Sn-hsc70 Gene in Sipunculus nudus: Cloning and Expression Analysis

doi:10.11924/j.issn.1000-6850.casb2020-0268

Abstract

Abstract:

The purpose is to explore the role of heat shock protein 70 (Hsc70) from Sipunculus nudus in the process of oogenesis, and to accumulate basic data for understanding the molecular mechanism of S. nudus oocyte development. With S. nudus as the samples, the full-length cDNA of Sn-hsc70 gene was obtained with the method of RACE technology. According to the bioinformatics analysis, Sn-hsc70 held the full-length of 2508 bp and encoded 656 amino acids; Sn-Hsc70 protein was a hydrophilic protein with molecular weight of 71.65 kDa. The conserved sequence, nuclear localization sequence, cytoplasmic localization sequence, continuous repeating tetrapeptide sequence, advanced structure, phylogenetic tree and domain of the protein were also analyzed. RT-PCR results showed that the expression level of Sn-hsc70 was significantly different in various stages of oocyte development (P <0.05) and showed a single peak. During the development of coelomic fluid (O1-O4), the expression of Sn-hsc70 increased rapidly, especially in the vigorous synthesis stage and mature stage of yolk (O3-O4). The expression of Sn-hsc70 was significantly down-regulated after leaving the coelomic fluid (O5-O6). The study indicates that Sn-hsc70 might play an important role in the accumulation of yolk, which is related to the weakening of oocyte transcription and protein synthesis.

Key words: Sipunculus nudus, heat shock response, heat shock protein 70, gene cloning, expression analysis, oocytes

CLC Number:

Huang Jianqiang, Wei Wenlu, Zhong Ruzhuo, Zhang Jiawei, Yang Chuangye, Wang Qingheng. Sn-hsc70 Gene in Sipunculus nudus: Cloning and Expression Analysis[J]. Chinese Agricultural Science Bulletin, 2021, 37(9): 95-102.

Figures/Tables 7

References 35

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引物	引物序列(5′-3′)	用途
5'-outer	ATCGTTGAAGTATGCTGGTA	5′RACE
5'-inner	TTGTCGCTGATGTCCTTC	5′RACE
3'-outer	CCACGGTTGAAGATGAGAA	3′RACE
3'-inner	GGAAGAATACGAGCACCAA	3′RACE
M13-F	CGCCAGGGTTTTCCCAGTCACGAC	菌落PCR
M13-R	AGCGGATAACAATTTCACACAGGA	菌落PCR
qPCR-S	CTCTGATGACCTGCCACTGAA	荧光定量
qPCR-A	TGTGCTCCTGCTTCTCCTTC	荧光定量
60SL7-S	CAGGCTAACAACTTCTTATGG	荧光定量(内参)
60SL7-A	TGGCTTGACAGATAACACTT	荧光定量(内参)

引物	引物序列(5′-3′)	用途
5'-outer	ATCGTTGAAGTATGCTGGTA	5′RACE
5'-inner	TTGTCGCTGATGTCCTTC	5′RACE
3'-outer	CCACGGTTGAAGATGAGAA	3′RACE
3'-inner	GGAAGAATACGAGCACCAA	3′RACE
M13-F	CGCCAGGGTTTTCCCAGTCACGAC	菌落PCR
M13-R	AGCGGATAACAATTTCACACAGGA	菌落PCR
qPCR-S	CTCTGATGACCTGCCACTGAA	荧光定量
qPCR-A	TGTGCTCCTGCTTCTCCTTC	荧光定量
60SL7-S	CAGGCTAACAACTTCTTATGG	荧光定量(内参)
60SL7-A	TGGCTTGACAGATAACACTT	荧光定量(内参)