欢迎访问作物学报,今天是

作物学报 ›› 2014, Vol. 40 ›› Issue (01): 45-53.doi: 10.3724/SP.J.1006.2014.00045

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

三个耐冻性不同的马铃薯野生种中FAD2基因的克隆及表达分析

李飞1,2,徐建飞1,刘杰1,段绍光1,卞春松1,Jiwan P. PALTA3,金黎平1,*   

  1. 1 中国农业科学院蔬菜花卉研究所, 北京 100081; 2 贵州省马铃薯研究所, 贵州贵阳 550006; 3 Department of Horticulture, University of Wisconsin, Madison 53706, WI, USA
  • 收稿日期:2013-07-01 修回日期:2013-09-16 出版日期:2014-01-12 网络出版日期:2013-10-22
  • 通讯作者: 金黎平, E-mail: jinliping@caas.cn, Tel: 010-82109543
  • 基金资助:

    本研究由“十二五”国家科技支撑计划项目(2012BAD02B05), 国家现代农业产业技术体系建设专项(CARS-10)和黔农科院院专项[2008]022号项目资助。

Molecular Cloning and Expression Analysis of FAD2 Gene from Three Wild Potato Species with Different levels of Freezing Tolerance

LI Fei1,2,XU Jian-Fei1,LIU Jie1,DUAN Shao-Guang1,BIAN Chun-Song1,Jiwan P. PALTA3,JIN Li-Ping1,*   

  1. 1 The Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China; 2 Institute of Potato in Guizhou Province, Guiyang, 550006, China; 3 Department of Horticulture, University of Wisconsin, Madison 53706, WI, USA
  • Received:2013-07-01 Revised:2013-09-16 Published:2014-01-12 Published online:2013-10-22
  • Contact: 金黎平, E-mail: jinliping@caas.cn, Tel: 010-82109543

摘要:

3个耐冻性和冷驯化能力不同的马铃薯野生种Solanum commersoniiS. acauleS. cardiophyllum为材料, 利用RT-PCR技术克隆出不饱和脂肪酸合成途径中的关键酶基因ω-6脱氢酶基因(FAD2), 分别命名为Cmm-FAD2 (GenBank登录号为KF214782)Aca-FAD2 (KF214781)Cph-FAD2 (KF214783)。序列分析表明该基因在3个马铃薯野生种中核苷酸长度都为1326 bp, 编码441个氨基酸残基。3个野生种FAD2基因核苷酸序列和蛋白序列比对结果表明, 18个位点的差异核苷酸中有8个位点的差异导致对应的氨基酸残基变化, 其中第11和第44氨基酸残基处, 耐冻且有冷驯化能力的S. commersoniiS. acaule有相同的氨基酸, 而冷冻敏感S. cardiophyllum的氨基酸却不同。二级结构预测结果表明S. commersoniiS. acauleFAD2蛋白与S.cardiophyllum FAD2蛋白的α-螺旋、延伸链和随机卷曲存在明显差异。蛋白多序列比对和进化树分析表明, 3个马铃薯野生种的FAD2基因与番茄的亲缘关系最近, 其次是与马齿笕。qPCR分析表明, 12 d冷驯化使FAD2基因在3个马铃薯野生种中都上调表达, S. commersoniiS. acauleFAD2基因表达水平均高于S. cardiophyllum, 且差异显著。

关键词: 马铃薯野生种, 耐冻, 冷驯化, FAD2, 基因表达

Abstract:

Using reverse transcription polymerase chain reaction (RT-PCR), three new full-length cDNAs of ω-6 desturases (FAD2) were obtained from three wild potatospecies with different levels of freezing tolerance and cold acclimation capacity, Solanum commersonii, S. acaule,and S. cardiophyllum, designated Cmm-FAD2 (GenBank accession No. KF214782), Aca-FAD2 (KF214781), and Cph-FAD2 (KF214783). Theresults of sequence analysis indicated that their nucleotide length is all 1326 bp, coding 441 amino acids. It’s detected that eight nucleotides resulted in corresponding change of amino acids among the eighteen difference nucleotides by nucleotide and protein sequences alignment of FAD2 genes from three potato species. At the amino acid residue 11 and 44, sequencesof S. commersonii and S. acaule are different from that of S. cardiophyllum. The results of secondary structure prediction indicated that there are obvious differences in alpha-helix, extend chain and random crimp of S. commersonii and S. acaule as compared with S. cardiophyllum. By protein multiple sequence alignments and phylogenetic tree analyses, the results showed that FAD2 genes from three species are highly similar to these of tomato and purslane. The results of qPCR showed that FAD2 genes expression was up-regulated in the three potato wild species, there existed significant differencein relative expression level of FAD2

Key words: Wild potato species, Frost tolerance, Cold acclimation capacity, FAD2, Gene expression

[1]Estrada R N. Breeding frost-resistant potatoes for the tropical highlands. In: Li P H, Sakai A, eds. Plant cold hardiness and freezing stress. New York: Academic Press, 1978. pp 333–341



[2]Li P H. Palta J P. Frost hardening and freezing stress in tuber bearing Solanum species. In: Li P H, Sakai A, eds. Recent advances in plant cold hardiness and freezing stress: Mechanism and crop implications. New York: Academic Press, 1978. pp 49–71



[3]李飞. 野生马铃薯植株耐冻性鉴定及耐冻机理研究. 中国农业科学院研究生院硕士学位论文, 2008.



Li F. Assessment and mechanism study for freezing tolerance in Solanum acaule seedling. MS Theses of the Graduate School of Chinese Academy of Agriculture Sciences, 2008 (in Chinese with English abstract)



[4]Steponkus P L. Role of plasma membrane in freezing injury and cold acclimation. Annu Rev Plant Physiol, 1984, 35: 543–584



[5]Riken A, Dill J W, Bergman D K. Correlation between the circadian rhythm of resistance to extreme temperatures and changes in fatty acid composition in cotton seedlings. Plant Physiol, 1993, 101: 31–36



[6]Steponkus P L, Uemura M, Webb M S. A contrast of the cryostability of the plasma membrane of winter rye and spring oat. In: Steponkus P L ed. Advances in Low-Temperature Biology. London: JAI Press, 1993. pp 211–312



[7]Upchurch RG. Fatty acid unsaturation, mobilization, and regulation the response of plants to stress. Biotechnol Lett, 2008, 30: 967–977



[8]Teixeira M C, Coelho N, Olsson M E, Brodelius P E, Carvalho I S, Brodelius M. Molecular cloning and expression analysis of three omega-6 desaturase genes from purslane (Portulaca oleracea L.). Biotechnol Lett, 2009, 31:1089–1101



[9]Kargiotidou A, Deli D, Galanopoulou D, Tsaftaris A, Farmaki T. Low temperature and light regulate delta 12 fatty acid desaturases (FAD2) at a transcriptional level in cotton (Gossypium hirsutum). J Exp Bot, 2008, 59: 2043–2056



[10]Niu B, Guo L, Zhao M, Luo T, Zhang R, Zhang F, Hou P, Zhang Y, Xu Y, Wang S, Chen F. Molecular cloning, characterization, and expression of an omega-3 fatty acid desaturase gene from Sapium sebiferum. J Biosci Bioeng, 2008, 106: 375–380



[11]Murata N, Sato N, Takahashi N, Hamazaki Y. Composition and positional distributions of fatty acids in phospholipids from leaves of chilling-sensitive and chilling-resistant plants. Plant Cell Physiol, 1982, 23: 1071–1079



[12]Lemieux B, Miquel M, Somerville C, Browse J. Mutants of Arabidopsis with alterations in seed lipid fatty acid compositon. Theor Appl Genet, 1990, 80: 234–240



[13]Vega S E, Del Rio A H, Bamberg J B, Palta J P. Evidence for the up-regulation of stearoyl-ACP (Δ9) desaturase gene expression during cold accliamtion. Am J Potato Res, 2004, 81: 125–135



[14]Yin D M, Deng S Z, Zhan K H, Cui D Q. High-oleic peanut oils produced by HpRNA-mediated gene silencing of oleate desaturase. Plant Mol Biol Rep, 2007, 25: 154–163



[15]Georgios B, Anastassios M, Nikos N, Polydefkis H. Spatial and temporal expressions of two distinct oleate desaturase from olive (Olea europaea L.). Plant Sci, 2005, 168: 547–555



[16]Rolletschek H, Borisjuk L, Sanchez-Garcra A, Gotor C, Romero LC, Martinez-Rivas J M, Mancha M. Temperature dependent endogenous oxygen concentration regulates microsomal oleate desaturase in developing sunflower seeds. J Exp Bot, 2007, 58: 3171–3181



[17]Li LY, Wang X L, Gai J Y, Yu D Y. Molecular cloning and characterization of a novel microsomal oleate desaturase gene from soybean. J Plant Physiol, 2007, 164: 1516–1526



[18]Mietkiewska E, Brost J M., Giblin E M, Francis T, Wang S, Reed D, Truksa M, Taylor D C. A Tropaeolum majus FAD2 cDNA complements the fad2 mutation in transgenic Arabidopsis plants. Plant Sci, 2006, 171:187–193



[19]崔红. 冬小麦东农冬麦1号抗寒生理特性及抗寒基因的克隆. 东北农业大学硕士学位论文, 2010



Cui H. Analysis in cold-resistant physiological characteristics and cloning genes of winter wheat dongnongdongmai1. MS Theses of Northeast Agricultural University, 2010 (in Chinese with English abstract)



[20]Van Berkel J, Salamini F, Gebhardt C. Transcripts accumulating during cold storage of potato (Solanum tuberosum) tubers are sequence related to stress-responsive genes. Plant Physiol, 1994, 104: 445–452



[21]Rorat T, Grygorowicz W J, Bcrbczy P, Irzykowski W. Isolation and expression of cold specific genes in potato (Solanum sogarandinum). Plant Sci, 1998, 133: 57–67



[22]Stone J M, Palta J P, Bamberg J B, Weiss L S, Harbage J F. Inheritance of freezing resistance in tuber-bearing Solanum species : Evidence for independent genetic control of nonacclimated freezing tolerance and cold acclimation capacity. Genetics, 1993, 90: 7869–7873



[23]Vega S E, Del Rio A H, Jung G, Bamberg J B, Palta J P. Marker-assisted genetic analysis of non-acclimated freezing tolerance and cold acclimation capacity in a backcross Solanum population. Am J Potato Res, 2003, 80: 359–3691



[24]李飞, 徐建飞, 刘杰, 段绍光, 雷尊国, Palta J P, 金黎平. 冷驯化前后野生马铃薯S. acaule内参基因的筛选. 西南农业学报, 2012, 25: 1592–1595



Li F, Xu J F, Liu J, Duan S G, Lei Z G, Palta J P, Jin L P. Selection of reference genes from wild potato Solanum acaule before and after cold acclimation.   Southwest China J Agric Sci, 2012, 25: 1592–1595 (in Chinese with English abstract)



[25]Kargiotidou A, Deli D, Galanopoulou D, Tsaftaris A, Farmaki T. Low temperature and light regulate delta 12 fatty acid desaturases (FAD2) at a transcriptional level in cotton (Gossypium hirsutum). J Exp Bot, 2008, 59: 2043–2056



[26]Hernandez M L, Padilla M N, Sicardo M D, Mancha M, Martinez-Rivas J M. Effect of different environmental stresses on the expression of oleate desaturase genes and fatty acid composition in olive fruit. Phytochemistry, 2011, 72: 178–187



[27]Matteucci M, Dangeli S, Errico S, Lamanna R, Perrotta G, Altamura M M. Cold affects the transcription of fatty acid desaturases and oil quality in the fruit of Olea europaea L. genotypes with different cold hardiness. J Exp Bot, 2011, 62: 3403–3420



[28]Zhang Y M, Wang C C, Hu H H, Yang L. Cloning and expression of three fatty acid desaturase genes from cold-sensitive lima bean (Phaseolus lunatus L.). Biotechnol Lett, 2011, 33: 395–401



[29]Yang L, Ye J, Guo W D, Wang C C, Hu H T. Differences in cold tolerance and expression of two fatty acid desaturase genes in the leaves between fingered citron and its dwarf mutant. Trees, 2012, 26: 1193–1201



[30]Shi J L, Cao Y P, Fan X R, Li M, Wang Y F, Ming F. A rice microsomal delta-12 fatty acid desaturase can enhance resistance to cold stress in yeast and Oryza sativa. Mol Breed, 2012, 29:743–757



[31]周洲. 转脂肪酸去饱和酶基因PtFAD2和PtFAD3银腺杨84K的抗寒性研究. 中国林业科学研究院博士学位论文, 2007



Zhou Z. The cold tolerance of transgenic Populus alba × Populus glandulossa 84K with fatty acid desaturase genes PtFAD2 and PtFAD3 .PhD Dissertation  Chinese of Academy of Forestry, 2007 (in Chinese with English abstract)



[32]Honjoh K, Machida T, Hagisako T, Suga K, Yonekura M, Shimizu H, Ohashi N, Miyamoto T, Hatano S, Lio M. Molecular Cloning and characterization of a cDNA for low-temperature indueible cytosolic glueose 6-PhosPhate dehydrogenase gene from Chlorella vulgaris and expression of the gene in Saceharomyces cerevisiae. Plant Sei, 2007, 172: 649–658



[33]Rodriguez-Vargas S, Sanchez-Garcia A, Martinez-Rivas J M, Prieto J A, Randez-Gil F. Fluidization of membrane lipids enhances the tolerance of Saccharomyces cerevisiae to freezing and salt stress. Appl Environ Microbiol, 2007, 73: 110–116

[1] 李海芬, 魏浩, 温世杰, 鲁清, 刘浩, 李少雄, 洪彦彬, 陈小平, 梁炫强. 花生电压依赖性阴离子通道基因(AhVDAC)的克隆及在果针向地性反应中表达分析[J]. 作物学报, 2022, 48(6): 1558-1565.
[2] 姚晓华, 王越, 姚有华, 安立昆, 王燕, 吴昆仑. 青稞新基因HvMEL1 AGO的克隆和条纹病胁迫下的表达[J]. 作物学报, 2022, 48(5): 1181-1190.
[3] 渠建洲, 冯文豪, 张兴华, 徐淑兔, 薛吉全. 基于全基因组关联分析解析玉米籽粒大小的遗传结构[J]. 作物学报, 2022, 48(2): 304-319.
[4] 陈新宜, 宋宇航, 张孟寒, 李小艳, 李华, 汪月霞, 齐学礼. 干旱对不同品种小麦幼苗的生理生化胁迫以及外源5-氨基乙酰丙酸的缓解作用[J]. 作物学报, 2022, 48(2): 478-487.
[5] 王艳朋, 凌磊, 张文睿, 王丹, 郭长虹. 小麦B-box基因家族全基因组鉴定与表达分析[J]. 作物学报, 2021, 47(8): 1437-1449.
[6] 宋天晓, 刘意, 饶莉萍, Soviguidi Deka Reine Judesse, 朱国鹏, 杨新笋. 甘薯细胞壁蔗糖转化酶基因IbCWIN家族成员鉴定及表达分析[J]. 作物学报, 2021, 47(7): 1297-1308.
[7] 解盼, 刘蔚, 康郁, 华玮, 钱论文, 官春云, 何昕. 甘蓝型油菜CBF基因家族的鉴定和表达分析[J]. 作物学报, 2021, 47(12): 2394-2406.
[8] 李鹏, 刘彻, 宋皓, 姚盼盼, 苏沛霖, 魏跃伟, 杨永霞, 李青常. 烟草非特异性脂质转移蛋白基因家族的鉴定与分析[J]. 作物学报, 2021, 47(11): 2184-2198.
[9] 黄素华, 林席跃, 雷正平, 丁在松, 赵明. 强再生力水稻品种碳氮营养与激素生理特征研究[J]. 作物学报, 2021, 47(11): 2278-2289.
[10] 米文博, 方园, 刘自刚, 徐春梅, 刘高阳, 邹娅, 徐明霞, 郑国强, 曹小东, 方新玲. 白菜型冬油菜温敏不育系PK3-12S育性转换的差异蛋白质组学分析[J]. 作物学报, 2020, 46(10): 1507-1516.
[11] 靳舒荣,王艳玫,常悦,王月华,李加纳,倪郁. 不同收获指数甘蓝型油菜β-淀粉酶活性及其基因家族成员的表达分析[J]. 作物学报, 2019, 45(8): 1279-1285.
[12] 冯韬,官春云. 甘蓝型油菜光敏色素互作因子4 (BnaPIF4)基因克隆和功能分析[J]. 作物学报, 2019, 45(2): 204-213.
[13] 赵晶,李旭彤,梁学忠,王志城,崔静,陈斌,吴立强,王省芬,张桂寅,马峙英,张艳. 陆地棉漆酶基因家族鉴定及在黄萎病菌胁迫下的表达分析 *[J]. 作物学报, 2019, 45(12): 1784-1795.
[14] 薛晓梦,李建国,白冬梅,晏立英,万丽云,康彦平,淮东欣,雷永,廖伯寿. 花生FAD2基因家族表达分析及其对低温胁迫的响应[J]. 作物学报, 2019, 45(10): 1586-1594.
[15] 谈欢,刘玉汇,李丽霞,王丽,李元铭,张俊莲. 马铃薯块茎花色素苷合成相关R2R3 MYB蛋白基因的克隆和功能
分析
[J]. 作物学报, 2018, 44(7): 1021-1031.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!