speculated a white birch OSCgene that for the coding of Beta-amyrin synthase temporarily nominated BpDS. Through the bioinformatics analysis, the open reading frame （ORF） length of this gene was 2211bp, encoded 736 amino acids, the 98-725 aa has a Isoprenoid_C2_like super family, contained conservative domain areasqualene of Oxidation squalene cyclase （SQCY₁）, and a repeat sequence of prenyltransferase and squalene epoxidase, the encoding protein was unstable and hydrophilic protein. GO analysis revealed that BpDS protein involved in pentacyclic triterpenoid biosynthetic processs including organic cyclic compound metabolic process, pentacyclic triterpenoid metabolic process, triterpenoid biosynthetic process.It also has catalytic activity activity,intramolecular transferase activity and isomerase activity.The phylogenetic tree analysis showed that the encoding amino acid sequence of BpDS, had the closest relationship with the surmised Oxidation squalene cyclase of Betula platyphylla var. japonica （OSC BPD BAB83089.1）, and the relationship with the beta-amyrin synthase in other species was far.2. The materials were one year Betula platyphylla Suk.The characteristic of BpDS gene were texted during the growing season （the middle ten days of May - the middle ten days of September） by quantitative PCR.The result show that in leaves, BpDS gene expression in May was the lowest; in September and July, BpDS gene expression were 31.5 times and 26.7 times of the expression in May; in June and August, BpDS gene expression were 3.8 times and 4.4 times higher than that in May. The expressions of BpDS gene showed up-regulated by various degrees in 10 μmol/L ABA induced by 1-72h.And the BpDS gene expression reached peak on the 12h-24h induced treatment. The expressions of BpDS gene showed up-regulated only in 5mmol/L ethylene treatment for 12h and was 5.4 times than the control, but the expressions of BpDS gene was inhibited in the other induced time.On of the most significant inhibitory effect for the treatment of lh,rhe expression was only 8% of control. The relative expression of BpDS in roots, stems and leaves of tissue culture seedlings showed that, in stem, expression of BpDS was the lowest, in leaves and roots, expression of BpDS were 4 times and 3.5 times of expression in stem. The expression of BpDS showed that, expression of BpDS was regulated by season, hormonal regulation and had tissue specificity.3. According to the sequence of BpDS gene,RNAi and pRNAi vector requirements, the 320bp of BpDS gene was selected as the interference fragment.The RNAi vector of pBI1303-BpDS was built by the method of traditional enzyme digestion-connect. pBI1303-BpDS was transferred into Agrobacterium tumefaciens successfully; pRNAi-GG-BpDS-CAS was built by the vector of pRNAi-GG and transferred into Agrobacterium tumefaciens. This study will lay the foundation for the genetic modification and biotechnology utilities in the triterpenoids metabolism pathway in white birch.4. The RNAi vectors contained BpDS gene, pBI13O3-BpDS, were transferred by Agrobacterium tumefaciens-mediated genetic transformation into clones of white birch, using the leaf-disc transformation method. Then acquired four transgenic white birch seedlings from 20 transformants were verified by PCR （NptⅡ and GUS gene） and Southern bloting with GUS and GFP fluorescence detection.qRT-PCR analysis showed that the gene expression of BpDS were suppressed in two seedlings （Y1, B3）with92% and58% of the wild-type gene expression,and BPY was suppressed with 2% and 9% of the control.