摘要
水稻是全球一半以上人口的主要粮食作物，农民种植水稻依赖于土地。土地盐碱化已经成为影响农作物生长和产量的最主要因素之一。土地碱化与盐化往往相伴发生，碱胁迫使作物不仅受到盐的胁迫作用，还要受到高pH值的胁迫作用，对作物的生长发育的影响更大，但是至今植物耐碱机制仍不明确。
培育耐盐耐碱作物品种无疑是最有效的盐碱地治理措施之一。通过回交导入方法可以改良现有品种不良性状，通常情况下比常规有性杂交稳定，年限时间短且效果较明显。基因表达芯片（Gene Expression Microarray）可以高通量的分析基因mRNA的表达量，发现重要基因，了解代谢途径，解读代谢机制。利用基因表达芯片进行功能基因组学研究已经成为目前最为活跃的研究热点之一。
本文通过回交导入的方法获得耐碱回交导入系K83，并且通过与其对碱胁迫耐性较差的轮回亲本吉粳88的比较来探讨水稻的抗碱机制。本实验使用基因表达芯片检测两种对碱胁迫耐性不同的材料的基因表达差异，并且以碳酸氢钠（NaHCO_3）和碳酸钠（Na_2CO_3）两种碱性盐处理水稻幼苗，检测两种材料对碱胁迫应答的差异基因。我们发现未处理的两种材料间有1226个基因存在着表达变异，其中572个基因在K83中表达上调，654个基因下调。碱处理后，K83中有503个基因发生了表达改变，其中347个表达上调，156个表达下调；吉粳88中有778个基因发生了表达改变，其中591个表达上调，187个表达下调。胁迫后在两种材料中表达上调的基因中，只有34个基因在未处理K83中的表达高于吉粳88，这说明不仅材料之间组成型基因表达差异在耐碱性状上起作用，其受胁迫诱导的基因同时也对植物的耐碱性起决定作用。对筛选出来的差异表达基因进行生物信息学分析，包括代谢，催化活性，转运和转录因子活性等通路与水稻耐碱胁迫相关。应用实时定量PCR方法对其中一部分基因的表达水平进一步分析，所得结果与基因芯片结果基本吻合，证明芯片分析数据可靠，为下一步耐碱相关基因的克隆、功能分析等研究提供了基础。
对苗期水稻进行非生物胁迫和外源激素处理，分析了基于芯片数据筛选出的差异表达候选基因的表达模式。结果显示，两个候选基因（Os01g0871600和Os12g0282000）受到非生物胁迫影响和激素诱导，此结果暗示候选基因可能在植物耐碱胁迫中起到一定作用。对候选基因进行了克隆和过表达载体的构建，并通过农杆菌介导转化到到水稻中，获得了大量的转基因阳性植株。经鉴定转基因植株中候选基因的表达量都有显著提高。苗期抗性实验结果表明，转基因植株抗性表现优于非转基因材料。本研究结果暗示根据芯片分析得到的耐性相关基因可能是潜在耐碱功能基因，可以用于水稻的耐碱性状改良。
Rice is a staple food for more than half of human population and to grow it,farmers largely depend on soil. Land salinization and alkalization is a majorenvironmental factor limiting plant growth and productivity. Alkalization is oftenassociated with salinization, alkaline stress not only included salt stress, but also withthe added influence of high-pH stress, so it affects plant growth badly, but so far onplant alkali resistance mechanism is not clear.
One of the most effective measures to improve land salinization and alkalizationis to develop new salt/alkali-tolerant varieties. The introgression by backcross methodis the most effective method in the development of improved crop varieties，andcompared with traditional hybridization, it has a much shorter time and moreeffectively usually. Gene expression microarray makes it possible to high-throughputanalysis of the mRNA expression, finding key genes, understanding metabolicpathways. Using gene expression microarray to detect the function of genes havebecome very popular.
To investigate the possible genetic basis of alkali tolerance in rice, we generatedan introgressed rice line (K83) with significantly greater alkali stress tolerance thanthe parental cultivar (Jijing88). Seedling treated with sodium bicarbonate (NaHCO_3)and sodium carbonate (Na_2CO_3), then we used microarray analysis to examine theglobal gene expression profiles of K83and Jijing88, and we found that more than1,200genes were constitutively and differentially expressed in K83in comparison toJijing88with572genes up-and654down-regulated. Upon alkali treatment, a total of347genes were found up-and156down-regulated in K83compared to591and187,respectively, in Jijing88. Among the up-regulated genes in both K83and Jijing88,only34were constitutively up-regulated in K83, suggesting that both the constitutivedifferentially expressed genes in K83and those induced by alkali treatment are mostlikely responsible for enhanced alkali tolerance. A gene ontology analysis based on allannotated, differentially expressed genes revealed that genes with expressionalterations were enriched in pathways involved in metabolic processes, catalyticactivity, and transport and transcription factor activities, suggesting that thesepathways are associated with alkali stress tolerance in rice. Further analysis of relativegene expression data using real-time quantitative PCR, real-time quantitative PCRresults basically agrees well with microarray results, it means microarray data arereliable. Our study results might have provided useful clues to gene cloning andfunctional studies of alkali tolerance in rice.
Based on the microarray data, we identified some candidate genes. Theexpression patterns of candidate genes were examined in rice under various abioticstress and hormone treatments. The results showed that two candidate genes (Os01g0871600and Os12g0282000) induced by abiotic stress and hormone, andsuggested these candidate genes may play a role in plant alkali stress. The two geneswere cloned, and their over-expression recombinant plasmids were constructed.Transformation of rice was conducted by agrobacterium tumefaciens infection. Alarge number of positive transgenic plants were obtained, and which showedhigh-expression of the candidate genes. Transgenic plants showed significantly moretolerance than the non-transgenic control based on seedling resistance assays. Theresults of this study suggest that genes identified from microarray analysis may serveas a potential gene pool to identify functional genes that can be used for rice geneticimprovement towards enhanced tolerance to alkalization.
引文
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