Study On Selection Pressure And The Structure Of CRY1 Gene’s Core Promoter In Hu Sheep

Sheep,short-day animals,usually come into heat in the autumn and winter and lamb intensively in the following spring.This seasonal nature of breeding limits its reproductive efficiency and greatly restricts the amount of meat sheep that can be slaughtered.It is essential to investigate the reasons inherent in seasonal reproduction.CRY genes were closely related to mammalian seasonal reproduction.In this study,we analyzed their origin and differentiation,as well as the selection pressure of CRY genes in the formation of seasonal reproductive rhythm in mammals,analyzed the changes of CRY genes during the puberty in Hu sheep at the transcriptional level,and studied the core promoter of CRY1gene.The main of all results were listed as follows:（1）Phylogenetic tree analysis showed that mammalian CRY family may be the product of the second round of genome-wide replication;Protein structure analysis showed that the CRY family had obvious differentiation,and the helix length of the C-terminal of CRY1protein was gradually shortened in the process of evolution.（2）The selection pressures of mammalian CRY genes were analyzed by the branch model and site model of codeml program.The results showed that both mammalian CRY1and CRY2 branches were subject to purification selection;the amino acid at 558^th site of CRY1 protein was positively selected in the seasonal breeding mammals,which was threonine in 77.8%of short-day mammals and methionine in 60%of long-day mammals;Bioinformatics analysis showed that the mutation from T to M at 558^th site would shorten theα-Helix length of the C-terminal and affect the hydrophobicity of CRY1 protein.Further more,FRAP experiments showed that the rate of entry into the nucleus sheep M558 protein was significantly higher than that of T558 site（p<0.01）.（3）Transcriptome analysis showed that the CRY family was expressed in all reproductive axes;the transcript level of CRY1 gene was significantly up-regulated in testicular tissues of Hu sheep during proestrus（p<0.05）.CRY1 protein might interact with PPP2R1B and USP2 proteins,and might be involved in spermatogenesis in sheep at early estrus.（4）The core promoter region of the Hu sheep CRY1 gene was inferred to be located at-1160/-958 bp in the upstream of the start codon by comparative genomics analysis and constructing the luciferase reporter vectors containing 5’-terminal deletion fragment in CRY1 gene and analyzing the double luciferase activities.（5）The results of mutation test of TCFL5/NRF1 binding sites showed that TCFL5/NRF1 binding sites（-1033/-1024 bp）could significantly affect the activity of CRY1core promoter（p<0.001）;After overexpression of TCFL5,the activity of its core promoter increased significantly（p<0.01）.The results of EMSA showed that the transcription factor TCFL5 might bind to the core promoter of CRY1 gene.In addition,the transcription levels of TCFL5 and CRY1 gene in testes of Hu sheep were positively correlated by quantitative PCR（p<0.01）...