水稻顺式还原酮加双氧酶基因的表达及功能分析

发布时间：2018-05-27 09:12

本文选题：水稻 + Os　；参考：《中国农业科学》2017年12期

【摘要】：【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。
[Abstract]:Objective: the tolerance of crops to stress is closely related to yield and is an important factor to be considered in crop breeding. In this paper, the gene Os ARD1 of cis-reducing ketone and dioxygenase in rice was studied, its expression pattern was analyzed, and the function of Os ARD1 in response to abiotic stress in rice was clarified. [methods] Total RNAs of different tissues and organs were extracted, and tissue specificity of Os ARD1 expression was analyzed by RT-PCR method. Wild type (Zhonghua 11) plants were treated with different abiotic stress for 14 days and total RNAs were extracted at different time points. The induction of Os ARD1 expression was analyzed by RT-PCR method. Transgenic rice callus was transformed by Agrobacterium tumefaciens genetic transformation. After a series of molecular tests, transgenic plants of stable T1 generation Os ARD1 were obtained, and wild type plants with empty vector were used as control. The wild type and over-expressed seedlings which were cultured in nutrient solution for 12 days were removed from the nutrient solution for water deficiency treatment and the recovery test was carried out. The seeds of wild type and overexpression transgenic plants were treated with osmotic stress in agar medium containing 5%PEG6000, and the agar medium without PEG6000 was used as control. [results] tissue specific expression analysis showed that Os ARD1 was mainly expressed in roots and mature tissues, especially in aging tissues. Under abiotic stress, the expression of Os ARD1 was induced by mechanical damage, high salt and osmotic stress. Stable and hereditary Os ARD1 transgenic plants of 6 independent lines were obtained. Transgenic plants and wild type control were treated with drought stress. After 5 hours of water stress treatment, leaf curls of wild type plants showed severe dehydration symptoms. The survival rate of wild type plants was only 10, while that of over-expressed plants was 80%, which was much higher than that of wild type plants. The results showed that the over-expression of Os ARD1 increased the tolerance of rice to water shortage. After simulated drought stress with PEG osmotic stress for 6 days, it was found that there was no significant difference between the growth of wild type seedlings and over-expressed plants in the control group without PEG6000, while in the PEG treatment group, the growth of wild type seedling roots was seriously inhibited. However, the growth of overexpressed seedlings was inhibited slightly, and the root length was significantly longer than that of wild type control plants. [conclusion] Os ARD1 was mainly expressed in roots and mature tissues of rice, and was induced by mechanical damage, high salt and osmotic stress. Overexpression of Os ARD1 improved drought resistance of rice.
【作者单位】：天津师范大学生命科学学院/天津市动植物抗性重点实验室;
【基金】：天津市自然科学基金重点项目(16JCZDJC33400) 天津市中青年骨干教师创新培养计划(ZX110GG017) 天津师范大学博士基金(52XB1612,52XB1611)
【分类号】：S511

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