海带<em>lhcf4</em>基因克隆和配子体差异表达研究

doi:10.7668/hbnxb.2011.06.009

摘要/Abstract

摘要： 根据糖海带(Laminaria saccharina)光捕获蛋白基因lhcf4的cDNA全长序列(GenBank登录号:AF₂26860)设计引物,通过RT-PCR方法获得海带(Saccharina japonica)配子体lhcf4基因的cDNA全长序列,共1 042 bp,编码一个含218个氨基酸的前体蛋白lhcf4,推测分子量为23.11 kDa,等电点为5.36。与其同源基因相似度分析表明,lhcf基因的开放阅读框(Open reading frame,ORF)在家族内及物种间高度保守,变异主要发生在非翻译区(Untranslated re-gion,UTR)。预测lhcf4成熟蛋白三级结构存在4个α螺旋区,其中第一、三螺旋较长,形成跨膜螺旋,第二个螺旋相对较短,完全镶嵌在膜中,另外一个为短螺旋,位于类囊体膜-类囊体腔界面上。杂色藻类、绿藻及豌豆光捕获蛋白氨基酸多序列比对显示叶绿素a结合位点及盐桥形成氨基酸残基在植物进化中高度保守;将预测的海带lhcf4蛋白3D结构与豌豆X-衍射晶体结构进行空间重叠,获得了结合在lhcf4蛋白上的5个叶绿素a的空间分布。Neighbor-joining分子进化树显示原绿球藻、绿藻和杂色藻类的光捕获蛋白基因是三个独立进化的系统,共同起源于蓝细菌的只结合叶绿素a的光捕获蛋白;海带lhcf4基因与糖海带lhcf4及巨藻的fcpb基因为直系同源;糖海带lhcf4、lhcf7及lhcF₁基因为旁系同源。实时荧光定量PCR结果证实lhcf4基因具有雌、雄配子体差异表达特征。

关键词: 海带, 配子体, lhcf4, 基因克隆, 序列分析, 差异表达

Abstract: lhcf4gene full length cDNA of Saccharina japonicawas cloned by using RT-PCR technique with primers designed based on Laminaria saccharinalhcf4full length cDNA sequence(accession number: AF₂26860). Its ORF encoded a precursor protein containing 218 amino acids with a molecular weight of 23． 11 kDa and an isoelectric point of 5． 36． ORFs of lhcf4gene and its homologs were conserved，while their UTRs were diverse according to results of similarity analysis． The predicted 3D structure of lhcf4contained 4 α-helixes in which the first and third helix were transmembrane helices， the second one was a little shorter and embedded in the thylakoid membrane， the fourth one was a short helix on the lumenal side． Chlorophyll a binding sites and amina acid residues forming salt-bridge between the first and third helix were conserved in chromophytes，green algae and higher plants． The spatial distribution of chlorophyll a in lhcf4protein was determined by overlapping its 3D structure with Xray diffraction structure of pea LHC-II monomer at 2． 5 resolution． The Neighbor-joining phylogenetic tree constructed by MEGA4． 0 revealed three significantly different lineages from lhc genes: chromophytes，green alga and prochlorococcus，which might be have the same ancestor from cyanobacteria． Otherwise， it also revealed that lhcf4genes of S． japonicaand L． saccharina， fcpb gene of Macrocystis pyrifera were orthologs while lhcf4， lhcF₁and lhcF7 genes of L． saccharina were Paralogs． Real-time PCR results confirmed that lhcf4gene of S． japonicadifferentially expressed between in female and male gametophytes．

Key words: Saccharina japonica, Gametophytes, lhcf4, Gene cloning, Sequence analysis, Differential expression

中图分类号:

毕燕会, 周志刚. 海带lhcf4基因克隆和配子体差异表达研究[J]. 华北农学报, 2011, 26(6): 41-49. doi: 10.7668/hbnxb.2011.06.009.

BI Yan-hui, ZHOU Zhi-gang. Molecular Cloning and Differential Expression of lhcf4Gene from Gametophytes of Saccharina japonica[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2011, 26(6): 41-49. doi: 10.7668/hbnxb.2011.06.009.

http://www.hbnxb.net/CN/Y2011/V26/I6/41

参考文献

[1] 方宗熙，欧毓麟，崔竞进，等. 海带配子体无性繁殖系培育成功[J]. 科学通报， 1978， 23: 115-116.
[2] Yabu H. Early development of several species of Laminariales in Hokkaido[J]. Memoirs of The Faculty of Fisheries Hokkaido University， 1964， 12: 1-72.
[3] Hsiao S I C，Druehl L D. Environmental control of gametogenesis in Laminaria saccharinaⅠ. The effects of light and culture media[J]. Canadian Journal of Botany， 1971， 49: 1503-1508.
[4] Hsiao S I C，Druehl L D. Environmental control of gametogenesis in Laminaria saccharina II. Correlation of nitrate and phosphate concentrations with gametogenesis and selected metabolites[J]. Canadian Journal of Botany， 1973， 51: 829-839.
[5] 周志刚，吴超元. 海带无性繁殖系的形成及孢子体诱导[J]. 生物工程学报， 1998， 14(1): 111-113.
[6] 史西志，毕燕会，周志刚. 海带雄性配子体差异表达基因片段的克隆及筛选[J]. 水产学报， 2005， 29(5): 666-669.
[7] 陆广琴，欧阳珑玲，周志刚. 海带雄配子体抑制消减cDNA 文库的生物信息学分析[J]. 中国水产科学，2009， 16(20): 221-229.
[8] Katoh T，Mimuro M，Takaichi S. Light-harvesting particles isolated from a brown alga，Dictyota dichotoma. A supramolecular assembly of fucoxanthin-chlorophyll-protein complexes[J]. Biochimica et Biophysica Acta(BBA)- Bioenergetics， 1989， 976(2-3): 233-240.
[9] Dion G D， Julie A P. Light-harvesting complex gene expression is controlled by both transcriptional and posttranscriptional mechanisms during photoacclimation in Chlamydomonas reinhardtii[J]. Physiologia Plantarum， 2003， 118(2): 193-205.
[10] Eppard M，Kühlbrandt W E，von Haeseler A，et al. Characterization of fcp4 and fcp12， two additional genes encoding light harvesting proteins of Cyclotella cryptica (Bacillariophyceae) and phylogenetic analysis of this complex gene family[J]. Plant Biology， 2000，2 : 283- 289.
[11] Dolganov N A M，Bhaya D，Grossman A R. Cyanobacterial protein with similarity to the chlorophyll a /b binding proteins of higher plants: evolution and regulation[J]. Proceedings of the National Academy of Sciences of the United States of America， 1995， 92(2): 636-640.
[12] Green B R，Kühlbrandt W. Sequence conservation of light-harvesting and stress-response proteins in relation to the three-dimensional molecular structure of LHC[J]. Photosynthesis Research， 1995， 44: 139-148.
[13] De Martino A，Douady D，Quinet-Szely M，et al. The light-harvesting antenna of brown algae: Highly homologous proteins encoded by a multigene family[J]. European Journal of Biochemistry，2000，267(17): 5540- 5549.
[14] 邹丹燕，毕燕会，周志刚. 光对海带雌、雄配子体lhcf5和lhcf6 基因相对转录量的影响[J]. 中国水产科学，2009， 16(6): 850-858.
[15] Standfuss J，Terwisscha van Scheltinga A C，Lamborghini M， et al. Mechanisms of photoprotection and nonphotochemical quenching in pea light-harvesting complex at 2. 5 resolution[J]. The EMBO Journal， 2005， 24(5): 919-928.
[16] Wheeler D L，Barrett T，Benson D A， et al. Database resources of the National Cent er for Biotechnology Information[J]. Nucleic Acids Research， 2007， 35: 5-12.
[17] HigGins D G，Sharp P M. CLUSTAL: a package for performing multiple sequence alignments on a microcomputer[J]. Gene， 1988， 73(1): 237-244.
[18] Chenna R，Sugawara H，Koike T，et al. Multiple sequence alignment with the Clustal series of programs[J]. Nucleic Acids Research，2003，31(13): 3497- 3500.
[19] Hall T A. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95 / 98 /NT[J]. Nucleic Acids Symposium Series，1999，41: 95-98.
[20] Tamura K，Dudley J，Nei M，et al. MEGA4: Molecular evolutionary genetics analysis(MEGA) software version4. 0[J]. Molecular Biology and Evolution，2007，24 (18), 1596-1599.
[21] Nicholas K B，Nicholas H B. Jr: GeneDoc: a tool for editing and annotating multiple sequence alignments[EB/ OL]. [http: / /www. nrbsc. org /gfx /genedoc /index. html ]distributed by the authors 1997.
[22] Kelley L A，MaCallum R M，Sternberg M J. Enhanced genome annotat ion using structural prof iles in the program 3D-PSSM[J]. Journal of Molecular Biology， 2000， 299(2): 499-520.
[23] Schrdinger L L C. The PyMOL Molecular Graphics System
[M]，Version 1. 3. Schrdinger，LLC， 2010.
[24] La Roche J， van der Staay G W M，Partensky F， et al. Independent evolution of the prochlorophyte and green plant chlorophyll a /b light-harvesting proteins[J]. Proceedings of the National Academy of Sciences of the United States of America， 1996， 93(26): 15244-15248.
[25] Grabowski B，Cunningham F X，Gantt E. Chlorophyll and carotenoid binding in a simple red algal light-harvesting complex crosses phylogenetic lines[J]. Proceedings of the National Academy of Sciences of the United States of America， 2001， 98(5): 2911-2916.
[26] Pascal A A，Caron L，Rousseau B， et al. Resonance Raman spectroscopy of a light-harvesting protein from the brown alga Laminaria saccharina [J]. Biochemistry， 1998， 37(18): 2450-2457.
[27] Bhaya D，Grossman A R. Characterization of gene clusters encoding the fucoxanthin chlorophyll proteins of the diatom Phaeodactylum tricornutum[J]. Nucleic Acids Research， 1993， 21(19): 4458-4466.
[28] Grossman A，Manodori A，Snyder D. Light-harvesting proteins of diatoms: their relationship to the chlorophyll a /b binding proteins of higher plants and their mode of transport into plastids[J]. Molecular ＆ General Genetics， 1990， 224(1): 91-100.
[29] Kroth-Pancic P G. Nucleotide sequence of two cDNAs encoding fucoxanthin chlorophyll a /c proteins in the diatom Odontella sinensis[J]. Plant Molecular Biology， 1995， 27(4): 825-828.
[30] Wolf G R，Cunningham F X，Grabowski B， et al. Isolation and characterization of photosystems II and I from the red alga Porphyridium cruentum[J]. Biochimica et Biophysica Acta(BBA)-Bioenergetics， 1994， 1188(3): 357 -366.
[31] van der Staay G W，Yurkova N，Green B R. The 38 kDa chlorophyll a /b protein of the prokaryote Prochlorothrix hollandica is encoded by a divergent pcb gene[J]. Plant Molecular Biology， 1998， 36(5): 709 – 716.
[32] Green B R，Durnford D G. The chlorophyll-carotenoid proteins of oxygenic photosynthesis[J]. Annual Review of Plant Physiology and Plant Molecular Biology， 1996， 47: 685-714.
[33] LaRoche J，Henry D，Wyman K， et al. Cloning and nucleotide sequence of a cDNA encoding a major fucoxanthin-， chlorophyll a /c-containing protein from the chrysophyte Isochrysis galbana: implications for evolution of the cab gene family[J]. Plant Molecular Biology， 1994， 25(3): 355-368.

选择文件类型/文献管理软件名称

选择包含的内容

海带lhcf4基因克隆和配子体差异表达研究

Molecular Cloning and Differential Expression of lhcf4Gene from Gametophytes of Saccharina japonica

PDF

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	金一锋, 高岩松, 王琦, 王萌萌, 赵迪, 熊毅, 陈阳. *草地早熟禾蛋白激酶SnRK2.4* 基因克隆及非生物胁迫响应分析**[J]. 华北农学报, 2022, 37(5): 9-15.
[2]	张冬梅, 冯亚艳, 修志君, 杨春芳, 杜美娥, 李得宙, 张笑宇. *硅酸钠诱导的马铃薯抗黑痣病StWRKY11基因克隆及生物信息学分析*[J]. 华北农学报, 2022, 37(5): 194-203.
[3]	郑玉斯, 王培, 郭颖, 白丽蓉, 喻达辉, 赵森. *合浦珠母贝PfCLEC17A* 基因的克隆及表达分析**[J]. 华北农学报, 2022, 37(5): 230-238.
[4]	王燕宁, 黄涛, 吴光亮, 黄诗颖, 钟奇, 王鹏, 杨朦朦, 李才敬, 程琴, 贺浩华, 金伟, 郭凌, 边建民. 水稻芽期响应镉胁迫的转录组分析[J]. 华北农学报, 2022, 37(5): 25-35.
[5]	肖士奎, 李芳, 张文婷, 吕淑芳, 史国安, 吴疆, 范丙友. *芍药ACS基因的克隆及其蛋白质原核表达*[J]. 华北农学报, 2022, 37(5): 36-44.
[6]	侯瑞泽, 侯玉翔, 许小勇, 李璿, 李梅兰. 普通白菜CYP同源基因克隆与表达分析[J]. 华北农学报, 2022, 37(5): 45-51.
[7]	王亚男, 梁岩岩, 严文培, 张银萍, 李强, 吴秀菊. *北细辛AhOMT1基因的克隆及原核表达分析*[J]. 华北农学报, 2022, 37(4): 62-70.
[8]	安清明, 王星, 吴震洋, 孟金柱, 赵园园, 宋兴超. 基于转录组挖掘不同性别贵州白山羊肌肉生长发育的关键基因及PI3K/ATK信号通路分析[J]. 华北农学报, 2022, 37(3): 213-222.
[9]	程春红, 游经番, 蔡兆明, 叶春宏, 陈丽. *茎瘤芥BjuEAR1-1的克隆与表达分析*[J]. 华北农学报, 2022, 37(3): 37-43.
[10]	张敬敬, 李冰, 史宇凡, 高秀瑞, 潘秀清, 宋雪, 武彦荣. 不同硬度西瓜果皮的转录组测序及相关基因表达分析[J]. 华北农学报, 2022, 37(3): 44-52.
[11]	唐忠丽, 逯晓楠, 赵蕊, 刘庆华, 李梅兰, 雷逢进, 许小勇. *黄皮西葫芦类胡萝卜素合成酶基因的鉴定及PSY1和LCYE2克隆分析*[J]. 华北农学报, 2022, 37(3): 60-67.
[12]	贾志强, 许云玉, 高雪, 陶宏征, 陈增敏, 刘雅婷, 李永忠. *辣椒CaWRKY30转录因子的克隆、亚细胞定位及番茄斑萎病毒侵染下的表达分析*[J]. 华北农学报, 2022, 37(3): 186-192.
[13]	史建磊, 熊自立, 苏世闻, 王克磊, 宰文珊. 基于RNA-seq的番茄青枯病响应基因鉴定与表达分析[J]. 华北农学报, 2022, 37(2): 171-182.
[14]	韩晓勇, 蒋璐, 殷剑美, 金林, 张培通. 山药品种苏蓣8号抗炭疽病基因的转录组测序与表达分析[J]. 华北农学报, 2022, 37(2): 211-222.
[15]	赵惠英, 杨光凯, 高燕, 张小军, 郝燕燕. *苹果MdSGR2基因克隆及植物超表达载体构建*[J]. 华北农学报, 2022, 37(2): 42-48.

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

海带lhcf4基因克隆和配子体差异表达研究

Molecular Cloning and Differential Expression of lhcf4Gene from Gametophytes of Saccharina japonica

PDF

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价