| 构建番茄红素β/ε环化酶基因RNAi植物表达载体及其表达分析 |
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| 作者单位： | 石河子大学农学院园艺系，新疆 石河子 832003 |
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| 基金项目： | 国家自然科学基金项目 (30460081)，新疆兵团博士资金项目 (ZD2007JC06) |
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| 摘 要： | 根据GenBank中Lyc-β基因 (X86452) 及Lyc-ε基因 (Y14387) 设计引物，通过高保真PCR从番茄cDNA中克隆到两段长度为302 和330bp的Lyc-β基因片段和两段长度为302 和288bp的Lyc-ε基因片段。从质粒pCAMBIA-2301 (AF234316) 克隆出gusA基因长度为168 和235bp的2个内含子片段。根据RNAi原理将正、反向序列与内含子连接，之后插入启动子与终止子之间构建成4组植物表达载体。利用农杆菌介导的叶盘法转化烟草植株，通过PCR鉴定共获得107株转基因植株。利用荧光定量PCR分析干扰效果，结果显示经4组干扰载体干扰后，转基因植株中目的基因mRNA含量分别为对照的3.4%、16.4%、15.2%和21.8%。进一步用HPLC对应分析转化株的番茄红素含量，结果表明转基因植株中番茄红素平均增长量分别为3.4、2.1、3.4和2.0μg/g。同时测定转基因植株中的β-胡萝卜素与叶黄素含量，发现它们根据干扰的基因不同呈现对应的变化。
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| 关 键 词： | 番茄红素 β-胡萝卜素 叶黄素 番茄红素β/ε环化酶 RNAi |
CONSTRUCTION OF RNAI PLANT EXPRESSION VECTORS FOR INTERFERING LYCOPENE CYCLASE-β/ε GENES AND ITS EXPRESSION ANALYSIS |
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| Institution: | Agricultural College, Shihezi University, Shihezi, xinjiang 832003 |
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| Abstract: | According to the Lyc-β gene (X86452) and Lyc-ε gene (Y14387) sequences published in the GenBank, four pairs of primers were designed to amplify the fragments of 302 and 330 bp (Lyc-β gene),302 and 288 bp (Lyc-ε gene) from tomato cDNA. The gusA gene intron sequences of 168 and 235 bp were cloned from the plasmid pCAMBIA-2301 (AF234316). Four plant expression vectors interfering Lyc-β and Lyc-ε genes were constructed by ligation the intron fragments of gusA gene within two target gene fragments, which were designed in opposite direction. The transformed tobacco plants were identified by PCR, and a total of 107 transgenic plants were obtained. The interference effects were analyzed by real time PCR, and the results showed that the target genic relative abundance in the transgenic plants were 3.4%, 16.4%, 15.2% and 21.8% respectively compared with wild type plants. Further analysis of the pigment content in transgene plants showed that the average accumulating rate of lycopene content in transgenic plants were 3.4, 2.1, 3.4, 2.0μg/g, respectively. The β-carotene and lutein contents in transgenic plants had the corresponding change. |
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| Keywords: | Lycopene &beta -carotene lutein lycopene &beta /&epsilon -cyclase RNAi |
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