水稻顺式还原酮加双氧酶基因的表达分析及功能研究

doi:10.3864/j.issn.0578-1752.2017.12.001

摘要/Abstract

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OsARD1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OsARD1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OsARD1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OsARD1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OsARD1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OsARD1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OsARD1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OsARD1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OsARD1增强了水稻耐受干旱胁迫的能力。【结论】OsARD1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OsARD1提高了水稻抗旱性能。

关键词: 水稻, OsARD1, 过量表达, 干旱胁迫, 乙烯

Abstract: 【Objective】 The tolerance to biotic/abiotic stress is highly associated with grain yield, which is a very important goal in crop breeding. The objective of this study was to analyze the expression pattern of OsARD1, to investigate its function to abiotic stress, and to provide a theoretical basis for rice breeding. 【Method】 To analyze the expression of OsARD1 in different tissues, total RNAs were isolated from different organs. To analyze the expression pattern of OsARD1, RNAs were isolated from wild type seedlings at 14-day-old under different abiotic stress, including PEG, high salinity and mechanical damage. RT-PCR method was used to analyze the expression of OsARD1 in different organs and under abiotic stress. An overexpressing vector of OsARD1 was constructed and OsARD1 overexpression transgenic plants were produced following the Agrobacterium-mediated transformation procedure. Transgenic plants with empty vector were also generated as a control. For drought stress, T1 generation transgenic plants and wild type at 12-day-old were subjected to water deficit treatment and then cultured in nutrition solution for 3 days to recover. For PEG osmotic stress experiment, wild type and OsARD1-overexpressing (OsARD1-OV) seeds were planted in agar medium containing 5% PEG6000 whose osmotic potential was -0.05 MPa. For control group, no PEG was added into agar medium. The phenotypes of wild type and OsARD1-OV plants were observed and recorded. 【Result】The tissue-specific expression analysis showed that OsARD1 expressed highly in root and mature organs, especially in senescent organs. OsARD1 gene transcript levels were strongly induced by PEG6000, high salinity and mechanical damage. Six independent OsARD1-OV lines were generated by Agrobacterium-mediated transformation method. RT-PCR expression analysis showed that the expression level of OsARD1 was significantly increased in OsARD1-OV lines of T0 and T1 generation plant compared with that in wild type, suggesting that overexpression vector worked and inherited stably. The experiments of water deficit of OsARD1-OV of T1 generation and wild type plants were further carried out. The results showed that the wild type plants exhibited severe dehydration symptoms with rolled and withered leaves after five hours of water deficit treatment, while OsARD1-OV plants were growing normally. After 8 hours of water deficit treatment and recovery culture for 3 days, there were only 10% wild type plants survived, while 80% transgenic plants survived. In PEG6000 osmotic stress experiment, it was found that the shoot and root length of wild type and OsARD1-OV plants had no significant difference with that in control group. However, the growth of WT roots was strongly inhibited in 5% PEG6000 treatment group compared with OsARD1-OV lines, and the root length of wide type was shorter than that of OsARD1-OV lines, indicating that the overexpression of OsARD1 improved drought tolerance in rice.【Conclusion】OsARD1 displayed higher expression in roots and mature tissues of rice, and was strongly induced by PEG, high salinity and mechanical damage. Overexpression of OsARD1 significantly improved the tolerance to water deficit and osmotic stress in rice, suggesting that OsARD1 plays a key role in drought tolerance.

Key words: rice, OsARD1, overexpression, drought stress, ethylene

熊炜，杨波，刘薇茵，王荃，孔晓聪，靳亚军，梁闪闪，栾维江，张泗举. 水稻顺式还原酮加双氧酶基因的表达分析及功能研究[J]. 中国农业科学, 2017, 50(12): 2199-2208.

XIONG Wei, YANG Bo, LIU WeiYin, WANG Quan, KONG XiaoCong, JIN YaJun, LIANG ShanShan, LUAN WeiJiang, ZHANG SiJu. Expression and Functional Analysis of Acireductone Dioxygenase Gene in Rice[J]. Scientia Agricultura Sinica, 2017, 50(12): 2199-2208.

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