一个棉花GDSL脂肪酶基因的克隆与功能分析

doi:10.3724/SP.J.1006.2013.01164

摘要/Abstract

摘要：

GDSL脂肪酶与GXSXG脂肪酶是2个重要的脂肪酶亚家族。其中, GDSL家族脂肪酶具有水解酶活性, 能水解多种酯类物质。本试验根据新乡小吉无绒无絮(XinWX)和无绒有絮(XinFLM)近等基因系纤维起始期29K芯片竞争杂交结果, 选择了一个在纤维起始期具有极显著表达差异的EST序列(GenBank登录号为DR458916), 以该序列信息为探针, 利用电子克隆方法并进行cDNA及基因组全长基因PCR扩增、测序验证, 克隆获得一个陆地棉GDSL脂肪酶基因(GhGDSL；GenBank登录号为KC186125)。该基因ORF长1065 bp, 编码354个氨基酸, 含有5个外显子和4个内含子。该基因在二倍体棉种基因组中含一个拷贝, 在四倍体棉种基因组中含2个拷贝。序列比对显示该基因在四倍体棉种的2个亚组中独立进化, 且D亚组比A亚组变异大。染色体定位显示该基因2个拷贝分别位于A4 (Chr. 4)和D4 (Chr. 22)染色体上。定量RT-PCR结果表明, GhGDSL在开花后3~10 d的纤维组织中表达量高, 其中在海7124中表达高峰在8DPA, 在TM-1中表达高峰从5DPA持续到10DPA。利用[(TM-1×Hai7124)×TM-1]的BC₁S₁群体开展GhGDSL功能与纤维、种子品质性状关联分析, 发现该基因A亚组的拷贝与种子脂肪含量存在显著相关(P=0.048)；D亚组的拷贝与种子蛋白含量存在极显著相关(P=0.008)。推测GhGDSL基因功能与种子中脂肪、蛋白代谢相关, 同时也参与纤维伸长过程。

关键词: GDSL脂肪酶, 克隆, 表达, 基因定位, 关联分析, 棉花

Abstract:

GDSL and GXSXG lipases are two important sub-families of lipases. Unlike the GXSXG motif-containing lipases, GDSL lipases have a GDSL motif and are active in hydrolysis and synthesis of abundant ester compounds. In this experiment, a EST sequence (GenBank accession No: DR458916) was screened based on extremely significant expression differences at fiber initiation stages between lintless-fuzzless XinWX and linted-fuzzless XinFLM isogenic lines by 29K cotton genome array hybridization. Using the EST sequence as queries, the Gossypium EST database (http://www.ncbi.nlm.nih.gov/) was screened and the corresponding cDNA sequences containing a complete ORF were assembled. Further, the ORF information was reconfirmed in transcriptional and genomic level. As a result, a novel gene encoding GDSL lipase was cloned, and the gene was designated as GhGDSL (Gossypium hirsutum GDSL; GenBank accession number: KC186125). GhGDSL included an open reading frame of 1 065 bp that encoded a polypeptide of 354 amino acids. The genome sequence indicated that GhGDSL had four introns and five exons. The homolog of GhGDSL had one copy in diploid cotton species G. herbaceum and G. raimondii and two copies in tetraploid cotton species G. hirsutum acc. TM-1 and G. barbadense cv. Hai 7124, respectively. Sequence alignment from different cotton species showed GhGDSL homoelogs in tetraploid were evolved independently, with more variations in D-subgenome than in A-subgenome. GhGDSL homoelogs in tetraploid were located on chromosome 4 (Chr. A4) and chromosome 22 (Chr. D4) by developing subgenome-specific SNP marker, respectively. Q-PCR analysis showed that GhGDSL was accumulated highly in ovule and fiber tissue at 3–10 days post anthesis (DPA), with expression peak at 8DPA in Hai7124 and high expression from 5DPA to 10 DPA in TM-1. The association analysis between GhGDSL and the traits related to fiber and seed qualities in corresponding [(TM-1×Hai7124)×TM-1] BC1S1 individuals showed that GhGDSL homolog in A-subgenome was significantly correlated with the seeds’ fat content (P=0.048), and GhGDSL homolog in D-subgenome was very significantly correlated with the seeds’ protein content (P=0.008). These results suggested that GhGDSL might be functionally important in the metabolic process of lipid and protein in seeds and in the process of fiber development.

Key words: GDSL lipase, Cloning, Expression, Gene mapping, Association analysis, Cotton

佟祥超,王丽曼,胡文静,张雪颖,张天真,郭旺珍. 一个棉花GDSL脂肪酶基因的克隆与功能分析[J]. 作物学报, 2013, 39(07): 1164-1171.

TONG Xiang-Chao,WANG Li-Man,HU Wen-Jing,ZHANG Xue-Ying,ZHANG Tian-Zhen,GUO Wang-Zhen. Molecular Cloning and Functional Analysis of a GDSL Lipase Gene from Gossypium hirsutum L.[J]. Acta Agron Sin, 2013, 39(07): 1164-1171.

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://zwxb.chinacrops.org/CN/10.3724/SP.J.1006.2013.01164

https://zwxb.chinacrops.org/CN/Y2013/V39/I07/1164

参考文献

[1]Kim Y. Cloning and expression of a lipase gene from rice (Oryza sativa cv. Dongjin). Mol Cells, 2004, 18: 40–45

[2]Roberts M R, Foster G D, Blundell R P, Robinson S W, Kumar A, Draper J, Scott R. Gametophytic and sporophytic expression of an anther-specific Arabidopsis thaliana gene. Plant J, 1993, 3: 111–120

[3]Pringe D, Dickstein R. Purification of ENOD8 proteins from Medicago sativa root nodules and their characterization as esterases. Plant Physiol Biochem, 2004, 42: 73–79

[4]Brick D J, Brumlik M J, Buckley J T, Cao J X, Davies P C, Misra S, Tranbarger T J, Upton C. A new family of lipolytic plant enzymes with members in rice, Arabidopsis and maize. FEBS Lett, 1995, 377: 475–480

[5]Oh I S, Park A R, Bae M S. Kwon S J, Kin Y S, Lee J E, Kang N Y, Lee S, Cheong H, Park O K. Secretome analysis reveals an Arabidopsis lipase involved in defense against Alternaria brassicicola. Plant Cell, 2005, 17: 2832–2847

[6]Rupperl M, Woll J, Giritch A, Genady E, Ma X Y, Stochigt J. Functional expression of an ajmaline pathway-specific esterase from Rauvolfia in a novel plant-virus expression system. Planta, 2005, 222: 888–898

[7]Basra A, Malik C P. Development of the cotton fiber: International fibers. In: Basra A M ed. Cotton Fibers. New York: Hawthorne Press, 1984. pp 231–267

[8]Ji S J, Lu Y C, Feng J X, Wei G, Shi Y H, Fu Q, Liu D, Luo J C, Zhu Y X. Isolation and analyses of genes preferentially expressed during early cotton fiber development by subtractive PCR and cDNA array. Nucl Acids Res, 2003, 31: 2534–2543

[9]Song X L, Wang K, Guo W Z, Zhang J, Zhang T Z. A comparison of genetic maps constructed from haploid and BC1 mapping populations from the same crossing between Gossypium hirsutum L. and Gossypium barbadense L. Genome, 2005, 48: 378–390

[10]Van Ooijen J W, Voorrips R E. JoinMap Version 3.0: Software for the Calculation of Genetic Linkage Maps. Wageningen, the Netherlands: CPRO-DLO, 2001

[11]Guo W Z, Cai C P, Wang C B, Wang C B, Han Z G, Song X L, Wang K, Niu X W, Wang C, Lu K Y, Shi B, Zhang T Z. A microsatellite-based, gene-rich linkage map reveals genome structure, function, and evolution in Gossypium. Genetics, 2007, 176: 527–541

[12]Voorrips R E. MapChart: Software for the graphical presentation of linkage maps and QTLs. J Heredity, 2002, 93: 77–78

[13]Paterson A H, Brubaker C L, Jonathan F. Rapid method for extraction of cotton (Gossypium spp.) genomic DNA suitable for RFLP or PCR analysis. Plant Mol Biol Rep, 1993, 11: 122–127

[14]Wu Y-T(武耀廷), Liu J-Y(刘进元). A modified hot borate method for efficient isolation of total RNA from different cotton tissues. Cotton Sci (棉花学报), 2004, 16(2): 67–71 (in Chinese with English abstract)

[15]Jiang J-X(蒋建雄), Zhang T-Z(张天真). Tissues with CTAB acidic phenolic method. Cotton Sci (棉花学报), 2003, 15(3): 166−167 (in Chinese with English abstract)

[16]Licak K J, Schmittgen T D. Analysis of relative gene expression data using Real-time quantitative PCR and the 2(-delta delta C(t)) method. Method, 2001, 25: 402–408

[17]Ling H, Zhao J Y, Zuo K J, Qiu C X, Yao H Y, Q J, Sun X F, Tang K X. Isolation and expression analysis of a GDSL-like lipase gene from Brassica napus L. J Biochem Mol Biol, 2006, 39: 297–303

[18]Sun S-K(孙善康), Chen J-H(陈建华), Xiang S-K(项时康), Wei S-J(魏守军). Study on the nutritional quality of cotton seeds. Sci Agric Sin (中国农业科学), 1987, 20(5): 12–16 (in Chinese with English abstract)

[19]Beisson F, Arondel V, Verger R. Assaying Arabidopsis lipase activity. Biochem Soc Transact, 2000, 28: 773–775

[20]Mayfield J A, Fiebig A, Johnstone S E, Preuss D. Gene families from Arabidopsis thaliana pollen coat proteome. Science, 2001, 292: 2482–2485

[21]Hong J K, Choi H W, Hwang I S, Kin D S, Kim N H, Choi D S, Kim Y J, Hwang B K. Function of a novel GDSL-type pepper lipase gene, CaGLIP1, in disease susceptibility and abiotic stress tolerance. Planta, 2008, 227: 539–558

[22]Qin Y M, Hu C Y, Pang Y, Kastaniotis A J, Hilunen J K, Zhu Y X. Saturated very-long-chain fatty acids promote cotton fiber and Arabidopsis cell elongation by activating ethylene biosythesis. Plant Cell, 2007, 19: 3692–3704

[23]Shi Y H, Zhu S W, Mao X Z, Feng J X, Qin Y M, Zhang L, Cheng J, Wei L P, Wang Z Y, Zhu Y X. Transcriptome profiling, molecular biological, and physiological studies reveal a major role for ethylene in cotton fiber cell elongation. Plant Cell, 2006, 18: 651–664

[24]Gou J Y, Wang L J, Chen S P, Hu W L, Chen X Y. Gene expression and metabolite profiles of cotton fiber during cell elongation and secondary cell wall synthesis. Cell Res, 2007, 17: 422–434

[25]Wei M-Y(魏明玉), Zhou J(周佳), Yan S-Z(闫素珍), Wang Z(王政), Tan X-L(谭小力). Cloning and expression analysis of an oil-content related lipase (BnSDP1) gene in Brassica napus. Chin Oil Crop Sci (中国油料作物学报), 2011, 33(4): 338–343 (in Chinese with English abstract)

相关文章 15

[1]	习千辉, 徐梓瑗, 刘梦梦, 王宏艺, 郎凯琳, 井震海, 陈锋, 赵磊. 小麦籽粒铜含量的全基因组关联分析及候选基因预测[J]. 作物学报, 2026, 52(6): 1604-1617.
[2]	毛嘉琦, 黄朋雨, 赵佳佳, 郑兴卫, 武棒棒, 郝宇琼, 屈非, 刘成, 马朋涛, 郑军. 山西小麦品种白粉病抗性评价及抗病基因分子检测[J]. 作物学报, 2026, 52(6): 1669-1681.
[3]	陈雪燕, 何华川, 李政嘉, 董新盼, 李藕琪, 刘小云, 李丹萍, 陈志伟, 刘国霞, 吕胜源, 吴印莹, 赵振东, 曹新有, 万何平. 水培盐碱复合胁迫下‘济麦60’苗期根系有机酸分泌动态变化及其转录调控机制[J]. 作物学报, 2026, 52(6): 1859-1875.
[4]	左同鸿, 张贺翠, 曾静, 朱利泉. 甘蓝自交不亲和相关基因BoPUB3L的克隆与表达分析[J]. 作物学报, 2026, 52(6): 1698-1710.
[5]	唐宽强, 李公允, 宋美毅, 赵雪, 常春玲. 大豆株高性状全基因组关联分析及预测模型构建[J]. 作物学报, 2026, 52(6): 1743-1756.
[6]	王文辕, 燕雪嘉, 刘玉霖, 孙晓彤, 李亚楠, 唐鑫华, 石瑛. 耐弱光马铃薯品种筛选及转录因子编码基因StPIF3的克隆与功能分析[J]. 作物学报, 2026, 52(6): 1631-1645.
[7]	胡赵, 钱润, 谢丰璞, 应素平. 水稻SPX基因家族鉴定及响应磷处理的表达分析[J]. 作物学报, 2026, 52(6): 1902-1912.
[8]	赵佳雪, 周龙昊, 郭岂源, 尚伦霄, 王涵, 刘志涛, 陈曦, 张晓佩, 宋宪亮, 毛丽丽. 长期秸秆还田与深松通过改善土壤环境与棉花光合特性提高滨海盐碱地棉花产量[J]. 作物学报, 2026, 52(5): 1548-1560.
[9]	孙淑凤, 许振南, 黄嘉鑫, 翁建峰, 李新海. 玉米MAPK家族全基因组鉴定及其对拟轮枝镰孢菌感染的响应[J]. 作物学报, 2026, 52(5): 1291-1308.
[10]	杨欣雨, 崔文涛, 迪力尼格尔·阿力木, 汪凯翔, 吴鹏昊, 任姣姣. 玉米穗上叶片数全基因组关联分析和全基因组选择[J]. 作物学报, 2026, 52(5): 1573-1590.
[11]	张曦, 王广恩, 李邵琦, 刘祎, 李俊兰, 钱玉源. 基于转录组测序解析陆海杂交姊妹系马克隆值差异的形成机制[J]. 作物学报, 2026, 52(5): 1442-1458.
[12]	韩亚鑫, 何冠华, 张小琼, 张登峰, 李永祥, 刘旭洋, 王天宇, 黎裕, 邹华文, 李春辉. 基于RNA-Seq和BSA-Seq联合分析挖掘玉米侧根密度基因资源[J]. 作物学报, 2026, 52(5): 1341-1352.
[13]	闫安, 蒋昆炜, 王蓉圆, 田林, 张璐, 王韵, 徐建龙. 水稻剑叶小维管束数基因SVN7的鉴定与克隆[J]. 作物学报, 2026, 52(5): 1364-1372.
[14]	田立涛, 丁宁, 王树林, 齐恩芳, 张荣, 王蕊蕊, 马利雯, 李建武, 杨江伟. 马铃薯Argonaute基因家族鉴定及晚疫病诱导表达分析[J]. 作物学报, 2026, 52(4): 1116-1126.
[15]	汪玲, 胡好, 宋家凤, 程洁蓝, 陈颖, 郑婷婷, 吕钊彦, 朱晓彪, 侯华兰. 马铃薯UDP-糖基转移酶基因StUGT52的克隆及功能验证[J]. 作物学报, 2026, 52(3): 665-676.

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed