白桦环阿齐醇合成酶(CAS)基因与达玛烯二醇合成酶(DS)基因的双基因RNA干扰载体构建  

Construction of RNA interference vector carrying two genes of DS and CAS of Betula platyphylla Suk( Birch. )

作　　者：梁甜[1] 马泓思[1] 尹静[1] 詹亚光[1,2] 张梦岩[1] 

机构地区：[1]东北林业大学生命科学学院,哈尔滨150040 [2]东北林业大学林学院林木遗传育种与生物技术国家重点实验室,哈尔滨150040

出　　处：《植物研究》2015年第3期355-362,共8页Bulletin of Botanical Research

基　　金：国家自然基金资助项目(31200428);黑龙江省自然科学基金项目(C201110);哈尔滨市青年创新基金项目(2012RFQXN007)

摘　　要：环阿齐醇合成酶(Cycloartenol synthase,CAS)和达玛烯二醇合成酶(Dammarenediol synthase,DS)为白桦三萜合成过程分支途径关键酶基因,对CAS和DS基因的抑制或沉默将有利于三萜前体-2,3氧化角鲨烯向目标产物白桦脂醇和齐墩果酸合成。利用同源序列PCR方法及pRNAi-GG新型载体,进行DS单基因RNAi及DS与CAS双基因RNAi载体的构建。结果显示,分别克隆获得CAS和DS c DNA序列长度为300和509 bp,Blast同源序列比对与已知日本白桦BPX1和OSCBPD相似度为100%和92%。依据基因RNAi引物设计及pRNAi-GG使用原则,分别将CAS、DS的正向片段和反向片段有序地连接到pRNAi-GG载体PDK内含子两侧,并转化大肠杆菌Trans1-T1中,经PCR及克隆测序验证,获得白桦DS单基因和CAS、DS双基因干扰载体,并通过三亲杂交方法分别获得含有两个RNAi载体的工程农杆菌菌株。该研究为进一步通过基因工程手段阻断白桦三萜分支途径及促进前体物质向目标产物白桦酯醇和齐墩果酸方向合成奠定了基础。Cycloartenol synthase （CAS） and dammarenediol synthase （DS） are the key enzymes of branch pathway of birch in birch triterpenoid synthesis process. Silencing of DS gene and CAS gene help triterpenoid synthesis precursor-2,3-oxidesqualene to synthesize betulin and oleanolic acid. PCR method of homologous sequences and a new vector pRNAi-GG were used to construct RNAi expression vectors of CAS gene and DS gene. The cloning part cDNA sequence of DS and CAS gene, respectively, CAS gene fragment length was 300bp, DS gene fragment length was 509bp, and their similarity reached 100% and 92%, respectively, compared with BPX1 and OSCBPD of Betula platyphylla var. japonica. With the principle of primer design and pRNAi-GG instruction, we connected the fragments on forward and contrary direction to the both sides of PDK intron of pRNAi-GG. The recombinant vectors were transformed into competent E. coli cells Transl-T1. By PCR and sequence detection, the interference vector of DS gene and interference vector of DS ＋ CAS two genes were constructed successfully. Our study will lay the foundation for the genetic modification of blocking-up triterpenoid branch pathways and promote the precursor to synthetize betulin and oleanolic acid in the triterpenoids metabolism pathway of white birch.

关 键 词：白桦 三萜 RNA干扰载体 环阿齐醇合成酶(CAS) 达玛烯二醇合成酶(DS) 

分 类 号：S792.153[农业科学—林木遗传育种]