【作者】 杨迪；

【导师】 郑用琏；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 小麦温光敏雄性不育材料为我国特有资源,拥有完全独立的自主知识产权,近十年来发展极为迅速,已经成为国内外小麦杂种优势利用的主要育种途径。本实验以温光敏雄性不育系BS20为材料,进行均一化cDNA文库构建、大规模cDNA测序、EST序列生物信息学分析,以及小麦雄蕊发育过程中微管、微丝细胞骨架激光共聚焦荧光标记体系探索等方面的研究,旨在为更好的揭示温光敏雄性不育分子机理搭建平台。本实验所获得的主要研究结果如下:1.温光敏雄性不育小麦穗部发育时期均一化cDNA文库构建、鉴定和EST分析(1)按照雄蕊不同发育时期,即小花分化期,药隔形成期,小孢子母细胞时期,四分体时期,单核花粉期和成熟花粉期,构建了6个独立的温光敏雄性不育小麦穗部和花药组织cDNA文库。除了小孢子母细胞时期构建的文库之外,其余5个文库的重组率均为100%。每个独立文库的库容量在1.6×10~6-2.0×10~7之间,平均插入片段长度大于0.85 kb。(2)通过与基因组饱和杂交的方法混合6个独立cDNA文库构建穗部发育时期均一化cDNA文库。文库库容量为4.0×10~5,平均插入片段长度为1.3 kb。(3)从均一化cDNA文库中随机挑选3264个克隆进行5′单向测序,剔除长度小于100 bp序列之后,共获得了3223条去载体序列的EST序列,其平均测序长度为926bp。通过聚类和拼接,结果产生2175条非冗余EST聚类,其中包括423条contig和1752条singleton,我们将它们合称为温光敏雄性不育小麦穗部发育相关UniGene(TPGMSSUniGene)。(4)通过综合利用不同序列比对工具的优势,检索多个数据库对TPGMSSUni-Gene进行功能注释。结果显示有60%的UniGene有功能注释。其中有264条(占总数的12%)在CSRD数据库中存在高度同源序列。它们主要是编码初生代谢相关基因、信号转导相关基因和转录调节相关基因。(5)2715个UniGene进行简单重复序列扫描。结果有108个二至六核苷酸为重复基序的SSR位点被找到,其中三核苷酸重复基序的SSR数量最多。2.小麦减数分裂时期细胞骨架荧光标记体系的探索(1)多聚赖氨酸直接粘片法:虽然通过改进固定液配方、针对微管微丝采用不同的固定时间和酶解时间,但还是发现这种方法存在细胞脱落情况严重,组织细胞粘片效果不佳和组织碎片荧光背景干扰等问题,不适合进行小麦雄蕊发育过程中微管、微丝细胞骨架荧光标记。(2)低熔点石蜡切片法:微管、微丝荧光标记效果较好,无背景杂质荧光信号干扰,克服了细胞脱落的情况,可以观察不同发育时期的小孢子细胞骨架结构。通过优化改进实际操作步骤等方面,最终总结出适合小麦雄蕊发育过程中微管、微丝细胞骨架观察的低熔点石蜡切片荧光标记体系。更多还原

【Abstract】 Thermo-photoperiod-sensitive genie male sterile(TPGMS) wheat line is the unique resource in China,which has independent intellectual property fights.Through the rapid development over the past decade,the TPGMS has become the main means to potentially use heterosis in agricultural crops.In this study,we used the TPGMS wheat,line BS20,to engage a lot of research,including the construction of normalized cDNA library, large-scale EST sequencing,bioinformatics analysis of this EST sequences and exploring the fluorescent labeling system of eytoskeleton of wheat during the meiotic stages.The goal of this study is to establish a platform for functional genomics studies to reveal the molecular mechanism of TPGMS wheat.The main results are shown as following:1.Construction,characterization and EST analysis of normalized cDNA library of TPGMS wheat from spike developmental stages(1) Six individual cDNA libraries of the spikelet of TPGMS Wheat were constructed in the periods of floret differentiation stage,anther development stage,microspore mother cell stage,tetrad stage,mono-nucleate microspores stage and mature pollen stage.Except for one library constructed with microspore mother cell stage,others owned 100%recombinant ratio.The number of recombinant of individual libraries ranges from 1.6×10~6 to 2.0×10~7.The insert fragment size was large than 0.85 kb.(2) Based on the strategy of saturation hybridization with genomie DNA,we set up a normalized complementary DNA(cDNA) library.The normalized library consisted of 4.0×10~5 clones and had an average insert length of 1.3 kb.(3) A total of 3,264 clones randomly selected from normalized spike development cDNA library were sequenced,which generated 3,223 vector-trimmed EST sequences with an average sequencing read length of 926 bp.Clustering and assembly analysis resulted in 2,175 unique ESTs from 423 contigs and 1,752 singletons.This final dataset was denoted as the TPGMS wheat Spike development-related UniGene (TPGMSSUniGene) set.(4) Taking advantage of various tools and database,gene function classification of the 2,175 unique ESTs showed that 60%of the ESTs were predicted to have putative gene function,264(12%) displayed significant homology to genes previously reported to be involved in cold-response related processes.Among these,sequences encoding activities related to primary metabolism,signal transduction,and transcriptional regulation were observed.(5) The 2,715 unigenes set was searched for potential simple sequence repeats(SSRs).In total,108 di-to pentanucleotide SSRs that fulfilled the criteria of the search were identified,and tri-nucleotide repeats were the most.2.Exploring the fluorescent labeling system of cytoskeleton of wheat during the meiotic stages(1) Poly-lysine gluing section method:we improved the composition of stationary liquid, the time of stationary and enzymolysis,but we observed the tissues lose easily during the process of experiment.The anther wall debris disturbed the backdrop of fluorescence.The method is not suitable for labeling cytoskeleton of wheat during the meiotic stages.(2) Steedman’s wax sectioning method:This method was used successfully to labell both the microfilament and microtubule in this experiment,the backdrop of fluorescence was very clean,and only a few cells lost during the process of experiment.By optimizing the actual steps,the cytoskeleton structure of different developmental stages of microspore was observed.We concluded that this optimized method is more suitable for fluorescent labeling during microspore development than other method.更多还原