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当前位置: 首页 > 文章 > 赤霉素诱导甘蔗GA20-Oxidase基因实时荧光定量PCR分析 分子植物育种 2009,7 (5) 79-84
Position: Home > Articles > The Real-Time Quantitative Fluorescent PCR Analysis of GA20-Oxidase Gene GA3-Induced in Sugarcane Molecular Plant Breeding 2009,7 (5) 79-84

赤霉素诱导甘蔗GA20-Oxidase基因实时荧光定量PCR分析

作  者：
吴建明;李杨瑞;王爱勤;杨柳;杨丽涛;甘崇琨
单  位：
广西农业科学院经济作物研究所;广西崇左市江州区土肥站;广西作物遗传改良生物技术重点开发实验室
关键词：
甘蔗;GA20-氧化酶;赤霉素;定量PCR;基因表达
摘  要：
本研究以喷清水甘蔗幼茎作为对照(即未处理),经过赤霉素处理后6h、12h、24h和48h的甘蔗幼茎为处理,分别进行取样并提取RNA,反转录获得cDNA作模板,利用SYBR GreenⅠ成功构建了目的基因(GA20-oxidase)和内参基因(GAPDH)的标准品和标准曲线。结果表明:内参基因与目的基因的起始模板浓度与Ct之间呈良好线性关系,决定系数分别为r2=0.998、r2=0.995;溶解曲线均显单峰型,证明扩增的特异性比较好,可对基因表达进行相对定量。定量结果表明:目的基因未处理的表达量最大,赤霉素处理后表达量持续下降,在6h达到最低值,12h后逐渐上升,但到48h又明显下降,且所有处理的表达量均低于未处理,6h、12h、24h和48h比对照的表达量分别下降了24.75%、13.18%、10.23%和20.34%。本实验研究GA20-oxidase基因在赤霉素处理后的表达情况,为进一步克隆甘蔗GA20氧化酶基因全长序列、研究该基因在甘蔗节间伸长过程中的表达调控及甘蔗茎间伸长中的作用机理提供理论依据。
译  名：
The Real-Time Quantitative Fluorescent PCR Analysis of GA20-Oxidase Gene GA3-Induced in Sugarcane
作  者：
Wu Jianming 1,2,3,4 Li Yangrui 1,2,4 Wang Aiqin 2 Yang Liu 1 Yang Litang 1,4 Gan Conghun 5 1 Cash Crops Research Institute,Guangxi Academy of Agricultural Sciences,Nanning,530007;2 Guangxi Crop Genetic Improvement and Biotechnology Lab,Nanning,530007;3 Guangxi Key Laboratory of Subtropical Bioresources Conservation and Utilization,Guangxi University,Nanning,530004;4 Sugarcane Research Center,Chinese Academy of Agricultural Sciences,Nanning,530007;5 Soil and Fertilizer Station of Jiang Zhou,Chongzuo,Guangxi,Chongzuo,532200
关键词：
Sugarcane,GA20-oxidase,Gibberellic acid,Real-time PCR,Gene expression
摘  要：
In this research,the stemlets of sugarcane sprayed by gibberellic acid(GA3) after separately treatment at 6 h,12 h,24 h and 48 h,were employed to be as experimental materials as well as the stemlets of sugarcane sprayed only by water as reference.We extracted RNA from treated samples for obtaining cDNA template by reverse transcription,and then making the standard sample and standard curve of target gene GA20-oxidase and the reference gene GAPDH by using SYBR GreenⅠ system.The results showed that there is a well linear relationship between initial template concentration and Ct of the reference gene and the target genes,and the correlation coefficients(r2) were 0.998 and 0.995,respectively.The dissolution curves both exhibited single-peak curve which proving that the amplification systems were specific and the relative quantitative analysis of gene expression could be carried out.The quantitative expression results showed that the expression of target gene got the highest level in the references,that in the GA3 treatments declined continuously,it reached the minimum level in 6 h,subsequently increased gradually after 12 h,and again decreased significantly at 48 h,the expression level of target gene in all the treatments were lower than that in the references,the target gene expressions level at 6 h,12 h,24 h and 48 h in treatments were scaled down 24.75%,13.18%,10.23% and 20.34% contrast with that in references,respectively.These results offer the theoretical foundation for cloning full-length sequence of GA20 oxidase gene and researching the gene expression regulation in internode growth and mechanism in stems elongation of sugarcane.

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