Resveratrol synthase (RS) is a key enzyme that plays a critical role in the resveratrol synthesis pathway. In this study, six RS genes were isolated and characterized from peanut variety “Zhenzhu Hong” by silico clo...Resveratrol synthase (RS) is a key enzyme that plays a critical role in the resveratrol synthesis pathway. In this study, six RS genes were isolated and characterized from peanut variety “Zhenzhu Hong” by silico cloning and RT-PCR. Bioinformatics analysis showed that deduced amino acid sequences of the six cloned RS genes were highly conserved with a similarity from 95% to 99% when compared to the RS genes which had been deposited at the GenBank. The results of amino acid sequences analysis showed six RS proteins contained the Chal_Sti_Synt_N and ACP_Syn_III_C domains and can be classified to same family but with different evolutionary distance. Expression pattern analysis by QRT-PCR provided evidence indicating that the mRNA of six RS genes were primarily expressed in the peanut shell at different developmental stages with different expression levels, but only lower levels of them were evident in the peanut kernel. The subcellular localization of RS protein in onion epidermal cell was performed by Agrobacterium tumefaciens-mediated transformation and the green fluorescent was monitored by confocal fluorescence microscopy. The results indicated that, RS1 and RS5 were located in the nucleus and plasma membrane respectively, while the RS2, RS3, RS4 and RS6 were located in both nucleus inner membrane and plasma membrane. The data will provide basic information for elucidating the regulatory mechanisms and enzyme kinetics underlying the RS genes in the resveratrol synthase pathway.展开更多
[Objectives] To obtain a resveratrol synthase gene of Vitis vinifera and make bioinformatics analysis. [Methods] Taking total RNA of V. vinifera as the template,by RT-PCR method,a complete c DNA sequence of resveratro...[Objectives] To obtain a resveratrol synthase gene of Vitis vinifera and make bioinformatics analysis. [Methods] Taking total RNA of V. vinifera as the template,by RT-PCR method,a complete c DNA sequence of resveratrol synthase gene was amplified from V. vinifera,and the resveratrol synthase gene was named as RS. The nucleic acid and protein sequences were analyzed using bioinformatics software.[Results]This sequence was 1179 bp in length,the similarity with reported resveratrol synthase gene reached 94%-99%,and the similarity with amino acid sequence reached 96%-99%; the RS gene encoded 392 amino acids,and amino acid sequence contained complete characteristic sequence GVLFGPGLT and active center sequence GCYAGGTVLR of stilbene synthase family; the predicted molecular weight was42. 78 k Da,the theoretical isoelectric point was 6. 57,the instability parameter was 35. 92,and it belonged to stable protein in the classification; the secondary structure was mainly α-helix,random coil and β-folding,α-helix content was 44. 13%,the random coil content was26. 53%,and β-folding content was 17. 66%. [Conclusions] The isolated RS gene is a resveratrol synthase gene from V. vinifera. This experiment is expected to lay a certain foundation for biosynthesis of resveratrol by the genetic engineering method.展开更多
文摘Resveratrol synthase (RS) is a key enzyme that plays a critical role in the resveratrol synthesis pathway. In this study, six RS genes were isolated and characterized from peanut variety “Zhenzhu Hong” by silico cloning and RT-PCR. Bioinformatics analysis showed that deduced amino acid sequences of the six cloned RS genes were highly conserved with a similarity from 95% to 99% when compared to the RS genes which had been deposited at the GenBank. The results of amino acid sequences analysis showed six RS proteins contained the Chal_Sti_Synt_N and ACP_Syn_III_C domains and can be classified to same family but with different evolutionary distance. Expression pattern analysis by QRT-PCR provided evidence indicating that the mRNA of six RS genes were primarily expressed in the peanut shell at different developmental stages with different expression levels, but only lower levels of them were evident in the peanut kernel. The subcellular localization of RS protein in onion epidermal cell was performed by Agrobacterium tumefaciens-mediated transformation and the green fluorescent was monitored by confocal fluorescence microscopy. The results indicated that, RS1 and RS5 were located in the nucleus and plasma membrane respectively, while the RS2, RS3, RS4 and RS6 were located in both nucleus inner membrane and plasma membrane. The data will provide basic information for elucidating the regulatory mechanisms and enzyme kinetics underlying the RS genes in the resveratrol synthase pathway.
基金Supported by Construction and Expression of Heterologous Biosynthesis Pathway for Resveratrol(BAK:201502bsh)
文摘[Objectives] To obtain a resveratrol synthase gene of Vitis vinifera and make bioinformatics analysis. [Methods] Taking total RNA of V. vinifera as the template,by RT-PCR method,a complete c DNA sequence of resveratrol synthase gene was amplified from V. vinifera,and the resveratrol synthase gene was named as RS. The nucleic acid and protein sequences were analyzed using bioinformatics software.[Results]This sequence was 1179 bp in length,the similarity with reported resveratrol synthase gene reached 94%-99%,and the similarity with amino acid sequence reached 96%-99%; the RS gene encoded 392 amino acids,and amino acid sequence contained complete characteristic sequence GVLFGPGLT and active center sequence GCYAGGTVLR of stilbene synthase family; the predicted molecular weight was42. 78 k Da,the theoretical isoelectric point was 6. 57,the instability parameter was 35. 92,and it belonged to stable protein in the classification; the secondary structure was mainly α-helix,random coil and β-folding,α-helix content was 44. 13%,the random coil content was26. 53%,and β-folding content was 17. 66%. [Conclusions] The isolated RS gene is a resveratrol synthase gene from V. vinifera. This experiment is expected to lay a certain foundation for biosynthesis of resveratrol by the genetic engineering method.
文摘为了获得含有白藜芦醇的转基因番茄,从葡萄雷司令中克隆到芪合酶基因,以之构建了含有组成型启动子的植物表达载体pBS2,用于农杆菌介导对番茄品种TXOO14的遗传转化.通过对诱导愈伤、出芽、生根、再生植株的筛选,得到5株再生小苗.经PCR、Southern检测证实,有3株为真正的转基因植株.用HPLC对3株转基因植株叶片进行白藜芦醇含量鲜重分析,它们中白藜芦醇的含量分别为12.45μg g,5.35μg g,4.55μg g.
