光裸星虫Sn-hsc70基因克隆及表达分析

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

中国农学通报 ›› 2021, Vol. 37 ›› Issue (9): 95-102.doi: 10.11924/j.issn.1000-6850.casb2020-0268

所属专题：生物技术

光裸星虫Sn-hsc70基因克隆及表达分析

黄剑强¹(), 魏雯璐¹, 钟如卓¹, 张家炜¹, 杨创业¹^,²(), 王庆恒¹^,²

¹广东海洋大学水产学院,广东湛江 524088
²广东省海水养殖生物育种工程实验室,广东湛江 524088

收稿日期:2020-07-20 修回日期:2020-09-28 出版日期:2021-03-25 发布日期:2021-04-09
通讯作者: 杨创业
作者简介:黄剑强,男,1995年出生,广东信宜人,硕士,研究方向：海水无脊椎动物增养殖。通信地址：524088 广东省湛江市麻章区海大路1号,Tel：13414950265,E-mail： 1204987259@qq.com。
基金资助:
广东省科技计划“光裸星虫高效健康养殖关键技术集成与应用示范”(2016A020209010,“光裸星虫沙虫优质苗种规模化繁育技术研究与示范”163-2019-XMZC-0009-02-0059);广东省大学生创新创业训练计划项目“光裸星虫卵子发生关键因子的筛选和功能研究”(201810566049)

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

Huang Jianqiang¹(), Wei Wenlu¹, Zhong Ruzhuo¹, Zhang Jiawei¹, Yang Chuangye¹^,²(), Wang Qingheng¹^,²

¹Fisheries College Guangdong Ocean University, Zhanjiang Guangdong 524088
²Guangdong Provincial Engineering Laboratory for Mariculture Organism Breeding, Zhanjiang Guangdong 524088

Received:2020-07-20 Revised:2020-09-28 Online:2021-03-25 Published:2021-04-09
Contact: Yang Chuangye

摘要/Abstract

摘要：

旨在研究光裸星虫热休克蛋白70 (Hsc70)在卵子发生过程中发挥的作用,为深入认识光裸星虫卵母细胞发育的分子机制积累基础资料。以光裸星虫为材料,用RACE技术获得光裸星虫Sn-hsc70基因的cDNA全长。进行生物信息学分析得出,Sn-hsc70全长2508 bp,编码656个氨基酸;Sn-Hsc70蛋白为71.65 kDa亲水蛋白。同时还分析了该蛋白的保守序列、核定位序列、胞质定位序列、连续重复的四肽序列、三级结构、系统进化树及结构域。RT-PCR结果显示,Sn-hsc70在光裸星虫卵母细胞发育的各个时期的表达量差异显著(P < 0.05),整体呈单峰型;在体腔液发育时期(O1-O4),Sn-hsc70表达量迅速上升,尤其是卵黄旺盛合成期和成熟期(O3-O4)显著高表达;脱离体腔液后(O5-O6),Sn-hsc70表达量显著下调。研究结果表明Sn-hsc70可能在卵黄积累中发挥重要作用,与卵母细胞转录与蛋白质合成减弱等有关。

关键词: 光裸星虫, 热休克反应, 热休克蛋白70, 基因克隆, 表达分析, 卵母细胞

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

中图分类号:

黄剑强, 魏雯璐, 钟如卓, 张家炜, 杨创业, 王庆恒. 光裸星虫Sn-hsc70基因克隆及表达分析[J]. 中国农学通报, 2021, 37(9): 95-102.

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.

图/表 7

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)

表1. Sn-hsc70基因克隆及荧光定量的引物序列

图1. Sn-hsc70 cDNA序列全长及推导的氨基酸序列 5′和3′UTR用小写字母表示;编码区以及推导的氨基酸序列用大写字母表示。Hsp70蛋白家族的特征序列用矩形框表示;四肽重复序列用双下划线表示;胞质定位序列用圆点表示;核定位序列用单划线表示

图2. Sn-Hsc70蛋白残基亲水性分布图

图3. Hsc70同源蛋白多序列比对 Hsp70蛋白家族特征序列用双划线标注;多次连续重复四肽序列用矩形框标注,胞质定位序列以三划线标注;两个核定位序列用单划线标注

图4. 光裸星虫(a)、双齿围沙蚕(b)和栉孔扇贝(c)的Hsc70蛋白三级结构

图5. 同源Hsc70的系统发育树及结构域

图6. Sn-hsc70在卵母细胞各发育时期的相对表达量

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光裸星虫Sn-hsc70基因克隆及表达分析

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

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