摘要
根据Slightom等RiA4TL DNA序列分析结果 ,设计了一对引物 ,以Ri 质粒DNA为模板 ,利用PCR方法对rolC基因进行了扩增。利用pGEM T载体对rolC基因进行了克隆和测序。结果 ,克隆的DNA片段序列与报道的ORF1 2阅读框内的rolC序列一致。将pGEM T rolC用限制性内切酶SacI酶切 ,与经SmaI酶切CIAP去磷酸化的pUC1 9质粒重组 ,经蓝白斑筛选得到正向重组的pUC1 9 rolC。再用Xbal SacI双酶切pUC1 9 rolC与pBI1 2 1 ,将rolC基因定向克隆到具有CaMV35S启动子的pBI1 2 1表达质粒上 ,构建成植物表达载体pBI rolC。用pBI rolC转化农杆菌LBA440 4构建成的LBA440 4 (pBI rolC)工程菌转化人参子叶 ,获得发根的表达。经PCR扩增分子检测 ,证明rolC基因确已整合到人参发根基因组中。经对转化的人参发根中单体皂苷含量测定 ,发现发根中含有所检测的 7种人参单体皂苷 ,总皂苷含量达 1 8 5 5mg g。
According to Bulgakov′s studies,the rolC gene is in the ORF12 of T-DNA of Ri plasmid.A pair of primers were designed and synthesized according to the analytical results of RiA 4T L-DNA sequence by Slightom et al.Both of 0.8kb PCR product was obtained by PCR using the DNA of the plasmid Ri of A 4 and the genome DNA of hairy root R9923 as template.The PCR product was ligased into pGEM-T vector and sequenced.The result of sequencing showed that the PCR product was the same as the sequence of rolC of ORF12.The vector pGEM-T-rolC digested by restriction enzyme SacI and vector pUC-19 digested by SmaI restriction enzyme was recombined into the pUC19-rolC plasmid that was screened by blue-white selection.Both of pUC19-rolC and pBI121 were degested by the restriction enzyme XbaI and SacI,the rolC gene was inserted into pBI121 to construct an expression vector pBI-rolC.After being sterilized,the cotyledon of Panax ginseng was cultured overnight together with LBA4404 (pBI-rolC).After the bacteria suspension was absorbed by sterile filter paper,the cotyledon transferred onto minimal medium (1/2MS) to be cultured in darkness at 22℃.After culture of about four weeks,the hairy roots were produced on the cotyledon of Panax ginseng,which was the result of rolC transformation.The content of monomer ginsenoside of Rg1、Re、Rf、Rb1、Rc、Rb2 and Rd in hairy roots were determined by the HPLC.The total ginsenosides content in the hairy roots came up to 18.55mg/g.
出处
《中国生物工程杂志》
CAS
CSCD
2004年第9期58-62,共5页
China Biotechnology
基金
吉林省自然科学基金资助项目 ( 2 0 0 0 0 5 5 4)
关键词
C基因
人参
发根
UC
皂苷
表达
栽体
酶切
子叶
粒重
rolC gene Ginsenosides PCR amplification Ginseng hairy roots Plant expression vector
