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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

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