南方农业学报2012，Vol.43Issue(8)：1079-1085,7.DOI:10.3969/j:issn.2095-1191.2012.08.1079
籼稻GAD基因的克隆、序列分析及其植物表达载体构建
Cloning, sequence analysis of GAD gene from indica rice and its plant expression vector construction
摘要
Abstract
[Objective]The current experiment was conducted to clone the full length cDNA of glutamate decarboxy-lase (GAD) gene from indica rice,and then analyze its sequence followed by the construction of plant expression vector to provide references for improving rice nutrition and healthcare quality. [Method]Full length cDNA of GAD gene from indica rice 'Xin-9-4' rich in GABA was cloned by RT-PCR and RACE based on the specific primers of the conserved domain of other GAD genes. Coding sequence was amplified and double T-DNA plant expression vector was constructed. [Result] The amplified full length cDNA (1962 bp) of GAD gene denominated as OsiGAD contained a 1479 bp ORF encoding a 492-amino-acid polypeptide. OsiGAD shared 67.1,69.4 and 98.3% homology with nucleotide sequence of rice OsGADl,OsGAD2 and 0sGAD3,respectively and the deduced amino acid sequence identity was 77.6,70.1 and 100.0%,respectively. Amino acid alignment indicated OsiGAD had a pyridoxal-5'- phosphate (PLP) binding domain in the middle region of the peptide and a calmodulin-binding domain at the carboxyl terminus. Double T-DNA vector with rice endosperm-specific expression was constructed,of which selectable marker hpt gene and OsiGAD gene were located in different T-DNA regions of the vector. [Conclusion]The full length of the cloned indica rice GAD gene (OsiGAD) was 1962 bp,hence the plant expression vector of this gene was successfully constructed.关键词
籼稻/谷氨酸脱羧酶(GAD)/γ-氨基丁酸(GABA)/基因克隆/序列分析/载体构建Key words
indica rice/glutamate decarboxylase (G AD) /γ-amino butyric acid (GABA)/gene cloning/sequence analysis/vector construction分类
农业科技引用本文复制引用
黄志伟,许明,林忠辉,蔡小玲,郑金贵..籼稻GAD基因的克隆、序列分析及其植物表达载体构建[J].南方农业学报,2012,43(8):1079-1085,7.基金项目
国家自然科学基金项目(30471073) （30471073）
国家转基因重大专项项目(2009ZX08001-032B) （2009ZX08001-032B）
福建省科技合作计划重点项目(2010I0001) （2010I0001）
福建省科技计划重点项目(2010N0003) （2010N0003）
