应用RNA干扰技术创造低脂肪氧化酶活性大豆新种质（副本）.pdfVIP

中国农业科学 2009,42(11):3804-3811 Scientia Agricultura Sinica doi: 10.3864/j.issn.0578-1752.2009.11.006 应用RNA干扰技术创造低脂肪氧化酶活性大豆新种质 马 建，张 君，曲 静，王云鹏，魏益凡，王丕武 （吉林农业大学农学院生物技术中心，长春 130118） 摘要：【目的】通过 RNA 干扰技术抑制大豆籽粒中脂肪氧化酶基因表达，改良大豆品质，创造优良大豆新种 质，探索大豆品质育种新途径。【方法】采用RT-PCR技术克隆大豆籽粒脂肪氧化酶基因（ Lx1、Lx2、Lx3）核心保 守序列357 bp片段，通过重组PCR构建大豆脂肪氧化酶基因RNA干扰表达载体，利用花粉管通道法转化大豆品种 吉农 18。【结果】获得了 12 个转基因株系，其中 10 个株系发生明显变化，SDS电泳和脂肪氧化酶活性测定 表明转基因植株籽粒中脂肪氧化酶活性明显降低，平均减低 64.2%；经近红外谷物分析仪测定，转基因植株脂肪 含量平均提高0.8%～1.5%，总蛋白含量降低1.2%～3.0%；RT-PCR结果表明转基因株系中脂肪氧化酶mRNA积累受 到明显抑制。转基因植株 2 代及 3 代检测结果表明，转基因植株脂肪氧化酶活性均明显降低，脂肪含量均有所提 高。【结论】应用RNA干扰技术可有效的抑制大豆籽粒中脂肪氧化酶基因表达，降低脂肪氧化酶活性，提高大豆油 份含量，改良大豆品质，创造优良大豆种质。 关键词：大豆；种质资源；脂肪氧化酶；RNA干扰；重组 PCR Development of Novel Soybean Germplasms with Low Activity of Lipoxygenases by RNAi Method MA Jian, ZHANG Jun, QU Jing, WANG Yun-peng, WEI Yi-fan, WANG Pi-wu (The Biology Center of Agronomy College, Jilin Agricultural University, Changchun 130118) Abstract: 【Objective 】 The objective of this study was try to find a new avenue of breeding technique in soybean to improve the quality of soybeans and create new superordinary germplasm resource of soybeans by RNAi, which prohibits the gene expression of lipoxygenase in soybean seed. 【Method 】 The consensus sequence of lipoxygenase gene (Lx 1, Lx2, Lx3) in soybean seeds was cloned by RT-PCR, the length of the sequence was 357 bp. An RNAi expression vector of soybean lipoxygenase gene was reconstructed by recombinant PCR, and transformed the vector in soybean (Jinong 18) by pollen tube pathway. 【Result 】 Twelve transgenic strains were obtained and 10 of