籼稻GAD基因的克隆、序列分析及其植物表达载体构建

[目的]克隆籼稻谷氨酸脱羧酶(GAD)基因的全长cDNA,并进行序列分析和植物表达载体构建,为改良水稻的营养保健品质奠定基础.[方法]根据已知植物GAD基因的保守区域设计引物,以高GABA籼稻品系“新-9-4”的胚芽为材料,利用RT-PCR和RACE技术克隆GAD基因的全长cDNA;扩增其编码序列,构建双T-DNA植物表达载体.[结果]扩增获得长1962 bp的籼稻GAD基因全长cDNA(OsiGAD),包含1479 bp的开放阅读框,编码492个氨基酸.OsiGAD与已报道的水稻GAD基因OsGAD1、OsGAD2、OsGAD3的核苷酸序列同源性分别为67.1%、69.4%、98.3%,推导氨基酸序列的同源性分别为77.6%、70.1%、100.0%.OsiGAD蛋白序列具有磷酸吡哆醛结合位点和与钙调蛋白结合的C端延伸区域;构建了其具有水稻胚乳特异表达特性的双T-DNA植物高效表达载体pCDMAR-OsiGAD-hpt,选择标记基因和OsiGAD-因分别位于此载体的两个不同T-DNA区.[结论]克隆获得的籼稻GAD基因OsiGAD长1962 bp,并成功构建了其双T-DNA植物高效表达载体.

Cloning, sequence analysis of GAD gene from indica rice and its plant expression vector construction

[Objective]The current experiment was conducted to clone the full length cDNA of glutamate decarboxylase (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 OsGAD1, OsGAD2 and OsGAD3, 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.