通过RNAi技术下调OsLOX提高水稻种子耐储性

Improving the storage tolerance of rice seeds by down-regulating OsLOX by RNAi

[目的]本文旨在通过RNAi技术下调OsLOX提高水稻种子耐储性,增强其在老化处理后的种子活力。[方法]利用RNA干扰(RNAi)技术,下调水稻品种‘宁粳1号’脂氧合酶基因(LOX)的表达,对转基因后代进行多年分子鉴定及生理生化特征和农艺性状考察。[结果]获得了稳定含有目的片段且不含潮霉素基因的2个改良家系XO3和XO6,与‘宁粳1号’相比,改良系中OsLOX2和OsLOX3表达量显著降低;经10个月自然放置和7 d人工老化处理,改良系脂氧合酶和丙二醛含量均显著低于受体亲本‘宁粳1号’,其脂氧合酶含量为0.094~0.111 ng·mL-1、丙二醛含量低于4 nmol·mg^-1,而‘宁粳1号’分别为0.115 ng·mL-1和6 nmol·mg^-1;老化后‘宁粳1号’发芽率只有31%,而改良系发芽率在70%以上;TTC染色后还原得到的2,3,5-三苯基四唑氯化物(TTCH)含量和发芽速率也表明改良系的种子活力显著提高;老化后,改良系的种子淀粉含量和胶稠度变化幅度小于‘宁粳1号’。此外,改良系的分蘖数和有效穗数明显增加但结实率降低。[结论]通过RNA干扰技术降低‘宁粳1号’中OsLOX2和OsLOX3基因的表达,提高了‘宁粳1号’的耐储性,为耐储藏水稻分子育种提供材料基础。

[Objectives]The paper aimed to improve the storage tolerance of rice seeds by down-regulating OsLOX by RNAi and enhance its seed vigor after aging treatment. [Methods]The RNA interference(RNAi)technique was used to down-regulate the expression of lipoxygenase gene(LOX)in the rice variety‘Ningjing 1’,and the molecular identification,physiological and biochemical characteristics and agronomic traits of the transgenic offspring had been investigated for several years. [Results]Two stable improved families XO3 and XO6 containing the target fragment and no hygromycin were obtained. Compared to‘Ningjing 1’,the expression levels of OsLOX2 and OsLOX3 significantly reduced in the improved families. After 10 months of natural storage and 7 days of artificial aging,the lipoxygenase and malondialdehyde contents of improved lines were significantly lower than those of the recipient parent‘Ningjing 1’,the lipoxygenase content ranged from 0.094 to 0.111 ng·mL-1,and the malondialdehyde content of improved lines was less than 4 nmol·mg^-1,however,the lipoxygenase and malondialdehyde content in‘Ningjing 1’was 0.115 ng·mL-1 and 6 nmol·mg^-1,respectively. After aging,the germination rate of‘Ningjing 1’was only 31% and the improved lines were still above 70%. The content of 2,3,5-triphenyltetrazoliumchloride(TTCH)after reduction by TTC staining and germination rate also showed a significant increase in seed vigor of the improved lines. The variational range of seed starch content and gel consistency of the improved lines was smaller than that of ‘Ningjing 1’after aging. In addition,the number of tillers and effective panicles of the improved lines increased significantly but the seed setting rate decreased. [Conclusions]The expression of OsLOX2 and OsLOX3 reduced in the improved lines and the storage stability of ‘Ningjing 1’was improved by RNA interference technology. This research provides material basis for molecular breeding of storage-resistant rice.