欢迎访问作物学报,今天是

作物学报 ›› 2014, Vol. 40 ›› Issue (08): 1371-1379.doi: 10.3724/SP.J.1006.2014.01371

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

结球甘蓝BoLH01BoLH02基因的克隆与表达

张林成1,高启国1,*,蒲全明1,任雪松1,刘豫东1,朱利泉2,王小佳1   

  1. 1 西南大学园艺园林学院 / 南方山地园艺学教育部重点实验室,重庆 400716;2 西南大学农学与生物科技学院,重庆 400716
  • 收稿日期:2013-12-02 修回日期:2014-06-16 出版日期:2014-08-12 网络出版日期:2014-06-10
  • 通讯作者: 高启国, E-mail: gaoqg031@swu.edu.cn, Tel: 023-68251974
  • 基金资助:

    本研究由国家重点基础研究发展计划(973计划)(2012CB113900)项目资助。

Cloning and Expression Analysis of BoLH01and BoLH02 Genes in Cabbage

ZHANG Lin-Cheng1,GAO Qi-Guo1,*,PU Quan-Ming1,REN Xue-Song1,LIU Yu-Dong1,ZHU Li-Quan2,WANG Xiao-Jia1,*   

  1. 1 College of Horticulture and Landscape Architecture, Southwest University / Key Laboratory of Horticulture Science for Southern Mountainous Regions, Ministry of Education, Chongqing 400716, China; 2 College of Agronomy and Biotechnology, Southwest University, Chongqing 400716, China
  • Received:2013-12-02 Revised:2014-06-16 Published:2014-08-12 Published online:2014-06-10

摘要:

bHLH类转录因子在植物叶片形态建成和发育过程中发挥重要的生理功能。本文通过结球甘蓝转录组数据分析,筛选出莲座期和结球期的茎尖间与叶片间均显著差异表达的2bHLH类转录因子,命名为BoLH01BoLH02基因,获得了2个基因的编码序列,其中BoLH01基因CDS全长为966 bp、编码321个氨基酸;BoLH02基因CDS全长870 bp、编码289个氨基酸;序列分析表明BoLH01BoLH02分别与拟南芥AtbHLH18AtbHLH19氨基酸同源相似性最高,达81%74%,均具典型bHLH结构域特点和完全保守的13E-16R9H-13E-17R以及Leu23对应的关键氨基酸位点;分子进化上BoLH01BoLH02AtTCP2/3/4/10/24亲缘关系较近,与AtTCP9/11/14/15较远;转录组和荧光定量PCR分析表明BoLH01BoLH02在平展的莲座叶中表达量较低,在卷曲的球叶中高量表达;序列分析BoHB7BoHB12BoILL6ATG上游均包含能被bHLH功能域识别的E-box序列,对BoHB7BoHB12BoILL6荧光定量PCR分析表明, 三者与BoLH01BoLH02在不同时期叶片中表达量的变化趋势完全一致;说明BoLH01BoLH02可能通过正向调控BoHB7BoHB12BoILL6基因的表达来参与甘蓝叶片卷曲的调控。

关键词: 甘蓝, 叶片卷曲, 基因克隆, 表达分析

Abstract:

Basic helix-loop-helix (bHLH) transcription factors play an important role in the leaf morphogenesis and leaf development. In this study, through transcriptome analyses, two bHLH transcription factor genes, differentially expressed in leaves and shoot tips between cabbage rosette stage and heading stage, were screened, named BoLH01 and BoLH02 genes. The cDNA sequences of BoLH01and BoLH02 were cloned from cabbage (Brasscia oleracea var. Capitata L). The CDS of BoLH01 was 966 bp, which encoded 321 amino acids, and the CDS of BoLH02 was 870 bp, which encoded 289 amino acids. Sequence analysis showed that the similarity of BoLH01 and BoLH02 with AtbHLH18 and AtbHLH19 was 81% and 74% respectively. They contained the typical bHLH domains, conservative 13E-16R, 9E-13R-17H and Leu23 corresponding key amino acid sites. Phylogenetic tree analysis displayed that BoLH01and BoLH02 were closest to AtTCP2/3/4/10/24, and had no close genetic relationship with AtTCP9/11/14/15. Moreover, the transcriptome and real-time fluorescence quantitative PCR analysis indicated that the relative expression levels of BoLH01 and BoLH02 were low in rosette leaves, and high in heading stage. Sequence analysis showed that the ATG upstream of BoHB7, BoHB12 and BoILL6 contained the E-box motif, and those motifs could been recognized by bHLH functional domains. The real-time fluorescence quantitative PCR analysis indicated that the variation in expression levels of BoHB7, BoHB12 and BoILL6 was similar with that of BoLH01 and BoLH02 in leaves (1 cm). These results meant that BoLH01 and BoLH02 maybe participate in the regulation of cabbage leaf curl by positively regulating BoHB7, BoHB12, BoILL6.

Key words: Brassica oleracea L., Leaf curl, Gene cloning, Expression analysis

[1]李曙轩. 中国农业百科全书. 北京: 农业出版社, 1990. pp 317–318



Li S X. Encyclopedia of China Agriculture. Beijing: Agriculture Press, 1992. pp 317–318 (in Chinese)



[2]Ito H. Effect of temperature and photoperiod on head formation of leafy head of Chinese cabbage. J Hort Assoc Japn, 1957, 26: 154–162



[3]Mart?´n-Trillo M, Cubas P. TCP genes: a family snapshot ten years later. Trends Plant Sci, 2010, 15: 31–39



[4]Nath U, Crawford B C, Carpenter R, Coen E. Genetic control of surface curvature. Science, 2003, 299: 1404–1407



[5]Palatnik J F, Allen E, Wu X L, Schommer C, Schwab R, Carrington J C, Weigel D. Control of leaf morphogenesis by microRNAs. Nature, 2003, 425: 257–263



[6]Koyama T, Mitsuda N, Seki M, Shinozaki K, Ohme-Takagi M. TCP transcription factors regulate the activities of ASYMMETRIC LEAVES1 and miR164, as well as the auxin response, during differentiation of leaves in Arabidopsis. Plant Cell, 2010, 22: 3574–3588



[7]Sarvepalli K, Nath U. Hyper-activation of the TCP4 transcription factor in Arabidopsis thaliana accelerates multiple aspects of plant maturation. Plant J, 2011, 67: 595–607



[8]Kieffer M, Master V, Waites R, Davies B. TCP14 and TCP15 affect internode length and leaf shape in Arabidopsis. Plant J, 2011, 68: 147–158



[9]Hervé C, Dabos P, Bardet C, Jauneau A, Auriac M C, Ramboer A, Lacout F, Tremousaygue D. In vivo interference with AtTCP20 function induces severe plant growth alterations and deregulates the expression of many genes important for development. Plant Physiol, 2009, 149: 1462–1477



[10]Viola I L, Uberti Manassero N G, Ripoll R, Gonzalez D H. The Arabidopsis class I TCP transcription factor AtTCP11 is a developmental regulator with distinct DNA-binding properties due to the presence of a threonine residue at position 15 of the TCP domain. Biochem J, 2011, 435: 143–155



[11]Aggarwal P, Das Gupta M, Joseph A P, Chatterjee N, Srinivasan N, Nath U. Identification of specific DNA binding residues in the TCP family of transcription factors in Arabidopsis. Plant Cell, 2010, 22: 1174–1189



[12]Makkena S, Lamb R S. The bHLH transcription factor SPATULA is a key regulator of organ size in Arabidopsis thaliana. Plant Signal Behav, 2013, 8: e24140



[13]Zhang L Y, Bai M Y, Wu J X, Zhu JY, Wang H, Zhang Z, Wang W, Sun Y, Zhao J, Sun X, Yang H, Xu Y, Kim S H, Fujioka S, Lin W H, Chong K, Lu T, Wang Z Y.Antagonistic HLH/bHLH transcription factors mediate brassinosteroid regulation of cell elongation and plant development in rice and Arabidopsis. Plant Cell, 2009, 21: 3767–3780



[14]Ikeda M, Fujiwara S, Mitsuda N, Ohme-Takagi M. A triantagonistic basic helix-loop-helix system regulates cell elongation in Arabidopsis. Plant Cell, 2012, 24: 4483– 4497



[15]Chandna R, Augustine R, Bisht N C. Evaluation of candidate reference genes for gene expression normalization in Brassica juncea using real time quantitative RT-PCR. Plos one, 2012, 7: e36918



[16]Massari M E, Murre C. Helix-Loop-Helix proteins: regulators of transcription in eucaryotic organisms. Mol Cell Biol, 2000, 20: 429–440



[17]Atchley W R, Terhalle W, Dress A. Positional dependence, cliques, and predictive motifs in the bHLH protein domain. J Mol Evol, 1999, 48: 501–516



[18]Toledo-Ortiz G, Huq E, Quail P H. The Arabidopsis basic/helix-loop-helix transcription factor family. Plant Cell, 2003, 15: 1749–1770



[19]Heim M A, Jakoby M, Werber M, Martin C, Weisshaar B, Bailey P C. The basic helix-loop-helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity. Mol Biol Evol, 2003, 20: 735–747



[20]Kong Q, Pattanaik S, Feller A, Werkmana J R, Chai C, Wang Y Q, Grotewold E, Yuan L. Regulatory switch enforced by basic helix-loop-helix and ACT-domain mediated dimerizations of the maize transcription factor R. Proc Natl Acad Sci USA, 2012, 109: E2091–E2097



[21]Payne C T, Zhang F, Lloyd A M. GL3 encodes a bHLH protein that regulates trichome development in Arabidopsis through interaction with GL1 and TTG1. Genetics, 2000, 156:1349–1362



[22]Efroni I, Blum E, Goldshmidt A, Eshed Y. A protracted and dynamic maturation schedule underlies Arabidopsis leaf development. Plant Cell, 2008, 20: 2293–2306



[23]Crawford B C, Nath U, Carpenter R, Coen E S. CINCINNATA controls both cell differentiation and growth in petal lobes and leaves of Antirrhinum. Plant Physiol, 2004, 135: 244–253



[24]Danisman S, Van der Wal F, Dhondt S, Waites R, de Folter S, Bimbo A, van Dijk A, Muino J M, Cutri L, Dornelas M C, Angenent G C, Immink R G. Arabidopsis class I and class II TCP transcription factors regulate jasmonic acid metabolism and leaf development antagonistically. Plant Physiol, 2012, 159:1511–1523



[25]Park J, Lee H J, Cheon C I, Kim S H, Hur Y S, Auh C K, Im K H, Yun D J, Lee S, Davis K R. The Arabidopsis thaliana homeobox gene ATHB12 is involved in symptom development caused by geminivirus infection. PLoS One, 2011, 6: e20054



[26]Widemann E, Miesch L, Lugan R, Holder E, Heinrich C, Aubert Y, Miesch M, Pinot F, Heitz T.The amidohydrolases IAR3 and ILL6 contribute to jasmonoyl-isoleucine hormone turnover and generate 12-hydroxyjasmonic acid upon wounding in Arabidopsis leaves. J Biol Chem, 2013, 288: 31701–31714



[27]Keller C P, Van Volkenburdh E V. Auxin-Induced epinasty of tobacco leaf tissues (A non-ethylene-mediated response).Plant Physiol, 1997, 113: 603–610

[1] 崔连花, 詹为民, 杨陆浩, 王少瓷, 马文奇, 姜良良, 张艳培, 杨建平, 杨青华. 2个玉米ZmCOP1基因的克隆及其转录丰度对不同光质处理的响应[J]. 作物学报, 2022, 48(6): 1312-1324.
[2] 陈松余, 丁一娟, 孙峻溟, 黄登文, 杨楠, 代雨涵, 万华方, 钱伟. 甘蓝型油菜BnCNGC基因家族鉴定及其在核盘菌侵染和PEG处理下的表达特性分析[J]. 作物学报, 2022, 48(6): 1357-1371.
[3] 秦璐, 韩配配, 常海滨, 顾炽明, 黄威, 李银水, 廖祥生, 谢立华, 廖星. 甘蓝型油菜耐低氮种质筛选及绿肥应用潜力评价[J]. 作物学报, 2022, 48(6): 1488-1501.
[4] 张以忠, 曾文艺, 邓琳琼, 张贺翠, 刘倩莹, 左同鸿, 谢琴琴, 胡燈科, 袁崇墨, 廉小平, 朱利泉. 甘蓝S-位点基因SRKSLGSP11/SCR密码子偏好性分析[J]. 作物学报, 2022, 48(5): 1152-1168.
[5] 周慧文, 丘立杭, 黄杏, 李强, 陈荣发, 范业赓, 罗含敏, 闫海锋, 翁梦苓, 周忠凤, 吴建明. 甘蔗赤霉素氧化酶基因ScGA20ox1的克隆及功能分析[J]. 作物学报, 2022, 48(4): 1017-1026.
[6] 晋敏姗, 曲瑞芳, 李红英, 韩彦卿, 马芳芳, 韩渊怀, 邢国芳. 谷子糖转运蛋白基因SiSTPs的鉴定及其参与谷子抗逆胁迫响应的研究[J]. 作物学报, 2022, 48(4): 825-839.
[7] 袁大双, 邓琬玉, 王珍, 彭茜, 张晓莉, 姚梦楠, 缪文杰, 朱冬鸣, 李加纳, 梁颖. 甘蓝型油菜BnMAPK2基因的克隆及功能分析[J]. 作物学报, 2022, 48(4): 840-850.
[8] 黄成, 梁晓梅, 戴成, 文静, 易斌, 涂金星, 沈金雄, 傅廷栋, 马朝芝. 甘蓝型油菜BnAPs基因家族成员全基因组鉴定及分析[J]. 作物学报, 2022, 48(3): 597-607.
[9] 靳容, 蒋薇, 刘明, 赵鹏, 张强强, 李铁鑫, 王丹凤, 范文静, 张爱君, 唐忠厚. 甘薯Dof基因家族挖掘及表达分析[J]. 作物学报, 2022, 48(3): 608-623.
[10] 王瑞, 陈雪, 郭青青, 周蓉, 陈蕾, 李加纳. 甘蓝型油菜白花基因InDel连锁标记开发[J]. 作物学报, 2022, 48(3): 759-769.
[11] 谢琴琴, 左同鸿, 胡燈科, 刘倩莹, 张以忠, 张贺翠, 曾文艺, 袁崇墨, 朱利泉. 甘蓝自交不亲和相关基因BoPUB9的克隆及表达分析[J]. 作物学报, 2022, 48(1): 108-120.
[12] 王艳花, 刘景森, 李加纳. 整合GWAS和WGCNA筛选鉴定甘蓝型油菜生物产量候选基因[J]. 作物学报, 2021, 47(8): 1491-1510.
[13] 尹明, 杨大为, 唐慧娟, 潘根, 李德芳, 赵立宁, 黄思齐. 大麻GRAS转录因子家族的全基因组鉴定及镉胁迫下表达分析[J]. 作物学报, 2021, 47(6): 1054-1069.
[14] 左香君, 房朋朋, 李加纳, 钱伟, 梅家琴. 有毛野生甘蓝(Brassica incana)抗蚜虫特性研究[J]. 作物学报, 2021, 47(6): 1109-1113.
[15] 许静, 潘丽娟, 李昊远, 王通, 陈娜, 陈明娜, 王冕, 禹山林, 侯艳华, 迟晓元. 花生油脂合成相关基因的表达谱分析[J]. 作物学报, 2021, 47(6): 1124-1137.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!