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在转基因植物及其产品的成分检测中,内参照基因(endogenous reference gene)起着重要的作用,木薯内参照基因的研究是转基因木薯成分定性PCR和定量PCR检测的基础。本研究选择木薯的亚麻苦苷酶(linamarase,LAM)和Polyubiquitin(PUBQ)基因为研究对象,运用定性PCR的方法在22种木薯的不同品种中进行稳定性分析,在12种大戟科其它植物、20种非大戟科植物及五类主要转基因作物的混合样品(玉米,大豆,水稻,棉花和油菜)中对LAM2和PUBQ1定性PCR引物进行特异性检测分析及灵敏度检测分析。结果显示:LAM2和PUBQ1引物在木薯的不同品种中均能得到稳定性扩增;在12种大戟科其它植物、20种非大戟科植物能够进行特异性扩增,没有发现非特异性扩增产物出现;LAM2在五类主要转基因作物的混合样品(玉米,大豆,水稻,棉花和油菜)中也没有非特异性扩增产物出现,而PUBQ1在五类主要转基因作物的混合样品(玉米,大豆,水稻,棉花和油菜)中却有很多的非特异性扩增产物出现。灵敏度检测发现定性PCR LAM2检测方法的灵敏度达到质量百分含量0.01%,SYBR GreenⅠ荧光定量PCR检测结果表明LAM2的灵敏度可达到0.001~0.000 1 ng/μL。选择部分品种的PCR扩增产物进行测序,序列分析表明LAM2在不同品种的木薯的基因组DNA均可以扩增到293 bp的核苷酸片段,比对分析表明所获得的木薯不同品种中LAM2 PCR扩增片段的核苷酸序列一致没有发现变异位点。研究结果表明LAM2引物扩增系统初步符合内参照基因的种内一致性,稳定性,种间特异性的特点,灵敏度检测的要求可应用于转基因木薯成分定性和定量PCR检测。
The endogenous reference gene plays an important role in the compositional analysis of transgenic plants and their products. The study of reference genes in cassava is the basis of qualitative PCR and quantitative PCR of cassava compositions. In this study, we selected the cassava linamarase (LAM) and Polyubiquitin (PUBQ) genes as the research object, using qualitative PCR analysis of stability in different varieties of 22 kinds of cassava in 12 kinds of other Euphorbiaceae plants , 20 non-Euphorbiaceae plants and five major GM crops (corn, soybean, rice, cotton and canola) were detected by specific PCR and sensitivity analysis. The results showed that both LAM2 and PUBQ1 primers could amplify stably in different varieties of cassava; among 12 species of Euphorbiaceae, 20 species of non-Euphorbiaceae could be amplified specifically and no nonspecific amplification was found LAM2 also showed no non-specific amplification products in the mixed samples of five major GM crops (corn, soybean, rice, cotton and canola), whereas PUBQ1 did not appear in the mixed samples of five major GM crops (corn, soybean, Rice, cotton and canola), many non-specific amplification products appear. The sensitivity test showed that the sensitivity of qualitative PCR LAM2 detection method reached 0.01% mass percentage. The results of SYBR Green Ⅰ fluorescence quantitative PCR showed that the sensitivity of LAM2 reached 0.001 ~ 0.000 1 ng / μL. The PCR products of some varieties were sequenced. The sequence analysis showed that the genomic DNA of LAM2 in different varieties of cassava could be amplified to 293 bp. The alignment analysis showed that LAM2 PCR amplification The nucleotide sequence of the fragment was unanimous and no mutation was found. The results showed that the amplification system of LAM2 primer preliminarily accorded with the intraspecific identity, stability and interspecificity of the internal reference genes. The sensitivity detection requirements could be applied to qualitative and quantitative PCR detection of transgenic cassava components.