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

doi:10.16409/j.cnki.2095-039x.2022.03.014

Abstract

Abstract: 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-actin＞NADH＞DIMT, 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.

Key words: Sitobion miscanthi, gene cloning, reference gene, expression profile, expression stability

CLC Number:

ZHANG Siyu, YU Miaomiao, WANG Wenkai, FAN Jia, CHEN Julian. Cloning and Expression Profiling of Two Reference Genes in Grain Aphid Sitobion miscanthi[J]. Chinese Journal of Biological Control, 2023, 39(1): 122-129.

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