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海带CRY-DASH基因的cDNA序列分离和表达分析
李璐1,2
学位类型硕士
导师段德麟
2017-05-20
学位授予单位中国科学院大学
学位授予地点北京
学位专业海洋生物学
关键词蓝光 海带 孢子体 Cry-dash 昼夜节律
摘要蓝光是能在海水中传播的主要光波,对潮下带生活的海带的生长发育起重要作用。隐花色素/光裂解酶家族(Cryptochrome/Photolyase Family, CPF)是一类广泛存在的能被蓝光诱导激活的蛋白家族,CRY-DASH是该家族的一类新成员,已知其能修复ssDNA和dsDNA环形结构中嘧啶酮二聚体损伤,但对其它功能了解较少。
本研究利用cDNA末端快速克隆(RACE)技术,获得海带CRY-DASH基因(SjCRY-DASH)全长序列,结构分析发现,其ORF区长1779 bp,编码592个氨基酸。进行氨基酸同源序列比对,其与其它藻类和高等植物间存在两个重要辅基MTHF和FAD结合的保守域。三维结构预测表明,SjCRY-DASH与目前已解析的CPF家族其他成员晶体结构的相似度较高。通过选择两种原核表达载体(pColdⅠ和pMAL-c5X),对SjCRY-DASH的ORF区进行体外表达及纯化,均得到纯度较高的蛋白。
通过不同光质处理海带幼孢子体,发现蓝光和白光诱导1 h,SjCRY-DASH转录水平均上升,且SjCRY-DASH对蓝光的响应更强烈,而红光对SjCRY-DASH无明显诱导效应。将海带幼孢子体培养于不同日长条件下,SjCRY-DASH转录变化均维持24 h的周期性,且在长、短日照培养下转录起始位点发生改变,表明其与昼夜节律调控密切相关。此外,SjCRY-DASH转录水平在不同温度处理下的节律变化具有温度补偿性。本研究相关结果对了解海带中的CRY-DASH功能具有重要意义,也对探讨海带生长发育受光、温调控的分子机制提供理论依据。
其他摘要Blue light is predominant wavelength below seawater surface, and it plays a critical role in regulating growth and development of photosynthetic stramenopile such as Saccharina japonica. The cryptochrome/photolyase family (CPF) is a diversified protein family which can be induced and activated by blue light. It has been confirmed that CRY-DASH, a new member of the cryptochrome/photolyase family, can repair the cyclobutane pyrimidine dimer (CPD) in ssDNA and loop structures of dsDNA, though its other functions are less investigated.
  Based on the transcriptome data, we characterized the full-length cDNA of CRY-DASH from S. japonica (designated as SjCRY-DASH) by rapid amplification of cDNA ends (RACE). Sequence analysis showed that the full-length ORF was 1,779 bp, encoding a protein of 592 amino acids. By multiple alignments of protein sequences, conserved residues that interact with two crucial cofactors, MTHF and FAD, were detected between SjCRY-DASH and several DASH-type proteins of other algae and higher plant. Prediction of protein structure showed SjCRY-DASH shared high similarity with other CPF members dissolved already. Two kinds of prokaryotic vectors were applied for SjCRY-DASH expression in vitro, and objective protein of high purity was obtained through affinity chromatography.
  Juvenile sporophytes were induced by light of different wavelengths. Both blue light and white light upregulated the transcriptional level of SjCRY-DASH in 1 h, though stronger response to blue light was found for SjCRY-DASH than white light. Nevertheless, red light had no notable influence on the transcriptional level. The SjCRY-DASH transcription variation persisted with 24-hour periodicity when juvenile sporophytes were grown under various daylength conditions. Besides, the time of onset of the transcription was reset by different daylengths, indicating SjCRY-DASH is closely related to circadian control. Furthermore, the rhythms of SjCRY-DASH transcription under different temperature treatments were temperature compensated, verifying that it was a crucial component of the circadian clocks. This study is expected to enrich our understanding of CRY-DASH function, and lays foundation for exploring the molecular mechanism of responses to light and temperature in S. japonica.
学科领域地球科学 ; 海洋科学 ; 海洋生物学
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/136680
专题实验海洋生物学重点实验室
作者单位1.中国科学院海洋研究所
2.中国科学院大学
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李璐. 海带CRY-DASH基因的cDNA序列分离和表达分析[D]. 北京. 中国科学院大学,2017.
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