水稻高叶绿素含量基因DET1 cDNA的克隆、生物信息学分析及超表达载体的构建

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中国生物工程杂志 ›› 2010, Vol. 30 ›› Issue (04) : 60-64.

研究报告

水稻高叶绿素含量基因DET1 cDNA的克隆、生物信息学分析及超表达载体的构建

黄俊丽^1**,刘太波¹,王贵学^1**,李贤勇^1,2

作者信息 +

cDNA Cloning, Bioinformatics Analysis and Construction of Overexpression Vector of High-chlorophyll Rice Gene DET1

HUANG Jun-li¹,LIU Tai-bo¹,WANG Gui-xue¹,LI Xian-yong^1,2

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文章历史 +

摘要

提高水稻叶片中叶绿素含量可以提高光合作用效率。以水稻高叶绿素含量突变体为材料，在前期精细定位的基础上，克隆了高叶绿素含量基因DET1（DE-ETIOLATED 1）cDNA。测序结果显示：DET1基因全长cDNA为1536 bp，由11个外显子组成，编码512个氨基酸。与野生型珍汕97B相比，DET1基因在第7个外显子上有T-C的转换，该转换引入了一个EcoR I酶切位点，并导致第328位的亮氨酸（L）改变为丝氨酸（S），且突变位点处于高度保守区域内。DET1基因的氨基酸序列与玉米等其它高等植物的氨基酸序列同源性高达62%以上。利用生物信息学方法对DET1基因的序列、进化树、理化性质及亲疏水性等进行分析，并构建了水稻超表达载体pCUPHN-DET1，为水稻高叶绿素含量突变基因DET1的功能验证奠定基础。

Abstract

The efficiency of photosynthesis can be improved by increasing chlorophyll content in rice leaves. In this study, DET1 （DE-ETIOLATED 1）gene related to high chlorophyll content has been cloned based on the fine mapping and sequence analysis of the DET1 gene was carried out in the high-chlorophyll mutants. The DET1 cDNA full-length of 1536 bp encoded 512 amino acids, and revealed a single T-to-C base transversion in the seventh exon of the DET1 gene which consisted of 11 exons, in comparison with the normal wild-type sequence. This transversion resulted in a conserved Leucine-to-Serine amino acid substitution at the position of 328, and introduced a recognition site for the EcoR I restriction endonuclease and the mutation located in the highly conserved region. Besides, the amino acid sequence of DET1 had more than 62% homology compared to corn and other higher plants. The phylogenetic tree, physical and chemical properties, hydrophilicity and hydrophobicity of the amino acid sequence have been analyzed by the bioinformatics technology. The overexpression vector of pCUPHN-DET1 has been constructed with the binary expression vector pCUPHN. The results laid good foundation for functional verification of DET1.

导出引用

黄俊丽刘太波王贵学李贤勇. 水稻高叶绿素含量基因DET1 cDNA的克隆、生物信息学分析及超表达载体的构建[J]. 中国生物工程杂志, 2010, 30(04): 60-64

HUANG Dun-Li, LIU Ta-Bei, WANG Gui-Hua, LI Xian-Yong. cDNA Cloning, Bioinformatics Analysis and Construction of Overexpression Vector of High-chlorophyll Rice Gene DET1[J]. China Biotechnology, 2010, 30(04): 60-64

参考文献

［1］李贤勇，王楚桃，李顺武，等.一个水稻高叶绿素含量基因的发现.西南农业学报，2002，15（4）: 122123. Li X Y, Wang C T, Li S W, et al. Southwest China Journal of Agricultural Sciences, 2002, 15（4）: 122123.
［2］李贤勇，王楚桃，李顺武，等.一个水稻高叶绿素基因的生物学特性研究.西南农业学报，2004，17（3）: 292294. Li X Y, Wang C T, Li S W, et al. Southwest China Journal of Agricultural Sciences, 2004, 17（3）: 292294.
［3］ Wang F H, Wang G X, Li X Y, et al. Heredity, physiology and mapping of a chlorophyll content gene of rice (Oryza sativa L.). Journal of Plant Physiology, 2008, 165(3):324330.
［4］ Mustilli A C, Fenzi F, Ciliento R, et al. Phenotype of the tomato high pigment2 mutant is caused by a mutation in the tomato homolog of DEETIOLATED1. Plant Cell, 1999, 11:145157.
［5］ Levin I, Frankel P, Gilboa N, et al. The tomato dark green mutation is a novel allele of the tomato homolog of the DEETIOLATED1 gene. Theor Appl Genet, 2003, 106:454460.
［6］ Pepper A, Delaney T, Washburn T, et al. DET1, a negative regulator of lightmediated development and gene expression in Arabidopsis, encodes a novel nuclearlocalized protein. Cell, 1994, 78:109116.
［7］ Song H R, Carre I A. DET1 regulates the proteasomal degradation of LHY, a component of the Arabidopsis circadian clock. Plant Molecular Biology, 2005, 57:761771.
［8］ Alexandrov N N, Brover W, Freidin S, et al. Insights into corn genes derived from largescale cDNA sequencing. Plant Mol Biol, 2009, 69:179194.
［9］ Paterson A H, Bowers J E, Bruggmann R, et al. The Sorghum bicolor genome and the diversification of grasses. Nature, 2009, 457 (7229):551556.
［10］范敏，金黎平，刘庆昌，等.马铃薯抗旱机理及其相关研究进展.中国马铃薯，2006，32（2）: 101107. Fan M, Jin L P, Liu Q C, et al. Chinese Potato, 2006, 32(2):101107.
［11］ Qiu D W, Mao J J, Yang X F, et al. Expression of an elicitorencoding gene from Magnaporthe grisea enhances resistance against blast disease in transgenic rice. Plant Cell Report, 2008, 28:925933.