摘要
根据GenBank中海州香薷(Elsholtzia haichowensis)液泡转化酶基因EhNvINV(JX500755)和EhCvINV(JX500756)的序列设计特异性引物,克隆cDNA全长序列,并通过生物信息学分析基因及推导的蛋白序列,利用SWISS-MODEL进行同源建模,并与蔗糖分子进行模拟对接,实时荧光定量PCR分析EhNvINV和EhCvINV在铜胁迫下的转录表达。结果表明,海州香薷非抗性和抗性种群液泡转化酶蛋白EhNvINV和EhCvINV在114和346处存在氨基酸趋异位点,模拟3D结构相似,仅在趋异位点Glu114/Gln114和Leu346/Pro346处有差别。EhNvINV、EhCvINV和模拟突变体(EhNvINV-E114Q和EhNvINV-L346P)与蔗糖分子对接形成的活性中心构象基本一致,但在空间位置上存在细微差异。实时荧光定量PCR结果表明铜胁迫7 d后,EhCvINV的表达受铜的诱导,而EhNvINV受铜的抑制。
Specific primers were designed to clone the cDNA sequences according to the EhNvINV（JX500755）and EhCvINV（JX500756）from Gen Bank. The DNA and deduced amino acid sequences were analyzed by bioinformatics methods. The three-dimensional structures were constructed by homologous modeling. The structures of vacuolar invertase from two populations of E. haichowensis and non-tolerant population with each single point mutation in complex with sucrose were simulated by Auto Dock 4.0. Transcript expression of EhNvINV and EhCvINV under copper tress was analyzed by real-time PCR. The results showed that there were two divergent amino acids at position 114 and 346 between EhNvINV and Eh Cc INV. The three-dimensional structures were exactly similar between EhNvINV and EhCvINV. It showed difference at Glu114/Gln114 and Leu346/Pro346,which were divergent sites. The structures of catalytic active center of EhNvINV,EhCvINV,and simulated mutants（EhNvINV-E114 Q,EhNvINV-L346P）binding with sucrose showed no significant differences. However,there were differences on spatial position. The result of real-time PCR indicated that the transcript expression of EhCvINV induced by copper stress after 7 days,however,the transcript expression of EhNvINV inhibited by copper stress after 7 days.
出处
《生物技术通报》
CAS
CSCD
北大核心
2016年第10期170-179,共10页
Biotechnology Bulletin
基金
国家自然科学基金项目(21477093
31270432)
生物质资源化学与环境生物技术湖北省重点实验室开放基金项目(HBRCEBL-2013-2014004)
遵义师范学院博士基金项目(遵师BS[2014]20号)
关键词
海州香薷
液泡转化酶
铜胁迫
转录表达
Elsholtzia haichowensis
vacuolar invertase
copper stress
transcript expression
