【作者】 孟凡超；

【导师】 王文泉；

【作者基本信息】 海南大学 ， 作物遗传育种， 2012， 硕士

【摘要】 木薯是大戟科木薯属,是世界三大薯类作物之一,全球六大粮食作物之一。在我国木薯块根90%用于制取淀粉,已经成为热区重要的能源作物之一。我国木薯种植区属于热带北缘,耐低温是重要的育种目标之一。目前关于植物低温胁迫的研究已有很多,而关于木薯抗寒机理的研究十分有限,采用表达谱测序技术对木薯抗寒性的研究尚未见报道。本研究选择较耐寒的木薯品种,通过高通量表达谱测序技术,数字表达谱分析对木薯在特定条件下、特定时期、特定部位的的转录本进行生物信息学分析和注释,以期对木薯耐寒性分子机理提供依据。选择抗寒性较好的华南124(SC124),在生长箱控制条件下,诱导后寒害和正常温度对照2个处理,分别在处理后6小时、24小时、48小时取幼叶、功能叶和块根样品。选取其中12个样品库进行Solexa表达谱测序,同时对全部样品混合进行了一个RNA-Seq测序,建立参考基因集。表达谱分析获得表达基因的Tag丰度信息,参考集转录组注释获得基因序列信息,将二者进行整合后,对表达谱进行了基因功能注释。所取得的主要结果如下：(1)通过对SC124低温处理及对照12个样品的表达谱测序,获得独立表达Tag序列种类总数在102804-189320之间,其中丰度大于100的高表达Tags平均占Tag总种类的4.45%,丰度为51-100的Tag总种类的平均约为3.84%,丰度为21-50的Tag平均约占总种类的8.08%,丰度为11-20的Tag种类平均约为9.43%,丰度为5-10的Tag种类平均约为14.12%,而拷贝数小于5的低表达Tags在种类上非常丰富,占mRNA总量近60.05%。(2)比较幼叶、功能叶及块根低温处理不同时间差异表达基因,发现在幼叶中三个时间点上调表达基因数量在506-715之间,三个时间点个下调基因数量在812-1425之间；功能叶中三个时间点上调基因表达数量在500-746之间,三个时间点个下调基因数量在553-1043之间；块根中三个时间点基因上调表达数量在439-806之间,三个时间点个下调基因数量在500-862之间；那些低温处理下高表达的基因,暗示着可能在低温胁迫时发挥重要作用。(3)通过表达模式分析,得到幼叶、功能叶、块根三个部位的表达模式聚类结果及重要基因的表达趋势。并通过比较三个部位间的表达模式发现,幼叶中下调趋势比其它两个部位明显,而功能叶上调趋势较其它两个部位显著。(4)GO分析结果,说明光合作用中的光吸收、叶绿素的生物合成及代谢、色素合成及代谢过程、调控的催化活性、渗透压响应、氧化应激反应、生长素刺激的反应、脂质的生物合成过程、脂质的代谢过程、氧化磷酸化等生物学过程受低温处理影响；(5)代谢途径pathway分析得到低温影响显著的代谢通路：光合作用天线色素蛋白,卟啉和叶绿素代谢、植物氧化主要在线粒体中进行磷酸化、在光合生物固碳、植物激素的生物合成、类胡萝卜素合成、α-亚麻酸的代谢、ABC转运。(6)通过对受低温影响显著的代谢途径中基因表达丰度分析,发现一批重要的低温胁迫响应基因：如富含甘氨酸蛋白、赤霉素调控蛋白、UDP-糖基转移酶、钙离子转运蛋白、CIPK同源基因、半乳糖转移酶、锌和铜离子转运蛋白、质膜H+-ATPase、APX2等。更多还原

【Abstract】 Cassava(Manihot esculenta Crantz), a species in Euphorbiaceae, the third potato in the world as well as rank the sixth staple food crop in the world, and it already became an important source of biofuel and industrial starch in China. Cold tolerance of cassava varieties is a special constraints to commercial plantation in higher latitude region of China. Up to now, there were a few reports on cold tolerance of cassava although there were many researches on cold stress mechanism in other crops. Expression profiling based on next-generation sequencing technology known as a powerful tool, here, we choice a cold tolerant variety SC124to take cold treatment in chamber with12samples. Expression profiling sequencing was used for screen of genes response to cold. Another transcriptome sequence data from mixture sample was added into the analysis. All tags and their expression richness of transcripts have been revealed by3’-end sequencing, and combining to RNA-seq annotation we performed the functional annotation of the genes for cold response. The essential results shown as the following:SC124, which has the better ability to cold stress, was cold injury induced, then, lobus cardiacus, function leaves and roots was sampling separately at6h,24h,48h. Also the lobus cardiacus, function leaves and roots without low temperature treatment was sampling. Every sample has a tag library which was sequenced with Solexa method separately. Every tag from each tag library was analyzed. We assembled the mixed samples’tag and got the transcripts that was annotated later. The statistic analysis result shows that there is difference not only in numbers but also in species between samples with low temperature treatment and those without. A large different expression exists in each part at different time. We select some genes that has high expression level in low temperature treatment, and these genes are related to cold stress.Through the bioinformatics analysis, we got conclusions as follow:(1) Throgh sequencing of the gene expression profile of12samples, including SC124in low temperature treatments and the control, distinct tagas were found between102804and189320, among which there are4.45%high expression level tags which copy number is above100,3.84%tags which copy number is between51and100,8.08%tags which copy number is21and50, around9,43%tags which copy number is between11and20, around14.12%tags which copy number is between5and10. Those tags which copy number is below5are very rich in species and represents60.05%of the total mRNA.(2) Compare the differential expression genes of tip leaves、function leaves and tuber roots at different time under cold stress, we found that506-715genes of tip leaves were up regulated at the three times, while812-1425were down regulated.500-746genes of function leaves were up regulated at the three times, while553-1043were down regulated.439-806were up regulated at the three times, while500-862were down regulated at the three times.That means when cassava is under cold stress, some genes that are in high expression level play a important role in cold stress.(3) Expression pattern analysis showed the expression pattern of clustering results of young leaves, function leaves and roots and the trend expression of important genes. By comparion of the expression clust result in three different parts of cassava, it is found that there are more down regulated expression in young leaves while more up regulated expression in function leaves(4) GO analysis of light absorption in photosynthesis, photosynthesis, light harvesting, chlorophyll biosynthetic process, pigment biosynthetic process, pigment metabolic process, regulation of catalytic activity, response to osmotic stress, response to oxidative stress, response to auxin stimulus, lipid biosynthetic process, lipid metabolic process, oxidative phosphorylation.(5) Genes that has different expression level in cold stress and low temperature stress were analyzed by pathway method and the significant metabolic pathways are Photosynthesis-antenna protein, porphyrins and chlorophyll metabolism, photosynthesis, metabolic pathway, the oxidation mainly in the mitochondria in photosynthetic organisms phosphorylation, solid carbon, plant hormones biosynthesis, carotenoids synthesis of alpha linolenic acid metabolism, ABC transshipment..(6) We got a series of differential expression genes that can express in cold stress and low temperature stress. They are in rich Glycine protein, gibberellic acid control protein, UDP-glycosyl transferase, calcium ions transporter, CIPK homologous gene, half of lactose shift enzyme, zinc ion and copper ions transporter, plasma membrane H+-ATPase, APX2.更多还原