【作者】 许振龙；

【导师】 肖凯；

【作者基本信息】 河北农业大学 ， 作物栽培学与耕作学， 2010， 硕士

【摘要】 RLP是一类类受体蛋白,在介导植物衰老信号和逆境信号转导中具有重要作用。迄今,有关水稻RLP的研究尚缺乏系统报道。鉴于此,本项研究通过对水稻基因组搜索,获得了41个水稻RLP基因。通过现代分子生物学和生物信息学技术,研究了上述水稻RLP基因的结构特征、部分基因在正常生长的自然衰老、6-BA和ABA处理下的表达特性,以及启动子及基因功能。通过保守序列克隆了棉花中的RLP同源基因。主要研究结果如下:1、水稻RLP基因在基因结构特征上存在明显差异。如不同成员之间在基因的DNA序列长度、cDNA的全长序列、开放阅读框(Open reading frame, ORF)和翻译的氨基酸序列等变幅较大。聚类分析发现水稻RLP基因在起源和进化上具有较高一致性,但个别基因在进化中变异较大。2、在正常生长的自然衰老及6-BA和ABA处理下,表达沉默的基因为OsRLP34;OsRLP06的表达受到自然衰老及6-BA和ABA的诱导,呈现明显的上调特异表达特征;OsRLP03、OsRLP37、OsRLP58、OsRLP59和OsRLP65在水稻叶片自然衰老的过程中均呈明显的上调表达特征,在6-BA处理下随处理进程上述基因表达呈单峰曲线特征,以处理10 d表达水平最高,ABA处理使上述基因(OsRLP03、OsRLP37、OsRLP58、OsRLP59和OsRLP65)表达明显增强。但OsRLP03和OsRLP37在10d后呈下调表达趋势,与其在水稻叶片6-BA处理下的表达特征相同。3、对融合OsRLP65启动子驱动报告基因表达的转基因烟草研究发现,OsRLP65启动子能驱动报告基因高水平表达。在该基因的启动子中,共鉴定出34个顺式调控元件,其中多与逆境胁迫有关。4、对OsRLP65启动子不同缺失长度片段的转基因烟草植株研究发现,全长及不同缺失片段长度启动子的转基因植株均能进行Gus组织化学染色,且染色程度随着启动子片段长度的增加而加深。启动子片段长度最短的D1(246 bp),驱动报告基因的表达能力很弱,D5(1427bp)驱动Gus的表达特征与OsRLP65全长启动子相似。5、在自然生长条件下,遗传转化反义GhRLP6基因和OsRLP65基因的转基因植株,与对照相比抗衰老能力明显改善。表现为转基因系植株叶片的丙二醛(MDA)含量降低;叶绿素a、b含量、类胡萝卜素含量和可溶蛋白含量明显增加。表明植株细胞保护代谢途径响应了RLP介导的衰老逆境信号过程。反义GhRLP6基因和OsRLP65基因在增强植株抵御衰老胁迫的能力中具有较重要的作用。更多还原

【Abstract】 Receptor-like proteins (RLPs) belong to one kind of receptor-like protein in plants. RLPs play an important role in mediating the signal transductions of senescence and the abiotic stresses. Update, it is still in lack of a thoroughly elucidation for the RLPs in rice. In this study, in total 41 RLP genes of rice were identified by searching the rice genome sequences which were released in the GenBank. Based on the approaches of modern molecular biology and bioinformatics, the gene structures of the rice RLP genes, the expression patterns of the rice RLP genes under normal growth (CK) and treatment of 6-BA, and ABA has been analyzed. The functions of several tested rice RLP genes were also studied. In the meantime, several RLP genes in cotton which are homologous to the corresponding ones in rice have been cloned. The main results were as follows:1. There were huge differences on the structure properties among the rice RLP genes. The DNA length, cDNA full length, open reading frame length, and the translated amino acid numbers among the rice RLP genes were dramatically diverse. The rice 41 RLP genes have high consistency in the origin and evolution based on phylogenetic tree analysis. However, part genes of them are variable on the gene structure characteristics.2. Under normal growth (CK) and 6-BA, ABA treatment, no transcripts of OsRLP34 gene were detected. The expression of OsRLP06 was specifically up-regulated in leaves under natural aging and 6-BA, ABA treatment. The expressions of OsRLP03, OsRLP37, OsRLP58, OsRLP59 and OsRLP65 were specifically up-regulated in leaves with the leaf natural aging progression. Meanwhile, the time-course changes of the transcripts of OsRLP03, OsRLP37, OsRLP58, OsRLP59 and OsRLP65 were shown to be a curve with a one peak, at the time point of 10 d after 6-BA treatment. OsRLP03, OsRLP37, OsRLP58, OsRLP59 and OsRLP65 were specifically up-regulated in leaves under ABA treatment, but OsRLP03 and OsRLP37 were specifically down-regulated in leaves after 10d treatment. The expressions of OsRLP03, OsRLP37, OsRLP58, OsRLP59 and OsRLP65 under 6-BA treatment were same as that of ABA treatment.3. It is found that the OsRLP65 promoter could drive the Gus to be transcribed at a high level. Totally 34 cis-regulatory elements were identified in the full promoter. Among them, some of cis-regulatory elements play an important role in mediating the abiotic stresses signallings.4. Based on the transgenic tobacco plants of genetic transformation OsRLP65 promoter, the functions of this promoter on driving the target gene expression, has been evuluated. It is found that the promoter of OsRLP65 and its deleted fragments could drive the Gus to be transcribed, with an increase pattern with the prolong of the promoter segments. D1 (246 bp) promoter could drive the Gus gene to be transcribed at a little level, D5 (1427bp) promoter could drive the Gus gene to be transcribed at high level same as that of the full length of OsRLP65 promoter.5. Under natural growth condition, the senescence tolerance in the transgenic lines with high expression of antisense GhRLP6 and OsRLP65 was obviously improved compared with the control (CK). Meanwhile, the malonaldehyde (MDA) contents were lower in the transgenic lines, and the contents of chlorophyll a, chlorophyll b, carotenoid, and soluble protein were all higher. Taken together, the cellular protection metabolic pathway was response to the senescence signal transduction mediated by antisense GhRLP6 and OsRLP65.更多还原