荻草谷网蚜两个内参基因的克隆及表达谱分析

Cloning and Expression Profiling of Two Reference Genes in Grain Aphid Sitobion miscanthi

利用染料法荧光定量PCR技术开展功能基因的表达量检测时,表达稳定内参基因的选择和引物的高特异性是确保检测结果准确可靠的前提。本文针对昆虫通用的内参基因NADH(nicotinamideadenine dinucleotide dehydrogenase)和DIMT(dimethyladenosine transferase)在荻草谷网蚜中进行了基因克隆、序列分析、不同龄期的时间表达谱分析和基因表达稳定性评估。两个基因的GenBank登录号分别为OL770461和OL770462。NADH和DIMT基因克隆序列与基于高通量测序技术预测结果高度一致,但仍有不同。以β-actin为内参分析两个表达谱,结果显示各自表达稳定、龄期间无显著差异;稳定值由高到低为β-actin,NADH,DIMT,稳定值彼此接近、远小于阈值1.5。以上研究结果说明,通常作为内参基因的持家基因虽然保守,在物种间仍可能存在较大变异,直接引用其他物种的内参引物不可行;不但如此,一代测序技术虽然通量低但准确度极高,与之相比,二代高通量测序则错误率较高,因此引物设计使用的基因应首做克隆测序验证;最后,NADH和DIMT在荻草谷网蚜各发育阶段表达稳定,适用于蚜虫基因、特别是不同发育阶段的基因表达差异分析。

Stable expression reference genes are critical for accurate quantification of target genes expression by SYBR Green quantitative PCR.Suitable reference genes were screened among different developmental stages in the grain aphid Sitobion miscanthi.Two reference genes NADH(Nicotinamide adenine dinucleotide dehydrogenase) and DIMT(Dimethyladenosine transferase) were cloned and analyzed based on genomic annotation.Expression patterns among different developmental stages were measured and the expression stabilities were evaluated.The results showed that the sequences of SmisNADH and SmisDIMT were almost consistent with the prediction except one nucleotide acid.Homologous alignment showed that the matching aphid sequences were generally based on high-throughput sequencing and not verified by cloning,and the sequence differences between species were large.The GenBank accession Nos are OL770461 and OL770462 respectively.Both of their expression levels showed no significant differences among different instars and adult.Their stabilities ranked β-actinNADHDIMT,but the values were close to each other and much lower than the threshold of 1.5.The above results show that although the housekeeping genes usually used as internal reference genes are conservative,there might still be large variations among species,and it is not feasible to directly use the internal reference primers of other species.In addition,although the first-generation sequencing technology has low throughput but high accuracy,the second-generation high-throughput sequencing has high error rate,so the genes used in primer design should be first verified by cloning sequencing.Further,the expression levels of NADH and DIMT are stable among instars as well as the adult stage,indicating that they are suitable as the reference genes to analyze functional gene expression in aphids,especially for analyzing the expression differences among developmental stages.