应用RNA干扰技术创造低脂肪氧化酶活性大豆新种质

Development of Novel Soybean Germplasms with Low Activity of Lipoxygenases by RNAi Method

【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357bp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-PAGE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。

[ 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 （Lxl, 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 which demonstrated significant differences. SDS-PAGE and activity of lipoxygenase demonstrated that the lipoxygenase activity of transgenic soybeans decreased significantly, the degree of decreasing was 64.2% on average. The near-infrared analyses demonstrated that the fat content of the transgenic soybeans increased by 0.8%-1.5% and 1.2%-3.0% the total protein decreased by 1.2%-3.0% compared with the control group. The results of RT-PCR demonstrated that the mRNA of lipoxygenase in transgenic soybeans was significantly inhibited. In addition, the activity of lipoxygenase decreased significantly in the progenies oftransgenic soybean seeds and the seeds had more fat. [Conclusion] The RNAi technology can effectively inhibit the expression of lipoxygenase gene in soybean seed, decrease the activity of lipoxygenase, increase the amount of oil content in its products, improve the quality of soybeans, and create new superordinary germplasm resources of soybean.