胚乳中不表达基因组片段的分离及磷代谢的品种差异分析

【作者】 魏君；

【导师】 张启发；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2003， 硕士

【摘要】 对水稻进行品质改良，转基因技术是目前被广泛使用的一种行之有效的方法。在外源基因上连上水稻自身的启动子不仅能够实现外源基因在受体体内的定向表达，还从某种程度上缓解了外源基因在受体体内表达沉默的问题。在本实验中利用本室构建的平衡化明恢63全生育期cDNA文库点阵膜来进行在胚乳中特异性不表达的cDNA克隆的筛选，分别用来自水稻不同器官中的RNA反转录制成放射性cDNA探针与cDNA点阵杂交，通过比较不同探针与点阵杂交所得到的杂交图像，得到了一批在胚乳中特异性不表达的克隆，对它们进行Northern检测和序列分析，从中挑选出编码水稻光系统Ⅱ10KDa蛋白前体的cDNA克隆EI#44P17来制备探针筛选58N BAC文库，对得到的阳性BAC119H12进行进一步的分析，从中分离得到长度为6.2 kb与EI#44P17同源的基因组片段，对其进行Shotgun测序，根据测序结果利用GENESCAN和GeneFinder对启动子分别进行了预测，两者的预测结果一致，都包含了编码该前体蛋白的完整ORF。 本实验的另一部分是通过cDNA芯片对两种低磷情况下的敏感材料(中早18和Lagrue)进行低磷胁迫后的表达分析，找到二者间的表达差异。从三叶期开始进行低磷胁迫处理，分别于胁迫后不同时间段不同器官进行取样并抽提RNA。来自不同材料和不同器官，取于不同时间的RNA分别与对应的非胁迫处理材料作为一组探针标记上不同的荧光标记来进行芯片杂交，芯片上包括明恢63全生育期cDNA文库和5种消减杂交cDNA文库，共计5760个unique-gene。“黄点实验”分析决定差异表达的极限值分别为1.78和0.55。来自不同材料，不同时期和部位的探针经过芯片杂交后得到的差异表达的克隆数各不相同。部分由芯片杂交得到的差异克隆经过Northern杂交得到了进一步的验证。对这些克隆进行序列分析发现它们编码了各种功能的酶和蛋白，涉及到植物生理生化的各个方面，其中部分已经被报道与低磷胁迫有关。对低磷胁迫消减杂交文库的序列分析也得到了相似的结果。根据Northern杂交和序列分析的结果，可以找到两种材料之间的表达差异：首先，中早18对低磷反应较Lagrue激烈；其次，中早18具有在一定时间内持续产生有机酸的机制，而在Lagrue中没有检测到；最后，低磷胁迫能够造成氮素代谢不正常，Lagrue受低磷胁迫的影响要远大于中早18，Lagrue中许多参与氮代谢的酶的表达大大降低。这些基因表达的差异很可能是造成中早18和Lagrue在低磷条件下生长发育状况，结实率以及干物质积累等重要生理参数不同的原因。更多还原

【Abstract】 Transgenic technology is wildly used in rice improvement. The foreign gene can express directional by linkage of foreign gene and rice promoter, and it could reduce the probability of foreign gene silence. The objective of this study was to identify genes that no expression in rice endosperm by cDNA array analysis. The cDNA array was made based on Minghui 63 normalized cDNA library. RNA samples isolated from different rice organs were used to synthesize cDNA as probes for screening the cDNA arrays. By analysis the expression result, several clones were identified that no expression in rice endosperm. After Northern assay and sequence analysis, cDNA clone EI#44P17 encoding photosystem II 10KDa protein precursor was used to screen 58N BAG library. The 6.2 kb fragment was found to be homolog of EI#44P17 in positive BAG clone 119H12 with further analysis. We have sequenced the whole BAC119H12 with Shotgun Method to find the candidate promoter. Sequence analysis with GENESCAN shows that it has the ORF of photosystem II lOKDa protein precursor, and the result of GeneFinder shows the same result.The object of this study is to identify the genes that are related to uptake or assimilation of phosphorus by analysis expression profiles in two rice cultivars. The two kinds of rice cultivars that have different sensitive in low-phosphorus were treated in two different solutions from 3-leaf-stage, one containing normal concentrations of phosphorous and the other containing one thirtieth of normal phosphorus respectively. mRNA was extracted from the shoots and roots at 1hr, 6hr, 12hr, 1day and 7days were used to synthesize probes for cDNA microarray hybridization. The fluorescently labeled probes were hybridized with the microarray of 5760 unique genes from the Minghui 63 normalized cDNA library and five subtractive cDNA libraries. The mRNA of the stressed and the control were reverse-transcribed and labeled with Cy3 and Cy5 respectively. The ratio of the thresholds were determined with statistical analysis the yellow-dots experiment, they are 1.78 and 0.55. Gene expression in response to low-phosphorus stressed wasinvestigated with microarray containing 5760 unique cDNA sequences. The number of this kind of genes is different in different cultivars, different organs and different period of time after low-phosphorus stresses. The significantly increased or decreased expressions were found and Northern-blot of part of clones showed the same results. Sequence analysis revealed the differentially expressed sequences encoded all kinds of proteins and enzymes that relate to all fields of plant physiology and biochemistry. Some of the genes identified in this study have been reported to be related to Pi-deprivation. It is easy to find the difference in gene expression after low-phosphorus stresses between the two rice cultivars: first, Zhongzao18 responses more drastically than Lagrue; second, Zhongzao18 has the ability to product organic acid continually in one period while Lagrue has not; last, Pi-deprivation can deduce abnormal in nitrogen metabolism, this abnormal in Lagrue is stronger than in Zhongzao18. This difference may lead that Zhongzao18 gowns better than Lagrue when Pi-deprivation in soil.更多还原