脊尾白虾隐花色素基因cry1的克隆及其功能分析
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Q 785;S 917.4

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国家重点研发计划(2018YFD0901302);江苏省高校优势学科建设工程资助项目(PAPD);江苏省“六大人才高峰”创新人才团队资助项目(2016-HYGC-CXTD-004);江苏省2018年度普通高校研究生科研创新计划(KYCX18-2571)


Cloning of cryptochrome 1 gene and its expression characteristics analysis in Exopalamon carinicauda
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This project was Funded by Natural Science Foundation of the Jiangsu Higher Education Institutions of China(Grant No. 17KJA240001 ),Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD) and "six talent summit" innovative talent team of Jiangsu (2016-HYGC-CXTD-004),Jiangsu Province University Graduate Research and Innovation Plan for 2018 (KYCX18-2571)

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    摘要:

    为探究隐花色素基因(cry1)在甲壳类中的节律调节功能,实验根据脊尾白虾转录组序列,利用RACE技术获得了脊尾白虾cry1的cDNA序列全长并对其进行功能分析。脊尾白虾cry1全长2 190 bp,开放阅读框1 845 bp,5'端非编码区为241 bp,3'端非编码区为104 bp,共翻译出614个氨基酸,预测蛋白质的分子质量为70.5 ku,理论等电点为5.09。同源性分析显示,脊尾白虾cry1与凡纳滨对虾的同源性最高,为71.6%。荧光定量分析结果显示,脊尾白虾cry1在眼柄、鳃、心脏、胃、肝胰腺、性腺、肌肉、肠道和腹索神经中均有表达,其中眼柄的表达量最高,性腺和心脏次之;不同时段的表达结果发现,其表达量在日节律(24 h)中表现出先下降再上升的趋势。不同光色条件下RNA干扰(RNA interference, RNAi)结果显示,注射小干扰RNA(siRNA)后3~6 h蓝光光照下脊尾白虾cry1的表达量显著高于白光光照,9~24 h蓝色和白色光照下的表达量无显著差异,而RNA干扰组的表达量显著低于对照组,此结果表明cry1可能主要响应蓝光周期节律。目前对甲壳类生物钟的研究较少,该研究为深入探究甲壳类生物钟基因的调控机制提供帮助。

    Abstract:

    Cryptochrome (Cry) is a kind of blue violet light receptor, which widely exists in animals, plants, bacteria and human body. At present, the research on cry mainlmainly focuses on its biological function, but rarely reported in crustacean biological rhythm. Exopalaemon carinicauda is a unique marine economic shrimp in China. Studying and understanding the circadian clock genes will help deepen the understanding of the biological clock regulation mechanism of crustaceans represented by E. carinicauda. The results showed that the full length of cry1 in E. carinicauda was 2 190 bp, the open reading frame was 1 845 bp, the 5 'noncoding region was 241 bp, and the 3' noncoding region was 104 bp. A total of 614 amino acids were translated. The predicted molecular weight of the protein was 70.5 ku and the theoretical isoelectric point was 5.09. After analyzing the amino acid sequence of Cry1, it was found that the Cry1 contains a structure homologous to DNA photolyase at 117-246 aa of N-terminal and a FAD binding domain at C-terminal. Homology analysis showed that the cry1 of E. carinicauda shared the highest homology with Litopenaeus vannamei and Euphausia superba (71.6% and 68.3%, respectively). Results of qRT-PCR analysis showed that cry1 of E. carinicauda was expressed in eyestalk, gill, heart, stomach, hepatopancreas, gonad, muscle, intestine and ventral cord nerve, and the expression level of eyestalk was the highest. The expression results at different time periods showed that the expression level of the cry1 in the eyestalks of E. carinicauda firstly decreased with the increase of the light time within 0-24 h, then began to increase after the lowest value at 9 h, and then decreased at 18 h. To the second trough, and finally re-entering the dark period, there is a significant increase again, and it was consistent with the more active rhythm of the E. carinicauda in the dark phase. After performing RNA interference under different light color conditions, the expression of cry1 in RNA interference group was significantly lower than that in control group under two light color conditions, which indicated that the expression of cry1 gene was successfully interfered in this study. The expression of cry1 in blue light was significantly higher than that in white light at 3-6 h after injection, but decreased at 9-24 h. The difference indicates that the cry1 is involved in the light signal transduction process under both blue and white light conditions, particularly involved in responding to the blue light periodic rhythm. This study provides a theoretical basis for in-depth exploration into the regulatory mechanism of crustacean circadian clock in the current situation where there is little research on crustacean circadian clock genes.

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朱佶轩,戴琴,高威,段健诚,宋崇阳,张攀,阎斌伦,高焕.脊尾白虾隐花色素基因cry1的克隆及其功能分析[J].水产学报,2021,45(2):170~178

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  • 收稿日期:2020-05-18
  • 最后修改日期:2020-06-09
  • 录用日期:2020-05-13
  • 在线发布日期: 2021-01-30
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