Study On The Differentially Expressed Genes In Male And Female Flower Buds Of Ginkgo Biloba L.

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.