QRT-PCR Reference Gene Screening And TERT Gene Expression Analysis Of Sepia Esculenta

Sepia esculenta is a representative species of the Coleoidea.It is an annual and only breeds once in a lifetime.It dies quickly after reproduction and belongs to one reproductive type.This phenomenon of reproduction and death that occurs in a short period of time can serve as an entry point for studying the intricate relationship between reproduction,aging and death.The telomere theory of aging suggests that telomeres play an important role in the process of cell aging.Each cell division will not completely replicate their chromosomes due to DNA polymerase dysfunction.The length of the telomeric DNA sequence will gradually shorten and eventually causes cell aging and die.Telomerase can extend telomere sequence,maintain chromosome stability,and ensure cell replication.The TERT(telomerase reverse transcriptase)gene is the key structure and major regulatory subunit of telomerase action.The TERT gene synthesizes and extends telomere length by reversing transcription of the telomerase RNA template sequence,synthesizing telomeric DNA repeats and adding them to the ends of the chromosome.In order to study the changes of TERT gene expression during the Sepia esculenta's reproductive to natural death.This study screened the TERT gene from the ovary transcript data of the golden squid obtained in the previous study group.We selected three key growth and development stages of the Golden Squid's Growth Stage(GS),Spawning Stage(SS),and Aging Stage(AS).In this study,qRT-PCR was used to analyze the expression of TERT genes in different developmental stages,different tissues and genders,which laid a foundation for revealing the relationship between reproduction and senescence of Sepia esculenta.1?In order to understand the molecular mechanism of the regulation of growth,reproduction and senescence of Sepia esculenta,real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR)is widely used for gene expression analysis.However,there is no reliable reference gene for standardization of qRT-PCR data.We selected 6 reference gene genes of glucose-6-phosphodehydrogenase(G6pd),glyceraldehyde 3-phosphodehydrogenase(GAPDH),18 S ribosomal RNA(18S),actin(ACTB),28 S ribosomal RNA(28S),and ?-tubule protein gene(TUB).This study used geNorm,Normfinder,and Bestkeeper algorithms to analyze the stability of six internal reference genes.The results showed that the most stable reference gene in the male and female gonads and liver was 18 SrRNA.These results laid the foundation for the analysis of qRT-PCR gene expression during the development,reproduction and senescence of golden squid.2 ? In this study,the suspected TERT gene sequences were screened from the transcriptome of Sepia esculenta.The TERT gene was identified by phylogenetic analysis and homologous sequence analysis.The sequence length was 2415 bp.We used real-time fluorescence quantitative PCR technology to detect the relative expression levels of TERT genes in the growth period,spawning period and dying period of the key tissues of Sepia esculenta,and analyzed the spatial and temporal differences and gender differences.The results showed that the expression of TERT gene in the heart,liver,pancreas,gill,gonad,and supraesophageal nerve group(excluding the optic gland and the brain tissue behind the optic lobe)showed a significant downregulation trend compared with the oviposition period.Therefore,it is speculated that it is related to the decreased ability of organ tissue division and proliferation in the dying period.The TERT gene has obvious tissue specificity in different tissues of the Sepia esculenta,indicating that the senescence state of the tissues and organs of the Sepia esculenta is not consistent.The results of this study can provide basic data for the in-depth study of the telomerase regulation mechanism of cephalopod reproduction and aging and other life processes.