| 摘要: |
| 为揭示类异黄酮还原酶(Isoflavone reductase-like, IRL)在烟草次生物质合成和生理功能中的作用, 从品种龙里红花烟中克隆普通烟草IRL(NtIRL)基因家族2个成员的全长cDNA和gDNA序列, 完成了生物信息学分析、Southern杂交和RT-PCR等分子鉴定。NtIRL1基因为1 946 bp, 全长cDNA为1 257 bp;NtIRL2基因为2 089 bp, 全长cDNA为1 261 bp。NtIRL1和NtIRL2蛋白均为310个氨基酸, 分子质量分别为34.652和34.650 ku, 等电点分别为5.58和5.78。2个蛋白可能发生磷酸化等翻译后修饰, 无信号肽, 定位于细胞质, 但有可能通过跨膜域与质膜发生联系。预测这2个蛋白的K8~G88为AdoHycase(cl09931)保守域, I9~T239为NmrA(pfam05368)保守域, 并具有PIP类型蛋白的所有保守性基序和活性残基。预测2个蛋白的二级结构以α螺旋和延伸链为主;三级结构具有PIP酶典型特征, 中间有1个催化裂口。BLAST和系统发生分析进一步证实NtIRL家族属于PIP大家族, 与IFR的关系比PCBER和PLR更近。Southern blot杂交也证明, 普通烟草基因组中有且只有2个IRL基因存在。半定量RT-PCR结果显示, NtIRL1和NtIRL2均在根中优势表达, 在地上部各器官中表达很弱或无表达。 |
| 关键词: 烟草 类异黄酮还原酶 分子克隆 基因家族 |
| DOI：10.11841/j.issn.1007-4333.2014.06.04 |
| 投稿时间：2014-02-21 |
| 基金项目:贵州省科学技术基金项目(黔科合J字[2011]2340号);贵州省烟草专卖局科技重大专项(2007-04);国家烟草专卖局科技重大专项(Ts-02-20110015) |
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| Molecular cloning of isoflavone reductase-like gene family from tobacco |
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CHEN Wei1, LIN Ye-chun1, GAO Wei-chang1, LI Chun-jian2, WANG San-gen3, LV Jun3, CHAI You-rong3
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| (1.Guizhou Academy of Tobacco Science, Guiyang 550081, China;2.College of Resources and Environmental Sciences, China Agricultural University, Beijing 100193, China;3.College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China) |
| Abstract: |
| In order to reveal the roles of isoflavone reductase-like (IRL) in biosynthesis of secondary metabolites and physiological functions in tobacco (Nicotiana tabacum), full-length cDNA and gDNA of 2 members of tobacco IRL (NtIRL) gene family were cloned from cultivar Longlihonghua, and molecular identifications such as bioinformatic analysis, Southern hybridization and RT-PCR were performed.NtIRL1 gene was 1 946 bp with full-length cDNA of 1 257 bp, while NtIRL2 gene was 2 089 bp with full-length cDNA of 1 261 bp.Both NtIRL1 and NtIRL2 proteins were of 310 amino acids, with MW of 34.652 and 34.650 ku and pI of 5.58 and 5.78 respectively.They might experience posttranslational phosphorylation.Without signal peptide, they were predicted to be located in the cytoplasm, but they might have association with plasma membrane via transmembrane domains.They both had a AdoHycase (cl09931) conserved domain at K8-G88 and a NmrA (pfam05368) domain at I9-T239, with typical conservative motifs and active residues of PIP-type proteins.Their predicted secondary structures were dominated by α-helices and extended strands.Their tertiary structures had typical features of PIP enzymes with a central catalytic cleft.BLAST and phylogenetic analysis further proved that NtIRL family belonged to PIP large family, with nearer relationship to IFR than to PCBER and PLR.Southern blot hybridization also verified that in tobacco genome there were only 2 IRL genes.Semi-quantitative RT-PCR results indicated that both NtIRL1 and NtIRL2 were dominantly expressed in roots, while above-ground organs had very weak or no expression of them. |
| Key words: tobacco (Nicotiana tabacum) isoflavone reductase-like (IRL) molecular cloning gene family |
