Selection of Reference Genes and Study of the Expression Levels of CIRP and Hsp70 Genes After Low Temperature Treatment on Japanese Flounder (Paralichthys olivaceus) Embryos
Abstract：Abstract Japanese flounder (Paralichthys olivaceus) is one of the important commercial marine fish in China. The embryo cryopreservation of Japanese flounder have been successful, however, there has been no study on the gene expression profiling in its embryos under low temperature. In this study, embryos at somite stage, tail bud stage and heart-beating stage were treated at 0 ℃, respectively, while embryos was cultured at 16.5 ℃ as the control. The stability of 4 candidate genes were analyzed by using GeNorm and NormFinder software, including 18S rRNA, ACTB, GAPDH and EF1α. Results showed that the stability of 4 genes were 18S rRNA=EF1α＞ACTB＞GAPDH by using the GeNorm software; while, the stability of 4 genes were 18S rRNA＞ACTB＞EF1α＞GAPDH by using NormFinder software. Therefore, 18S rRNA was supposed as the most stable housekeeping gene in Japanese flounder embryos incubated in low temperature. In order to study the influence of low temperature on gene expression of Japanese flounder embryos, embryos at tail bud stage were treated at 0 ℃ as embryos at this stage were more adaptive to temperature variations. Embryos culturing at 16.5 ℃ were used as control group. The expression variations of 2 genes, including cold-inducible RNA-binding protein gene (CIRP) and heat shock protein gene (Hsp70) were measured and analyzed by using qRT-PCR. It was found that CIRP mRNA expression decreased within 30 min, increased and reached the peak in 120 min, and then decreased again in 180 min to the level that was no significant different from the control. Hsp70 mRNA expression dropped in the first 30 min of treatment, then increased and reached the peak in 120 min; however, it decreased during 120~180 min, which showed significant difference from the control group (P＜0.05). These results indicated that low temperature would trigger the change of gene expressions of CIRP and Hsp70. Meanwhile, the variation trend suggested that there could be a protection mechanism in cells against low temperature. This study is a good basis for the further exploration on the adaption to low temperature of Japanese flounder embryos.
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