【作者】 杨惠；

【导师】 杨官品； 茅云翔；

【作者基本信息】 中国海洋大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 条斑紫菜是具有重要经济价值和科学研究价值的大型红藻,是潮间带藻类抗逆分子生理学研究的理想模式物种。但是,目前对于其基因组信息知之甚少,对其环境耐受机制以及分子育种研究也都非常欠缺。本论文通过对条斑紫菜功能基因组及重复序列的全面系统研究,深入了解条斑紫菜转录组特性和基因组结构特征,为其抗逆育种和栽培技术改良提供重要参考。本论文主要包括以下内容:利用Solexa技术结合生物信息学方法对胁迫条件下的条斑紫菜转录组及小RNA进行深度测序分析;利用软件筛查条斑紫菜基因组及转录序列中的重复序列并进行比较分析;利用富集文库法筛选条斑紫菜和坛紫菜基因组微卫星标记并用于纯系鉴定和遗传分析。主要结果如下:⑴利用Solexa技术对不同生理状态和不同胁迫处理的条斑紫菜混合样品进行测序,共获得13,333,334条高质量序列,碱基总量为12×10~9 nt,组装后得到31,538条平均长度为419 nt的Unigene。把获得的Unigene与数据库中EST进行比对分析,发现前者对后者有非常高的覆盖率,也证明了获得的序列绝大多数为条斑紫菜中的新序列。通过与数据库进行比对,发现条斑紫菜转录组中包含大量在其他物种中与光适应、失水适应以及抗氧化机制相关的功能基因,并且发现了红藻中未曾报道过的超氧化物歧化酶类型。通过KEGG分析,发现了条斑紫菜中的120条代谢途径,其中包括一条几近完整的与高等植物类似的C3/C4碳固定途径和其他与抗逆机制相关的代谢途径。与数据库比对之后,仍有56.6%的Unigene没有找到同源序列,说明测序结果中可能存在大量的功能未知的新基因。⑵通过对胁迫条件下条斑紫菜小RNA的Solexa深度测序,共获得高质量序列8,262,593条,其中以22 nt的RNA tags数量最多,筛选其中表达量大于1次的682,398条RNA tags进行后续分析。与数据库比对,找出其中的rRNA、tRNA、snRNA和snoRNA共计35,967条,保守的microRNA有3条,剩下的序列(94.7%)均没有注释。利用条斑紫菜的GSS数据进行miRNA前体预测,共找到8条符合前体标准的新类型miRNA,靶基因预测结果表明,其中6条miRNA参与调控10个靶基因的表达,而这些靶基因绝大部分参与生物代谢、物质转运及信号转导过程。⑶通过对基因组序列和转录序列中的重复序列进行筛查和比较分析发现:在条斑紫菜中,小卫星重复总长占基因组不到2%,拷贝数较低,以四碱基组成类型最为丰富,且明显富含GC。基因组序列中的小卫星与转录序列中的小卫星相比在拷贝数、数量及长度比例上均略低。微卫星序列在基因组中的长度比例不到0.4%,拷贝数主要分布在9次以下,以三碱基重复类型最为丰富,五碱基重复类型最少,CCG是最为丰富的基序。基因组序列中的微卫星与转录序列中的微卫星相比,出现频率低,长度比例高。在条斑紫菜中,散布重复序列均由RNA转座子和DNA转座子组成,且前者比例远高于后者。RNA转座子中LTR数目最多,LINE次之,SINE最少。在所有转座元件中gypsy数目最多。基因组序列中的转座元件与转录序列中的相比,出现频率高,长度比例也高。总之,条斑紫菜基因组序列中的重复序列总长度比例约为6.5%,明显高于转录序列中的相应比例,而出现频率却相对较低。⑷首次通过富集筛库法,获得24对条斑紫菜微卫星引物。利用其中12对引物对青岛汇泉湾野生群体进行微卫星位点评估,共获得29个等位基因,平均每个位点的等位基因数目为2.42,有效等位基因数目为1.81。利用24对引物分析11个不同地域来源的条斑紫菜丝状体,聚类结果显示这11个个体根据地域的差别被明显地分为两支。利用微卫星标记分析单个叶片的扩增条带,发现同一位点两条不同带型的序列差别在于微卫星重复次数的不同。利用数据库搜索法和富集文库法,分别得到9对和14对有较好扩增谱带的坛紫菜微卫星引物,并且通过实验证明了这些标记在遗传分析中的实用价值。更多还原

【Abstract】 Porphyra yezoensis Ueda is a genus of red macroalga considered to be of important economic and research value. It is also an ideal model species for studying on the stress-tolerance molecular physiology of intertidal seaweeds. However, the knowledge of their environment-tolerance mechanisms and molecular breeding are very lacking at present. In this thesis, comprehensive and systematic study of functional genomics and repetitive sequences will substantially improve the global view of the P. yezoensis transcriptome and its genome structure, and will be beneficial to improve the stress-tolerance breeding and cultivation techniques.Illumina/Solexa sequencing technology is used to study the transcriptome and small RNA of P. yezoensis with the aim to characterize its functional genome. Comparative analysis of the composition and distribution of repetitive sequences is performed in P. yezoensis. A group of microsatellite DNA markers are developed and used to determine the pure line and genetic diversity in Porphyra.The following are the major findings:The transcriptome of P. yezoensis was profiled with next-generation RNA sequencing technology. A total of 13,333,334 quality paired-end reads were obtained from the cDNA library constructed for a mixture of P. yezoensis at different growth stages and under different growing conditions, which generated 1.2×10~9 nt. The assembling of these reads yielded 31,538 unigenes with a mean length of 419 nt. The unigenes obtained overlapped the majority of the deposited in dbEST database at NCBI. A large set of annotated genes involved in stress tolerance and defense, and a C4-like carbon fixing pathway was profiled. Less than half of them can be annotated based on the similarity with the known proteins in diverse databases, while the remaining should be novel.Deep sequencing technology is used and 8,262,593 quality small RNAs are obtained from a small RNA library. The largest number of small RNAs are length of 22 nt. By blasting small RNAs cloned more than once (682,398) against known databases, all small RNAs were classified into different categories, including rRNA, tRNA, snRNA, snoRNA and unannotated RNAs. By blasting against miRBase, 3 small RNAs were found to be the same as plant miRNAs. Eight novel miRNA candidates were predicted from the GSS of P. yezoensis as well as from the sequencing results. By prediction, six novel miRNA candidates result in ten target genes, most of which encode proteins involved in metabolism, transportation and signal transduction.Comparative analysis of repetitive sequences show their characteristics of composition and distribution in P. yezoensis. The total length of minisatellite repeats in P. yezoensis is less than 2% of total sequences analized with low repeat times. Most of the minisatellite DNA is composed of four types of bases with GC-rich. The repeat times, number and length proportion of minisatellite repeats in genome sequences are lower than those in transcribed sequences. The total length of microsatellite repeats in P. yezoensis is less than 0.4% of total sequences analized. The repeat times of most microsatellite repeats are less than nine. Trinucleotide repeat types are the most abundant, and CCG is the most abundant motif. The frequency of microsatellite repeats in genome sequences is lower than that in transcribed sequences, whereas, the length proportion is higher. In P. yezoensis, RNA transposons are much more than DNA transposons. The most abundant type of RNA transposons is the LTR retrotransposons, followed by LINE elements and SINE elements. Among all elements, gypsy made up the largest fraction in number. The frequency and length proportion of transposable elements in genome sequences are higher than those in transcribed sequences. In short, the length proportion of repetitive sequences in genome is approximately 6.5%, which is much higher than that in transcribed sequences. However, the frequency of repetitive sequences in genome is lower than that in transcribed sequencesTwenty-four microsatellite DNA markers are developed from enriched library of P. yezoensis. Twelve primer pairs amplify two to four bands, whereas another 12 primer pairs produce monomorphic banding patterns. A total of 29 alleles are produced at 12 loci, with an average of 2.42 alleles (Na) and 1.81 effective alleles (Ne) per locus. These markers are used to analyze the genetic diversity within 11 geographically different lines of P. yezoensis. Overall, these lines are clustered into two divisions with those from close geographic locations clustering together. Further cloning and sequencing of size variant alleles at two microsatellite loci from one blade reveals that the variable numbers of motif repeats in different alleles are major sources of polymorphisms. In P. haitanensis, nine and fourteen microsatellite DNA markers are developed from EST and enriched library, respectively. These markers are also used to determine pure line and genetic diversity of P. haitanensis, which proved their value of practical applications.更多还原