木薯内标准基因定性PCR方法分析

Analysis of Cassava Endogenous Reference Genes by Using Qualitative Polymerase Chain Reaction(PCR)

内标准基因对于转基因植物及其产品的定性定量PCR检测起着决定性的作用,木薯内标准基因的系统研究是木薯转基因成分检测的基础。在木薯的DFCI(http://compbio.dfci.harvard.edu/tgi/plant.html)数据库中,选择了有可能作为木薯内标基因的一些EST序列,包括亲环蛋白2(cyclophilin type 2,DB934316)、3-磷酸甘油醛脱氢酶基因B亚单位(Glyceraldehyde-3-phosphate dehydrogenase B subunit,TC445)GAPDH、Actin基因(TC2158与TC3381的拼接序列)、Alpha-tubulin(TC8072)基因,Polyubiquitin(TC9946),亚麻苦苷酶(linamarase,TC1540)6个基因序列,设计了7对定性PCR的引物(CP2、Actin、GAPDH、PUBQ2、Tublinα1、Tublinα2和LAM1),并进行了种内一致性、稳定性和种间特异性测试分析。结果表明,只有木薯的3-磷酸甘油醛脱氢酶基因B亚单位的GAPDH定性PCR引物扩增系统在木薯的22个不同品种中都能够得到一致性稳定性的扩增结果,在大戟科其他植物及其他非大戟科植物没有发现非特异性扩增产物。另外6个基因(CP2、Actin、PUBQ2、Tublinα1、Tublinα2和LAM1)的定性PCR引物扩增系统在大戟科其他植物及非大戟科植物中都有非特异性的扩增产物。因此GAPDH定性PCR引物扩增系统初步符合内标准基因的种内一致性,稳定性,种间特异性的特点,可应用于转基因木薯成分定性PCR检测。

Endogenous Reference Gene is a determining factor in detection the transgenic plants and productsby using Qualitative Polymerase Chain Reaction（PCR） and Real- Time Quantitative methods. The systemresearch in cassava endogenous reference genes could lay a foundation for detection of genetically modifiedcassavas and derived products. Several possible endogenous reference genes of cassava including cyclophilintype 2（DB934316）, Glyceraldehyde- 3- phosphate dehydrogenase B subunit（GAPDH, TC445）, Actin（the edited sequences of TC2158 and TC3381）, Alpha- tubulin（TC8072）, Polyubiquitin（TC9946）, linamarase（TC1540） were selected from DFCI（http：//compbio.dfci.harvard.edu/tgi/plant.html） of cassava ESTS, and 7pairs primers（CP2, Actin, GAPDH, PUBQ2, Tublinα1, Tublinα2 and LAM1） were designed, then theirinternal consistency were assayed in 22 different cassava varieties and their species specific was also detectedin other 12 non-cassava euphorbiaceae species and 20 non-euphorbiaceae species. For GAPDH the identicalamplification products were obtained with the DNA samples from 22 different cassava varieties, noamplification products were observed in other 12 non- cassava euphorbiaceae species and 20 non-euphorbiaceae species. As for another 6 pairs primers（CP2, Actin, PUBQ2, Tublinα1, Tublinα2 and LAM1）,the non-specificity amplification products were observed in the non-cassava euphorbiaceae species or non-euphorbiaceae species. These results indicated the GAPDH PCR amplification system was species specificand internal consistency and could be used as the endogenous reference genes for the detection of geneticallymodified cassavas and derived products.