论文部分内容阅读
【目的】农作物对逆境胁迫的耐受能力与产量息息相关,是作物育种要考虑的重要因素。文中对水稻顺式还原酮加双氧酶基因Os ARD1进行研究,分析其表达模式,明确其在水稻应对非生物胁迫中的功能,为水稻耐旱品种的分子设计及育种提供参考依据。【方法】提取不同组织器官的总RNA,利用RT-PCR方法分析Os ARD1表达的组织特异性。利用不同的非生物胁迫处理14 d大小的野生型(中花11)植株,在不同时间点提取总RNA,利用RT-PCR方法分析Os ARD1表达的受诱导情况。通过农杆菌遗传转化法转化水稻愈伤组织,经过一系列分子检测后获得稳定遗传的T1代Os ARD1的过量表达转基因植株,以转入空载体的野生型植株作为对照。将在营养液中正常培养的12 d大小的野生型和过表达幼苗移出营养液进行缺水处理并进行恢复试验。将催芽后的野生型和过表达转基因植株种子种在含有5%PEG6000的agar培养基中进行渗透胁迫处理,以不含PEG6000的agar培养基作为对照,观察二者的表型。【结果】组织特异性表达分析表明Os ARD1主要在根及成熟的组织中表达,尤其在衰老的组织中有较高表达。非生物胁迫处理表明Os ARD1的表达明显受机械损伤、高盐和渗透胁迫的诱导。获得6个独立株系的可稳定遗传的Os ARD1过量表达转基因植株。对过量表达转基因植株及空载体野生型对照进行干旱胁迫处理,缺水处理5 h后,野生型植株叶片卷曲皱缩成针状表现出严重的缺水症状,但此时过表达转基因植株叶片仍处于舒展状态;缺水处理8 h后开始复水培养3 d,野生型植株的存活率仅为10%,而过表达植株存活率为80%,远远高于野生型,说明过量表达Os ARD1提高了水稻对缺水的耐受能力。用PEG渗透胁迫模拟干旱胁迫处理6 d后发现,不含PEG6000对照组中野生型和过表达植株的幼苗生长情况没有明显的差别;在PEG处理组中,野生型幼苗根的生长受到严重抑制,而过表达植株幼苗根的生长受到抑制较小,根长明显长于野生型对照植株,说明过量表达Os ARD1增强了水稻耐受干旱胁迫的能力。【结论】Os ARD1主要在水稻根及成熟的组织中表达,并且受机械损伤、高盐和渗透胁迫的诱导。过量表达Os ARD1提高了水稻抗旱性能。
【Objective】 The crop tolerance to stress is closely related to the yield, which is an important factor to be considered in crop breeding. In this paper, the gene of cis-reductone and dioxygenase Os ARD1 in rice was studied, its expression pattern was analyzed, and its function in coping with abiotic stresses in rice was clarified. This provided a reference for the molecular design and breeding of drought-tolerant rice varieties. 【Method】 Total RNA was extracted from different tissues and organs. The tissue specificity of Os ARD1 expression was analyzed by RT-PCR. The wild-type (Zhonghua 11) plants of 14 days were treated with different abiotic stress, total RNA was extracted at different time points, and the induction of Os ARD1 expression was analyzed by RT-PCR. The rice calli were transformed by Agrobacterium tumefaciens transformation method. After a series of molecular tests, the overexpression transgenic plants of T1 generation Os ARD1 with stable inheritance were obtained, and wild type plants transformed into empty vector were used as control. Wild-type and overexpressed seedlings of 12-day-old, which were normally cultured in nutrient solution, were removed from the nutrient solution and subjected to a water deficit treatment and recovery test. The germination of the wild-type and overexpression transgenic plant seedlings in osmotic stress agar medium containing 5% PEG6000 PEG6000 agar medium as a control to observe the phenotype. 【Results】 Tissue-specific expression analysis showed that Os ARD1 was mainly expressed in root and mature tissues, especially in aged tissues. Abiotic stress treatment indicated that Os ARD1 expression was significantly induced by mechanical injury, high salt and osmotic stress. OsRD1 overexpression transgenic plants of 6 independent lines were obtained. Wild-type control plants overexpressing and wild-type control were treated with drought stress. After 5 h of water deficit, the leaves of wild-type plants crimped and contracted acicular to show severe water-deficit symptoms. However, overexpression of transgenic leaves In a state of being stretched. When the water-deficit treatment was started for 8 days, rehydration culture was started for 3 days. The survival rate of wild type plants was only 10%, while that of overexpression plants was 80%, which was much higher than that of wild type plants, indicating that overexpression of Os ARD1 Increased water tolerance of rice. The results showed that there was no significant difference in seedling growth between wild-type and overexpressed plants without PEG6000. In PEG-treated group, the growth of wild-type seedlings was severely inhibited, However, the root growth of over-expressed plants was less inhibited and the root length was significantly longer than that of wild-type control plants, indicating that overexpression of Os ARD1 enhanced the ability of rice to tolerate drought stress. 【Conclusion】 Os ARD1 is mainly expressed in rice roots and mature tissues, and is induced by mechanical injury, high salt and osmotic stress. Overexpression of Os ARD1 enhanced rice drought resistance.