【作者】 潘腾飞；

【导师】 潘东明；

【作者基本信息】 福建农林大学 ， 果树学， 2010， 博士

【摘要】 琯溪蜜柚(Citrus grandis cv. Guanximiyou)是我国柚类优良品种之一,原产于福建省平和县,已有四百多年的栽培历史,具有很高的经济价值。在生产上,其果实的裂瓣与汁胞粒化造成了食用品质下降,直接影响果农的经济收入。目前,对具有重要经济价值的物种的基因组学研究已成为热点,这些研究成果为解决生产上的问题提供了理论依据,但对琯溪蜜柚基因组的研究尚属空白。因此,本研究首次构建了琯溪蜜柚BAC文库,并将文库应用于汁胞粒化相关基因的筛选和BAC末端测序;用生物信息学方法分析相关的基因组序列,初步研究了琯溪蜜柚基因组结构;为进一步开展琯溪蜜柚基因组学研究,揭示柑果汁胞粒化分子机制奠定了良好基础。取得的主要研究结果如下:1、本研究以琯溪蜜柚叶片为材料,比较了两种提取方法对高分子量核DNA(HMW-DNA)制备效果的影响。结果表明,改良Zhang法与Peterson法相比,前者更适合于琯溪蜜柚HMW-DNA的提取。改良Zhang法用温和的物理方法破碎细胞壁,在一定的渗透压下保证细胞核的完整性,运用miracloth和低速离心去除大部分的多糖和细胞壁碎片,经显微观察,所提取的细胞核完整、纯净、质量高;将这些细胞核用低熔点琼脂糖包埋,以琼脂糖小块的方式保存细胞核,以保持核的稳定;琼脂糖小块的最适消化时间为48 h,脉冲场凝胶电泳分析结果表明所获得的HMW-DNA大于1 Mb,这些核DNA能够被限制性内切酶消化,适合于琯溪蜜柚BAC基因组文库的构建。2、本研究改进了适合琯溪蜜柚BAC文库构建的方法,用一次电泳回收法获得酶切后的DNA片段,构建的琯溪蜜柚BAC文库含有26112个单克隆,空载率小于1%,叶绿体DNA的污染率不超过1%,插入片段平均大小约120 kb,覆盖8倍的琯溪蜜柚基因组。3、利用PCR法和Southern杂交法相结合,以琯溪蜜柚汁胞粒化相关EST M4为探针,从琯溪蜜柚BAC文库中筛选得到一个阳性BAC克隆。说明本研究所构建的BAC文库覆盖琯溪蜜柚基因组,从中能筛选得到目的片段。利用与琯溪蜜柚汁胞粒化相关的EST序列M4设计PCR引物筛选文库,得到一段大小为1088 bp的DNA序列,该序列含有两段大小分别为122 bp和172 bp的内含子。在M4序列的两端设计一对反向PCR引物,利用反向PCR技术,以自连接产物为模板,分别扩增得到约4000 bp和400 bp的DNA片段。将目的片段回收、克隆、测序,序列用Phred软件拼接,得到3984 bp的DNA序列,其中位于M4上游的序列3107 bp,位于下游的序列877 bp。分别用GENSCAN和FGENESH软件预测这部分DNA序列中可能包含的基因,两个软件预测的编码区基本一致。两个软件的预测编码区与NCBI上的核酸数据库进行BLAST,BLAST结果表明,该序列中部分序列与蓖麻(Ricinus communis)、杨树(Populus trichocarpa)多铜氧化酶(multicopper oxidase)cDNA序列分别有85%和71%的同源性;与毛叶番荔枝(Annona cherimola)和拟南芥(Arabidopsis thaliana)果胶酯酶(pectinesterase)cDNA序列分别有76%和73%的同源性。推测多铜氧化酶和果胶酯酶可能与琯溪蜜柚汁胞粒化相关。4、本研究从琯溪蜜柚BAC文库中共随机挑选了120个克隆,获得105664 bp BAC末端序列。根据末端序列信息,通过生物信息学分析,对琯溪蜜柚基因组进行初步的研究。结果表明,琯溪蜜柚基因组中SSRs占总基因组的0.2%,17.8%的琯溪蜜柚BAC末端序列含有转座元件,基因组中的转座元件以反转录转座子为主。琯溪蜜柚基因组GC含量约为39.17%,估算出琯溪蜜柚的编码区大约为64.02 Mb,预测其基因数大约为32001个,编码区的GC含量约为46.7%。更多还原

【Abstract】 Guanximiyou [Citrus grandis (L.) Osbeck cv.Guanximiyou], originated from Pinghe county Fujian Province, is one of the most famous pummelos with high economic value. It has been cultivated for 4 hundred years. Juice sac granulation effected edible quality severely and income of farmers. Genomics research mainly focuses on plants with significantly economical value, and the study results offer the theoretical basis to solve the issues on production. Few researches on Guanximiyou genomics have been reported.A BAC library was constructed for the first time in this study. The library was screened by probe EST M4. Some BAC end sequences were sequenced, and bioinformatics were used to analyze the BESs. The study revealed first insights into the organization of Guanximiyou genome.The main results were indicated as follows:1. High molecular weight nuclear DNA(HMW-DNA) from leaves of Guanximiyou was prepared by two methods (improved Zhang’s and Peterson’s method) and the extracting effects of the two preparing methods were compared, the results showed that improved Zhang’s was more suitable for preparation of HMW-DNA in Guanximiyou. In the improved Zhang’s method, the cell walls were broken down by gentle physical homogenization to keep integrity of nuclei under suitable osmolarity. Debris of cell walls and most of polysaccharides were removed by filtering with miracloth and centrifugation under slow speed, the isolated nuclei were proved to be intact, pure, and high-quality by observation under microscope and were embedded with low-melting-point agarose to preserve the isolated nuclei in plugs for keeping stability of nuclei. The optimum digestion time of plugs was 48 hours, the pulse field gel electrophoresis (PFGE) results showed that the isolated HMW-DNAs were more than 1 Mb in size and were digested completely or partially by restriction enzymes, therefore, Zhang’s method was suitable for construction of BAC library of Guanximiyou.2. Method for constructing Guanximiyou BAC library was improved. The result showed that DNA fragments obtained by one step electrophoresis protocol were suitable for library construction. The library consists of 26112 clones. A random sample of 100 clones indicated an average insert size of 120 kb, corresponding to 8 genome equivalents. Less than 1% of the clones do not contain inserts. Screening of BAC library with chloroplast DNA probes indicated that less than 1% of the clones contained organelle-derived DNA.3. The BAC library was screened with a pair of primers deprived from EST related to granulation by PCR and Southern blot. A positive clone was obtained, proving that the library had deep coverage of Guanximiyou genome. A 1088 bp DNA fragment was amplified by PCR with the primer from EST M4. The sequence contained two introns with the length 122 bp and 172 bp respectively. An about 4000 bp and an about 400 bp DNA fragments were amplified by I-PCR with a pair of I-PCR primers from EST M4 using self-ligations as DNA template. The 4000 bp DNA fragment was cloned and sequenced, and the exactly length was 3984 bp. The potential coding regions were predicted by GENSCAN and FGENESH. The results showed that the coding regions predicted by these two softwares were almost consistent. The coding sequence has 85% and 71% identities with multicopper oxidase cDNA sequences of Ricinus communis and Populus trichocarpa respectively, furthermore, it has 76% and 73% identities with pectinesterase cDNA sequences of Annona cherimola and Arabidopsis thaliana respectively. According to the characterization of BAC clones and the results of screening, the library is suitable for functional genome research on Guanximiyou .4. One hundred and twenty BAC clones were picked up from the library, and a total length of 105664bp end sequence was obtained. These sequences were analyzed by bioinformatics. The results showed that approximately 0.2% of Guanximiyou genome contained simple sequence repeats (SSRs), 17.8% of BESs contained transposable elements and most of these elements were retrotransposons. The average GC content of the Guanximiyou genome was 39.17%, and the content of the predicted coding regions of BESs was 46.7%. It was estimated that Guanximiyou genome contained 64.02 Mb coding regions, and gene number was 320001.更多还原