脊尾白虾隐花色素基因cry1的克隆及其功能分析
CSTR:
作者:
中图分类号:

Q 785;S 917.4

基金项目:

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
Author:
Fund Project:

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)

  • 摘要
  • | |
  • 访问统计
  • |
  • 参考文献 [35]
  • |
  • 相似文献 [20]
  • | | |
  • 文章评论
    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.

    参考文献
    [1] Liu B, Yang Z H, Adam G, et al. Signaling mechanisms of plant cryptochromes in Arabidopsis thaliana[J]. Journal of Plant Research, 2016, 129(2): 137-148
    [2] 吕垣澄, 吴晓晖. 动物隐花色素研究进展[J]. 水产学报, 2014, 36(9): 864-870
    Lv Y C, Wu X H. Research progresses in animal cryptochromes[J]. Hereditas, 2014, 36(9): 864-870(in Chinese)
    [3] 梁辉, 王文栋, 朱晓苏, 等. 家蚕隐花色素基因Cry1与Cry2的表达谱与功能研究[C]//2011年全国时间生物医学学术会议论文集. 南宁: 中国中西医结合学会时间生物医学专业委员会, 2011: 40-54.
    Liang H, Wang W D, Zhu X S, et al. Cloning and functional identification of Cry gene from Bombyx mori[C]//The Professional Committee of Time Biomedicine of the Chinese Society of Integrated Traditional Chinese and Western Medicine. Nanning: Time Biomedical Committee of Chinese Society of Integrated Traditional and Western Medicine, 2011: 40-54 (in Chinese).
    [4] 韦献雅, 牛应泽, 余青青, 等. 拟南芥CRY1基因C末端导入甘蓝型油菜的遗传及表达[J]. 高技术通讯, 2007, 17(6): 648-652
    Wei X Y, Niu Y Z, Yu Q Q, et al. Inheritance and expression of the C-terminal fragment of Arabidopsis Cry1 gene transferred into Brassca. napus[J]. Chinese High Technology Letters, 2007, 17(6): 648-652(in Chinese)
    [5] Zhang Y C, Gong S F, Li Q H, et al. Functional and signaling mechanism analysis of rice CRYPTOCHROME 1[J]. The Plant Journal, 2006, 46(6): 971-983
    [6] Hirose F, Shinomura T, Tanabata T, et al. Involvement of rice cryptochromes in de-etiolation responses and flowering[J]. Plant and Cell Physiology, 2006, 47(7): 915-925
    [7] 张小冰. 植物隐花色素及其功能研究进展[J]. 生物学教学, 2010, 35(7): 4-5
    Zhang X B. Research advances in plant cryptochrome and its function[J]. Biology Teaching, 2010, 35(7): 4-5(in Chinese)
    [8] 朱涵毅, 薛慧良, 徐金会, 等. 黑线仓鼠Cry1, Cry2克隆及生物信息学分析[J]. 曲阜师范大学学报(自然科学版), 2019, 45(4): 74-82
    Zhu H Y, Xue H L, Xu J H, et al. Clone and bioinformatics analysis of Cry1, Cry2 in Criecetulus barabensis[J]. Journal of Qufu Normal University (Natural Science), 2019, 45(4): 74-82(in Chinese)
    [9] Gegear R J, Foley L E, Casselman A, et al. Animal cryptochromes mediate magnetoreception by an unconventional photochemical mechanism[J]. Nature, 2010, 463(7282): 804-807
    [10] Hazlerigg D G, Andersson H, Johnston J D, et al. Molecular characterization of the long-day response in the Soay sheep, a seasonal mammal[J]. Current Biology, 2004, 14(4): 334-339
    [11] Van der Horst G T J, Muijtjens M, Kobayashi K, et al. Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms[J]. Nature, 1999, 398(6728): 627-630
    [12] 汪京京. Timeless通过结合Cry1aa负向调控斑马鱼生物钟[D]. 苏州: 苏州大学, 2015.
    Wang J J. Zebrafish Timeless negatively regulates the circadian clock through binding to Crylaa[D]. Suzhou: Soochow University, 2015 (in Chinese).
    [13] 张鹏. 中华绒螯蟹隐花色素EsCry和铁硫簇组装蛋白EsIscA2在生物节律和免疫调控中的作用初探[D]. 大连: 大连海洋大学, 2018.
    Zhang P. Role of EsCry and EsIscA2 in biological rhythm and immune regulation of Chinese mitten crab[D]. Dalian: Dalian Ocean University, 2018 (in Chinese).
    [14] 易健明, 屈武斌, 张成岗. 实时荧光定量PCR的数据分析方法[J]. 生物技术通讯, 2015, 26(1): 140-145
    Yi J M, Qu W B, Zhang C G. Data analysis methods of real-time fluorescent quantitative PCR[J]. Letters in Biotechnology, 2015, 26(1): 140-145(in Chinese)
    [15] 黄玲燕. 斜纹夜蛾光受体CRY1基因沉默及其对生殖相关基因表达影响研究[D]. 昆明: 云南大学, 2017.
    Huang L Y. Silencing of photoreceptor CRY1 gene and its effect on the expression of reproduction-relate genes in Spodoptera litura[D]. Kunming: Yunnan University, 2017 (in Chinese).
    [16] 朱春利, 张桂荣, 蔡爱军, 等. 植物隐花色素结构与功能研究进展[J]. 基因组学与应用生物学, 2009, 28(1): 174-178
    Zhu C L, Zhang G R, Cai A J, et al. Advances in the structure and function of cryptochrome in plants[J]. Genomics and Applied Biology, 2009, 28(1): 174-178(in Chinese)
    [17] 胡甘雨. 葱蝇隐花色素基因cry的克隆, 特征及昼夜表达模式[D]. 重庆: 重庆师范大学, 2014.
    Hu G Y. Molecular cloning, characteristics and expression of cryptochrome in the onion maggot, Delia antiqua[D]. Chongqing: Chongqing Normal University, 2014 (in Chinese).
    [18] Fanjul-Moles M L, Escamilla-Chimal E G, Gloria-Soria A, et al. The crayfish Procambarus clarkii cry shows daily and circadian variation[J]. Journal of Experimental Biology, 2004, 207(9): 1453-1460
    [19] 姜令绪, 王仁杰, 周莉, 等. 脊尾白虾在不同盐度条件下的行为变化[J]. 南方农业学报, 2011, 42(12): 1564-1567
    Jiang L X, Wang R J, Zhou L, et al. Behavioral analysis of Exopalaemon carinicauda under different salinity conditions[J]. Journal of Southern Agriculture, 2011, 42(12): 1564-1567(in Chinese)
    [20] 李红丹, 闫蕾, 孙蕾, 等. 玉米隐花色素CRY1bCRY2基因转录丰度对不同光质处理的响应[J]. 作物学报, 2018, 44(9): 1290-1300
    Li H D, Yan L, Sun L, et al. Transcription abundances of CRY1b and CRY2 genes in response to different light treatments in maize[J]. Acta Agronomica Sinica, 2018, 44(9): 1290-1300(in Chinese)
    [21] 闫蕾. 玉米隐花色素基因CRY1a的克隆及功能分析[D]. 太原: 山西大学, 2016.
    Yan L. Cloning and functional analysis of cryptochrome 1a in Maize[D]. Taiyuan: Shanxi University, 2016 (in Chinese).
    引证文献
    网友评论
    网友评论
    分享到微博
    发 布
引用本文

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

复制
分享
文章指标
  • 点击次数:1301
  • 下载次数: 1340
  • HTML阅读次数: 0
  • 引用次数: 0
历史
  • 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
文章二维码