矮牵牛DFR-A基因的克隆与表达分析

doi:10.11924/j.issn.1000-6850.2013-2373

摘要/Abstract

摘要： 通过分离矮牵牛DFR-A基因，研究其在不同部位的表达规律和酶活性变化，为花色素苷合成的结构基因分离及其他研究奠定基础。基于已公布的矮牵牛DFR序列，直接从矮牵牛花瓣中分离了DFRA基因序列，利用实时荧光定量PCR技术研究其在不同器官中的表达特性，同时测定了不同器官中的DFR酶活性。（1）矮牵牛DFR-A基因cDNA序列为1473 bp，该基因最大开放阅读框为1143 bp，编码380个氨基酸。（2）DFR-A基因在矮牵牛各种器官中均有表达，但不同组织中表达量存在差异，在花药中的表达量最高，其次是初开和盛开花瓣中，各期花蕾中的表达量都较低，而在叶中表达量最低。（3）DFR酶在叶片及花药中几乎无活性。在初开花瓣中达到最高峰。（4）除花药以外的部位，DFR酶活性与DFR mRNA浓度存在线性相关性。矮牵牛DFR酶活性主要受转录调控，活性在初开花瓣中最高。

关键词: 品质, 品质

Abstract: After isolation the DFR-A from Petunia hybrida, its expression characteristic and the DFR enzyme activity in different tissues were studied. It will be helpful for the isolation and study of other anthocyanin structural genes. Based on the petunia DFR gene sequences published by NCBI, the full length cDNA of DFR was isolated from the petals of petunia. Real-time quantitative PCR (qRT-PCR) was performed to determine the expression pattern of DFR in different tissues of petunia. The DFR enzyme activities in different tissues were measured also. (1) 1473 bp cDNA sequence of petunia DFR- A was cloned. Bioinformatics analysis showed that the open reading frame of this gene was 1143 bp, encoding 380 amino acids. (2) Expression analysis showed that the expression of DFR gene was detected in all tissues of Petunia. But the expression levels were different; the expression level of this gene was highest in anther, medium in petal of flower starting to open and flower fully opened, lower in flower buds, and the lowest expression level of DFR was in leaf. (3) DFR activity analysis revealed that DFR was almost no activity in leaf and anther, low activity in flower buds, the highest activity in flower starting to open. (4) There was correlation between DFR activity and DFR mRNA concentration except in anther. The DFR enzyme activity of Petunia was mainly controlled by the transcriptional regulation.

褚云霞陈海荣潘俊松吴爱忠蔡润邓姗. 矮牵牛DFR-A基因的克隆与表达分析[J]. 中国农学通报, 2014, 30(10): 163-169.

参考文献

[1] Jacobs M, Rubery P. Naturally occurring auxin transport regulators [J]. Science,1988,241:346-349.
[2] 李春雷,崔国新,许志茹,等.植物二氢黄酮醇 4-还原酶基因的研究进展[J].生物技术通讯,2009,20(3):442-445.
[3] 代色平,包满珠.矮牵牛育种研究进展[J].植物学通报,2004,21(4): 385-391.
[4] 李义龙,肇涛澜,陈立超,等.花色素苷生物合成及花色的调控[J].生命科学,2008,20(1):147-152.
[5] O'Reilly C, Shepherd N S, Pereira A, et al. Molecular cloning of the al locus of Zea mays using the transposable elements En and Mul [J]. The EMBO Journal,1985,4(4):877-882.
[6] Li H H, Qiu J, Chen F D, et al. Molecular characterization and expression analysis of dihydroflavonol 4- reductase (DFR) gene in Saussurea medusa[J]. Mol Biol Rep,2012,39(3):2991-2999.
[7] 李云,卢其能,赵昶灵,等.二氢黄酮醇-4-还原酶基因的结构与功能研究[J].安徽农业科学,2011,39(26):15858-15859.
[8] Gerats A G M, Huits H, Vrijlandt E, et al. Molecular characterization of a nonautonomous transposable element (dTphl) of Petunia[J]. Plant Cell,1990,2:1121-1128.
[9] 宋锋.毛白杨二氢黄酮醇 4-还原酶(DFR)基因在烟草中的功能验证[D].重庆:西南大学,2010.
[10] Meyer P, Heidmann I, Forkmann G, et al. A new petunia flower colour generated by transformation of a mutatnt with maize gene[J]. Nature,1987, 330(17):677-678.
[11] Tanaka K, Fukui Y, Fukuchi-Mizutani M, et al. Molecular cloning and characterization of Rosa hybrida dihydroflavonol 4- reductase gene[J]. Plant Cell Physiol,1995,36(6):1023-1031.
[12] Mol J N M, Holton T A, Koes R E. Floriculture: genetic engineering of commercial traits [J]. Trends in Biotechnology, 1995, 13(9):350-355.
[13] 李吉莲,姚宁涛,祝建波,等.矮牵牛花特异表达启动子 PchsA的克隆及蓝色基因 F3’ 5’ H表达载体的构建[J].安徽农业科学,2009,37 (17):7897-7899.
[14] Nakatsuka A, Izumi Y, Yamagishi M. Spatial and temporal expression of chalcone synthase and dihydroflavonol 4- reductase genes in the Asiatic hybrid lily [J]. Plant Science,2003, 165(4): 759- 767.
[15] Koes R E, Quattrocchio F, Mol J N M. The flavonoid biosynthetic pathway in plants: Function and evolution [J]. BioEssays,1994,16 (2):123-132.
[16] Gasteiger E, Hoogland C, Gattiker A, et al. Protein identifieation and analysis tools in the ExPASy server [M]. The Proteomics Protocols Handbook, Walker J M, Totowa:Humana Press Inc., 1999: 571-607,531-552.
[17] Combet C, Blanchet C, Geourjon C, et al. NPS@:network protein sequence analysis [J]. Trends Biochem Sci.,2000,25(3):147-150.
[18] Arnold K, Bordoli L, Kopp J, et al. The SWISS- MODEL workspace: a web- based environment for protein structure homology modelling [J]. Bioinformatics,2006,22(2):195-201.
[19] Hayashi M, Takahashi H, Tamura K. Enhanced dihydroflavonol-4- reductase activity and NAD homeostasis leading to cell death tolerance in transgenic rice [J]. Proc Natl Acad Sci USA, 2005,102 (19):7020-7025.
[20] 朱奇朗,李晓波,肖向文,等.不同色彩矮牵牛 DFR基因的克隆与生物信息学分析[J].生物技术通报,2013(9):68-76.
[21] 花庆,王蕾,韦灵林,等.矮牵牛 F3'5'H基因的克隆及其在不同着色花药中的表达[J].西北农业学报,2011,20(8):152-158.
[22] Huits H S, Gerats A G, Kreike M M, et al. Genetic control of dihydroflavonol 4-reductase gene expression in Petunia hybrida [J]. Plant J,1994, 6(3):295-310.
[23] Beld M, Martin C, Henk H, et al. Flavonoid synthesis in Petunia hybrida: partial characterization of dihydroflavonol- 4- reductase genes [J]. Plant Mol Biol,1989,13(5):491-502.