【作者】 张婷；

【导师】 杨代常；

【作者基本信息】 武汉大学 ， 遗传学， 2013， 博士

【摘要】 野生稻为改良栽培稻重要农艺性状提供了宝贵遗传资源。长雄野生稻具有和亚洲栽培稻相同的AA基因组,其地下茎无性繁殖特性是多年生性的理想供体。同时,长雄野生稻对低温等非生物胁迫的抗性也为采用遗传工程手段培育抗逆水稻新品种提供了基因资源。本研究论文以具地下茎的长雄野生稻和拟高粱、耐冷及冷敏感水稻品种丽江新团黑谷和IR29为材料,采用基因芯片及转录组测序等技术平台,对地下茎发育及耐冷性进行系统的功能基因组学和比较转录组学分析,发掘地下茎发生发育相关功能候选基因及解析水稻耐冷胁迫调控的分子遗传机制,为进一步克隆地下茎发育及耐冷基因打下基础。具体研究结果如下：1)构建了长雄野生稻地下茎茎尖均一化cDNA文库,并随机挑选10,283个克隆进行测序分析,获得原始ESTs序列10,136条,经高质量ESTs拼接得到4,419条非重复序列。在≥80%序列一致性情况下,分别有4,285(96.97%)和4,151(93.94%)条非重复序列定位到日本晴和9311基因组上。41条非重复序列表现为特异的可变剪接形式,516条非重复序列中共检测到666个简单序列重复(SSR).地下茎茎尖中表达的178条非重复序列共定位到10个地下茎相关QTLs区间。此外,还对OLRR1在五个组织中进行实时荧光定量PCR分析,组织原位杂交进一步验证其在地下茎茎尖的顶端分生组织中高表达。2)采用异源芯片杂交策略,利用Agilent水稻寡核苷酸长探针芯片对拟高粱五个组织进行全基因组表达谱比较分析,总共检测到548个组织高水平表达基因,其中有31和114个基因在地下茎茎尖和节间中高水平表达。在地下茎茎尖特异高表达基因中发现三个顺式调控元件可能在地下茎发生发育过程中起重要作用,即ABA响应的RY重复序列CATGCA.蔗糖抑制蛋白相关元件TTATCC和GA响应元件TAACAA.对比分析前人报道的长雄野生稻和拟高粱地下茎特异表达基因,发现包括脱落酸、生长素、赤霉素和水杨酸等植物激素在地下茎发生发育中起重要调控作用。3)鉴于异源芯片表达谱分析的局限性,本研究采用新一代RNA测序技术对拟高粱地下茎和地上茎表达谱进行比较分析。结果显示拟高粱基因组中超过70%的基因在地上茎和地下茎中检测到表达,同时发现1,963和599个基因在地上茎和地下茎特异或高表达,其中分别包括122和55个转录因子,功能聚类分析表明它们在地上或地下茎组织生长发育过程中起重要作用。进一步分析发现ACGT box、GCCAC、GATC和TGACG box等顺式调控元件在地上茎特异表达基因中显著富集；而MYB和ROOTMOTIFTAPOX1顺式元件、10-promoter element TATTCT及响应细胞分裂素元件TATTAG在地下茎特异表达基因中显著富集,表明组织特异及复杂的分子调控网络参与调控地上茎和地下茎的生长发育。此外,27.9%的拟高粱基因检测到可变剪接,其中60%的可变剪接具有组织特异性,表明可变剪接可能在组织特异细胞功能决定中起重要作用。对本研究中鉴定的地下茎高水平表达基因和已报道的地下茎高水平表达基因进行比较分析,共鉴定了111个基因在至少两个不同平台中的地下茎高水平表达。这些在至少两个不同平台中尤其是在三个不同平台中都是地下茎高水平表达的基因,不仅验证了其表达水平的可靠性,还表明这些基因在拟高粱和长雄野生稻中的功能可能是保守的,为地下茎发生发育相关基因克隆奠定基础。4)采用Affymetrix水稻全基因组芯片对水稻耐冷品种丽江新团黑谷(LTH)和冷敏感品种IR29进行冷胁迫转录组比较分析。结果表明,部分胁迫反应及信号传导相关基因的组成性高表达与LTH耐冷性相关：同时LTH和IR29在连续时间冷胁迫条件下,基因表达水平呈现基本相同的早期反应和品种特异的后期反应,早期反应主要表现为转录因子和信号传导相关基因上调表达；而在冷胁迫处理后期,不同品种由于对持续冷胁迫的功能性适应而表现为不同的差异表达,包括ROS相关基因在LTH特异高表达,而在IR29中抑制表达。在冷胁迫终止后的恢复过程中,LTH冷胁迫差异表达基因大部分都迅速而有效地回复到正常水平,而IR29冷胁迫差异表达基因则恢复缓慢或不能恢复。进一步分析表明包括CBF和MYBS3调控元在内的许多调控途径参与冷胁迫反应。5)采用RNA-seq分析长雄野生稻地上茎及地下茎冷胁迫全基因组表达谱。总测序数据量25gigabases (Gb),约覆盖长野基因组58倍。约10%的转录本不能定位于已测序水稻基因组或基因区域,暗示长雄野生稻与水稻基因组序列间存在差异。冷胁迫共导致913和884个基因在地上茎和地下茎中差异表达,包括共同上调表达的33个转录因子。在两组织中发现大量冷胁迫特异可变剪接事件,且这些可变剪接基因广泛参与信号转导、生物学调控、定位和细胞组分生物合成途径,表明可变剪接在冷胁迫信号转导与基因调控网络中起重要作用。此外,在地上茎和地下茎冷处理及对照中共鉴定8,005个新转录本和3,916个融合基因。进一步对长雄野生稻、LTH和IR29冷胁迫全基因组表达谱进行比较分析,鉴定了154个三个基因型共同冷胁迫诱导上调基因,其中包括34个转录因子,功能注释揭示这些转录因子在栽培稻和野生稻冷胁迫应答中的功能是保守的。另外56个基因(占总数36.4%)的启动子区域至少含有一个CRT/DRE核心元件A/GCCGAC,表明这些COR基因在栽培稻和野生稻中共同参与冷胁迫分子调控网络。更多还原

【Abstract】 Wild rice relatives have valuable sources of genetic variation for the improvement of agronomical traits in cultivated rice. Oryza longistaminata, a perennial wild rice species with the same AA genome as O. sativa, provides a model system for genetic and molecular dissection of the rhizomatous trait in grasses. Meanwhile, O. longistaminata possesses many important adaptive traits for cultivation such as tolerance to cold and can contribute to the development of more sustainable grain, forage, and bioenergy cropping systems.In present study, functional genomic and comparative transcriptomic techniques including microarray and RNA-seq were used to discover candidate genes and putative pathways specifically responsible for rhizome development in O. longistaminata and Sorghum propinquum, as well as for chilling tolerance among O. longistaminata, LTH and IR29. All those functional candidate genes identified in this study provided a basis for future cloning of genes associated with rhizome development and chilling stress tolerance in rice. The main results are as following:1) We sequenced10.283cDNA clones from a normalized cDNA library constructed from rhizome tip tissues of O. longistaminata and generated4,419unisequences with diverse functional categories. These unisequences were mapped onto the O. sativa genome, which revealed that4,285(96.97%) and4,151(93.94%) of the unisequences were alignable to the japonica and indica genomic sequences, respectively, with≥80%sequence identity. Additionally,41unisequences showed four typical types of alternative splicing patterns. More than600simple sequence repeats were identified. A subset of unisequences were physically co-localized onto rhizome-related QTLs intervals in rice and sorghum; and one gene, OLRR1, was further confirmed to be highly enriched in the rhizome tip and young leaf by quantitative PCR and in situ hybridization. 2) We used a whole rice genome oligonucleotide microarray to obtain tissue-specific gene expression profiles of S. propinquum. A total of548tissue-enriched genes were detected, including31and114unique genes that were expressed predominantly in the rhizome tips (RT) and internodes (RI), respectively. Further GO analysis indicated that the functions of these tissue-enriched genes corresponded to their characteristic biological processes. A few distinct cis-elements, including ABA-responsive RY repeat CATGCA, sugar-repressive TTATCC, and GA-responsive TAACAA, were found to be prevalent in RT-enriched genes, implying an important role in rhizome growth and development. Comprehensive comparative analysis of these rhizome-enriched genes and rhizome-specific genes previously identified in O. longistaminata and S. propinquum indicated that phytohormones, including ABA, GA, and SA, are key regulators of gene expression during rhizome development.3) We sequenced the RNAs of S. propinquum shoot and rhizome using an Illumina platform. More than70%of the genes in the S. propinquum genome were expressed in shoot and rhizome. The expression patterns of1963and599genes, including transcription factors, were found to be specific or enriched in shoot and rhizome respectively, indicating their possible roles in physiological processes in these organs. Comparative analysis revealed several cis-elements, ACGT box, GCCAC, GATC and TGACG box, which showed significantly higher abundance in shoot-specific genes. Cis-elements including MYB and ROOTMOTIFTAPOX1motifs, and10promoters and cytokinin-responsive elements were highly enriched in rhizome-specific genes. Of the S. propinquum genes,27.9%were identified as alternatively spliced and about60%of the alternative splicing events were organ-specific, suggesting that alternative splicing played a crucial role in determining organ-specific cellular function.Comprehensive comparative analysis of rhizome-enriched genes mentioned above and previously identified ones in O. longistaminata and S. propinquum resulted in a number of111genes were found to be commonly enriched in rhizome in at least two different platforms, which mutually confirmed the rhizome-enriched genes as well as their evolutionarily conserved expression pattern in both rice and sorghum.4) The temporal gene expression patterns of two rice genotypes (chilling-tolerant LTH and chilling-sensitive IR29) in response to chilling stress were compared. A set of genes with higher basal expression were identified in chilling-tolerant LTH compared with chilling-sensitive IR29, indicating their possible role in intrinsic tolerance to chilling stress. Under chilling stress, the major effect on gene expression was up-regulated in the chilling-tolerant genotype and strongly repressed in chilling-sensitive genotype. Early responses to chilling stress in both genotypes featured commonly upregulated genes related to transcription regulation and signal transduction, while functional categories for late phase chilling regulated genes were diverse with a wide range of functional adaptations to continuous stress. Following the cessation of chilling treatments, there was quick and efficient reversion of gene expression in the chilling-tolerant genotype, while the chilling-sensitive genotype displayed considerably slower recovering capacity at the transcriptional level. In addition, the detection of differentially regulated TF genes and enriched cis-elements demonstrated that multiple regulatory pathways, including CBF and MYBS3regulons, were involved in chilling stress tolerance.5) RNA sequencing was performed to profiling the gene expression level of rhizome and shoot of O. longistaminata under long term chilling stress. The total length of the reads was over25gigabases (Gb), representing about58-fold coverage of the O. longistaminata genome. However, about10%of total reads remained unmapped, indicating the existence of gaps and diversity between the genome sequences of O. sativa and O. longistaminata. There were913and884genes differentially regulated in shoot and rhizome under chilling stress, respectively, including33up regulated transcription factors in both tissues. Further analysis found a set of chilling specific alternative splicing (AS) transcripts, which fell into very broad functional categories such as signal transduction, regulation of biological process, localization and cellular component organization, indicating AS events could play an important role in chilling stress tolerance. Besides, we identified8,005novel transcripts and3,916fusion genes in shoot and rhizome during chilling stress and control conditions. And56chilling induced genes in O. longisataminata were colocalized onto the chilling tolerant QTLs intervals in rice.Comparative analysis of transcriptomic data in O. longistaminata, LTH and IR29under chilling stress, we identified154chilling induced genes sharing in three genotypes. Among them,34are transcription factors, indicating their conserved function between cultivaty and wild rice in response to chilling stress. Besides, the promoters of56(36.4%) of these commonly chilling induced genes contained at least one CRT/DRE core motif A/GCCGAC. These chilling responsive genes in three genotypes may play an important role in responding to chilling stress and their function need further elucidated.更多还原