【作者】 张晓文；

【导师】 邢世岩；

【作者基本信息】 山东农业大学 ， 森林培育， 2014， 博士

【摘要】 银杏原产中国，有世界“金色活化石”之称，为典型的雌雄异株植物，适应性强，种植范围广泛，集材、果、药、观赏多功能于一体，具有较高的经济价值和生态效益，在国内外市场供不应求。银杏雌雄株的生长习性、形态特征与用途各不相同，雄株常用作绿化中的行道树，而雌株则以果实的生产为主要目的。银杏雌雄株的选择往往受制于童期长、性别分化和性别表现较迟的影响，在其生长早期不能正确地区分。因此，准确的鉴定银杏性别，在生产实践中意义重大。二十世纪以来，随着分子生物学及生物技术的迅猛发展，已利用遗传标记对银杏的性别决定及遗传机制有了一些了解，但有关性别基因的确定及调控机理方面的研究少见报道，利用分子生物学的方法分离性别相关基因仍需大量的研究工作。抑制差减杂交（SSH）技术因其假阳性率低、高效灵敏、经济速度、重复性好等特点，近年来已作为一种高效便捷鉴别差异表达基因的方法，取得许多研究成果，并已广泛应用于分子遗传学及定向克隆的研究中。基于以上原因，本研究应用分子生物学、生物信息学及实时定量分析等方法技术，分别以银杏雌、雄花芽为材料，构建银杏雌雄花芽抑制性差减文库，通过对差减cDNA文库所包含的所有序列进行生物信息学功能注释和分类，筛选与银杏性别调控相关的ESTs，继而对这些ESTs进行表达分析及检测。集中研究这些ESTs在银杏雌雄株花器官发育中的差异表达，为进一步探讨银杏雌雄株性别分化的机理奠定理论基础，也为后续银杏花发育相关基因的克隆、表达和功能分析工作打下基础。主要研究结果如下：1.首次应用SSH技术成功构建了银杏雌雄花芽差异表达的正反向两个差减cDNA文库，文库片段插入率接近95%，插入片段的大小主要分布在300-1300bp之间，平均长度在750bp左右。差减文库的成功构建为进一步筛选及克隆与银杏性别相关的基因奠定了基础，对银杏性别鉴定研究的分子机制具有重要意义。2.利用NCBI网站BLASTn软件，将银杏雌雄花芽差减文库筛选得到的差异基因ESTs片段序列与GenBank数据库中的序列进行同源性比对，获得的功能注释序列通过GO分类体系进行初步生物信息学分析。结果雌花芽优势表达序列中有23.91%得到了同源序列，雄花芽优势表达序列中有58.42%比对得到了同源序列，总体超过半数的差异表达EST序列没有获得功能注释，参与光合作用、能量代谢等的保守基因序列占10%左右，同源基因获取率低。3.将差异基因ESTs与本实验室之前的银杏叶片RNA-seq测序的结果进行比对后，再一次提交BLASTn比对，结果显示，超过80%的序列获得了功能注释，明显提高了同源基因获取率。得到的雌雄花芽差异ESTs按照生物学功能分类共分为：功能未知、能量代谢、光合作用、信号转导、转录调控、蛋白质代谢和折叠、脂代谢、次生代谢、氨基酸代谢、细胞壁重塑、细胞骨架重塑、转运、抗性与逆境相应、糖代谢、核苷酸代谢和细胞周期与细胞生长，共16类。其中，核苷酸代谢为雌花芽特有，细胞周期与细胞生长为雄花芽特有。雌、雄花芽共有14个功能类别，说明同样作为花器官的前体和旺盛生长的组织，雌雄花芽在基因表达和代谢状态上具有一定的相似性，同时也存在特异性。由此可见，银杏雌雄花芽发育过程中，伴随着复杂多样的生理生化过程。4.差减文库中筛选到8个可能控制植物花器官发育及配子体形成相关基因的ESTs，如Dof家族转录因子通过抑制CO基因的转录可以延迟拟南芥植株开花；CRT蛋白参与某些植物花柱道的发育和双受精过程；PAE通过调节细胞壁的代谢，参与植物花粉发育过程；PLD具有促进细胞扩展的作用，参与植物花粉管极性生长等过程；PAL基因的突变会造成花粉活力丧失；SHEPHERD基因的突变会抑制花粉管的极性伸长等。另外，从差减文库中筛选到5类激素相关基因，如Beta-葡糖苷酶能够活化CTK分子； ETH信号转导相关序列；DWARF基因负责GA的合成等。这些ESTs功能的分析和验证，为分析银杏雌雄花基因的表达，以及进行转基因研究调控花的发育奠定基础。5.从雄性和雌性花芽优势表达序列中各鉴定到数个ATP合成酶编码基因，在雄性花芽优势表达序列中发现1个WD家族蛋白编码的基因。这些序列有可能作为SlY-1或者DD44的同源基因而定位于性染色体上，是在性别决定过程中发挥关键调控作用的基因。6.差减文库中筛选得到3个与动物性别决定基因相关的ESTs：Sex-lethal介导雌性特异剪接、起始和维持雌性特征的发育；导致小鼠雄性连锁的腭裂的CASK基因和小鼠维持雄性性征的H-Y基因。思考，植物性别决定和动物的性别决定是否存在部分共同的机制联系。7.采用Real-time PCR方法，从银杏雌雄花芽正反差减文库筛选的差异ESTs中，随机挑选15个，研究来自银杏雌雄花芽中差异基因的表达情况。结果表明，7个雌性花芽优势表达序列在雌性花芽中的表达量明显高于雄性花芽，1个雌性花芽优势表达序列表达量较低，且在雌雄花芽之间差异不明显。7个雄性花芽优势表达序列均表现出在雄性花芽中明显高于雌性花芽的表达模式。验证了抑制性差减杂交得到的差异表达序列的可靠性。8.检测差异表达序列在银杏雌性和雄性基因组中的分布情况发现，大多数序列没有在雌、雄基因组之间表现出分布差异。CH11和CH15在雄性基因组扩增得到的信号大致为雌性基因组的2倍，可能是定位在Z和W染色体上的基因，推测CH15和CH11为定位于W染色体的雌性特异区域，且在Z染色体上具有同源基因的序列。更多还原

【Abstract】 Ginkgo biloba L nicknamed as “golden living fossil” in the world is native to China,which is a typical dioecious plant with a strong adaptability. Moreover, Ginkgo biloba iswidely planted and has a multifunction of wood, fruit, medicine and ornament with a highereconomic values and ecological benefits so that the demands for such kind of plantexceeds the supply in domestic and international markets. The morphological characteristics,growth habits and use are different between female and male plants of Ginkgo biloba, maleplants are commonly used as street trees, while the female plants are planted for their fruits.The selections of male and female plants of Ginkgo biloba often subject to juvenility, sexdifferentiation and later sex expression, so the male and female plants are unable to bedifferentiated correctly in early growth stage. Therefore, accurate sex identification methodin Ginkgo biloba will be of great significance in production practices.Since the20th century, with the rapid developments of molecular biology andbiotechnology, the sex differentiation and genetic mechanism in Ginkgo biloba plants havebeen partially understood by using genetic marker, but few studies relevant to thedetermination of sex gene and regulation mechanism was reported, so the differentiation ofthe genes associated with sex by the methods of molecular biology still calls for lots of work.Suppression Subtractive Hybridization (SSH) technique as an efficient and convenient tool toidentify the differentially expressed genes recently for its lower false positive rate (FPR),higher efficiency and sensitivity and higher repeatability has been widely used in the studiesof molecular genetics and directional cloning, and numerous research findings have beenacquired.Based on the above reasons, the present study with male and female flower buds ofGinkgo biloba plants as experimental materials established a suppression subtractive libraryof male and female flower buds of Ginkgo biloba plants by the methods and techniques ofmolecular biology, bioinformatics and fluorescence quantitative analysis. The ESTs related tothe sex regulation of Ginkgo biloba plants were selected by bioinformatics functionannotation and classification conducted on all the sequences included in the subtractive cDNAlibrary and then expression analysis and detection were conducted on these ESTs. Furthermore, the present study focused on the differential expression of the the ESTs selectedin the male and female floral organ development in Ginkgo biloba plants in order to lay atheoretical basis for the further study on the sex differentiation mechanism of Ginkgo bilobamale and female plants and provide a basis for the following research work including cloning,expression and function analysis of the genes related to floral development in Ginkgo bilobaplants. The main results are as follows:1. For the first time, forward and reverse subtractive cDNA libraries in the differentialexpressions of male and female flower buds were established by application of SSHtechnology in Ginkgo biloba plants, library fragment insert rate was close to95%, the size ofthe inserted fragment are mainly distributed between300-1300bp with the average lengtharound750bp. The success of subtractive library building laid a solid foundation for furtherscreening and cloning of genes associated with sex of Ginkgo biloba plants and would be ofgreat importance for the molecular mechanism of sex identification in Ginkgo biloba plants.2. Homology alignment was conducted on ESTs fragment sequences in the differentialgenes selected in Ginkgo biloba male and female flower bud subtractive libraries and thesequences in GenBank database using BLASTn software on NCBI. Additionally, apreliminary bioinformatics analysis was conducted on the functional annotation sequencesacquired through the GO classification system. The result showed that:23.91%of the femaleflowers bud sequences that were expressed preferentially acquired homologous sequences,while58.42%of male flower bud sequences which were expressed preferentially obtainedhomologous sequences, and as a whole more than half of the differentially expressed ESTssequences did not obtain functional annotations. The conservative gene sequences involved inphotosynthesis, energy metabolism, accounted for about10%, homologous genes access ratewas low.3. After the comparison of differential gene ESTs and RNA-seq sequencing results onGinkgo biloba leaves previously obtained in our lab, the ESTs was again to be submitted toBLASTn. The result showed that more than80%of the sequences obtained functionalannotation, homologous genes access rate increased significantly. In addition, acquireddifferential ESTs in male and female flower buds were categorized a total of16kindsincluding unknown, energy metabolism, photosynthesis, signal transduction, transcriptionregulation, protein metabolism and folding, lipid metabolism, secondary metabolism, aminoacid metabolism, cell wall remodeling, cytoskeleton remodeling, transshipment, accordinglywith adversity resistance, sugar metabolism, nucleotide metabolism with cell cycle and cellgrowth, according to the biological functions. Among them, the nucleotide metabolism was special to female flowers bud, while cell cycle and cell growth was characteristic of maleflower bud. Furthermore, there were a total of14function categories in male and femaleflower buds, which indicated that there was a certain similarity in gene expression andmetabolic state between male and female flower buds, simultaneously there also existed somespecificities between them, indicating that there were various complicated physiological andbiochemical processes involved in the developments of male and female flower buds inGinkgo biloba plants.4.8ESTs of the related genes that possibly control plant flower organ development andgametophyte formation were selected from subtractive library, e.g. Dof family of transcriptionfactors could delay the blossoms of arabidopsis thaliana plants by inhibiting the transcriptionof CO gene; the CRT protein is involved in the development and double fertilization processof pistillar chord; PAE was participated in plant pollen development process by regulating themetabolism of the cell wall; PLD could promote cell expansion and participate in theprocesses such as polarity pollen tube growth process in plants; the mutations of PAL genecould lead a loss of pollen vitality; the mutations of SHEPHERD gene wound suppress thepolarity of the pollen tube elongation, etc. In addition,5types of the genes related to hormonewere selected from subtractive library, e.g. Beta-glucoside enzyme could activate CTKmolecule; ETH could transduct signals; DWARF genes are responsible for the synthesis ofGA. Analysis and validation of these ESTs functions could lay the foundation of the analysison the expression of male and female flower genes and the genetic modification studies on theregulation of flower development in Ginkgo biloba plants5. Several genes encoding the ATP synthase were identified from the male and femaleflower bud sequences that were expressed preferentially, respectively. In the male flower budsequences that were expressed preferentially, one WD family protein-coding gene wasdetected. These sequences as homologous genes of SlY-1or DD44might be positioned on thesex chromosomes, which were the genes that play a crucial role in the regulation of the sexdetermination process.6.3ESTs related to sex determination genes of animal were selected from subtractivelibrary: Sex-lethal which mediated female specific splicing, start and maintain thedevelopment of female characteristics; CASK gene leading to cleft palate in male mice andH-Y gene maintaining the male sexuality in mice. The present study investigated whetherthere exists a common relationship between the sex determination of plant and animal sexdetermination. 7.15ESTs were randomly chosen from the differential ESTs selected in the forward andreverse subtractive libraries of male and female flower buds to study the differential genesexpressions in male and female flower bud of Ginkgo biloba plants by the Real-time PCRmethod. The result demonstrated that: the expression quantity of7female flower budsequences that were expressed preferentially was significantly higher than those in maleflower bud, while the expression quantity of one female flower bud sequences that wasexpressed preferentially is lower, and there was no significant difference between male andfemale flower buds. In addition, the expression quantity of all the7male flower budsequences that were expressed preferentially was significantly higher than those in femaleflower bud. The results verified the reliability of differentially expressed sequences obtainedby suppression subtractive hybridization (SSH).8. The test of differentially expressed sequences distributions in male and female genomeof Ginkgo biloba plants found that there were no significant differences in the distributions ofmost sequences between female and male genome. The signal of CH11and CH15acquired byamplification in male genome is roughly twice as that in female genome, which may be thegenes positioned in the Z and W chromosomes, and it was speculated that CH15and CH11were sequences positioned in female specific areas of the W chromosome and had thehomologous genes on the Z chromosome.更多还原