摘要
类病毒是由247～600个核苷酸组成的单链、共价闭合环状RNA,无蛋白质外壳.采用酚-氯仿抽提法提取梨中类病毒的粗核酸,再用DEAE纤维素过柱纯化;通过往返电泳检测,对比ASSVd,可知梨类病毒基因长度在328bp左右.设计引物,RTP-CR扩增此类病毒,除去所得PCR产物中的酶蛋白、单核苷酸等,再转化大肠杆菌DH5α;挑取阳性转化子,用碱性裂解法小量提取转化子质粒DNA;进行EcoRⅠ和HaeШ酶切鉴定,结果与预期的梨皱果类病毒基因大小一致.
Viroids are single-strand,covalent and ringy RNA molecules which are composed of 247 to 600 nucleicacids and lack protien capsid.The nucleicacids from pear are obtained with phyenol-CHCl3 and are purified by DEAE.The length of the viroid gene is about 317 bp by round electrophoresis and ASSVd comparison.According to the sequence of ASSVd gene the primers are designed.Then the target gene was amplified by RT-PCR.After that the enzyme and single nucleiocide of the PCR production are deleted.The target gene is transformed into E.coli(DH5α). Judging from white-blue spot,the positive clones are selected.Then the plasmid DNA of transformants is obtained through SDS basic lysomic method.By using restrict endonucleotidase EcoRⅠand Hae Ⅲ,the result is consistent with the expected size of pear viroid gene.
出处
《生命科学研究》
CAS
CSCD
2006年第S2期85-91,共7页
Life Science Research
