Studies On The Expression Patterns Of Tissue-specific And Predominant Genes In The Growth And Development Of Rice

The process of growth and development in angiosperm included a series of ever-changing events such as root growth,leaf development,flower morphogenesis,meiosis,and double fertilization,ect.These events were regulated by a series of sophisticated networks of genes,and the screening of genes specifically expressed in different tissues was of great significance for revealing the process of growth and development of plants by searching for differences in those networks.Currently,Arabidopsis and rice were the most representative dicotyledonous and monocotyledonous pattern plants.In Arabidopsis,a series of tissue-specific expression genes had been obtained,such as EC1,DD22 and STK expressed at the reproductive growth stage,WUS,SCR and FAMA expressed at the vegetative growth stage.However,there were few researchs about genes specifically expressed in the tissues of rice compared with Arabidopsis.Since the lack of tissue-specific marker genes during the growth and development in rice,the research on the functions of genes were difficult and a large number of molecular regulatory mechanisms remained unclear.Therefore,the screening of genes specially expressed in the developmental process of rice,especially at the reproductive development stage,would promote the study on the molecular regulation mechanisms of rice.The spatiotemporal expression patterns of these genes also provided a reference for the later functional studies of such genes.Meanwhile,the promoter of specifically expressed gene can be used to characterize the developmental patterns of plant-specific tissues and can be used to assist in the functional studies of other genes.In this study,21 genes named OsSPE1-OsSPE21(Oryza sativa Specially or Predominantly Expressed Gene)were screened from a pistil transcriptome database and a ovule transcriptome database,which were specificly or preferentially expressed in the pistils and ovules of rice.Then some transgenic plants of 11 genes of them were obtained for further tissue observation and the characteristics of these genes were analyzed by bioinformatics.The promoter sequences of these genes were cloned into vector pCAMBIAI1300-GFP carrying a nuclear localization sequence to construct recombinant vectors,where GFP(Green fluorescent protein)was expressed by these promoters.Next,the recombinant vectors were transformed into the callus of rice by Agrobacterium-mediated transformation,producing transgenic plants.The GFPs expressed in tissue of transgenic positive plants were observed and analyzed under confocal microscopy.In this study,the main experimental results obtained were as follows:(1)The analysis of promoter sequences and predictions of signal peptide,transmembrane domain and subcellular localization of 12 genens OsSPE01,OsSPE02,OsSPE05,OsSPE06,OsSPE07,OsSPE08,OsSPE10,OsSPE11,OsSPE13,OsSPE15,OsSPE16 and OsSPE18 were carried out by bioinformatics websites.The results indicated that the promoter fragments of all genes contained transcriptional regulatory elements,hormone response elements and photoresponsive elements,et al.Five genes among the twelve genes analysed above were selected to be detailed illustrated: the promoter region of OsSPE01 contained seed-specific regulatory elements and cis-acting elements for endosperm expression.As a member of the defensin family,OsSPE05 contained meristem expression-related elements on its fragment of promoter.As a member of the expansin family,OsSPE06 contained a variety of hormone-related response elements on its promoter.The promoter region of OsSPE07 contained aCAT-box element associated with meristem expression and a related element HD-Zip Ⅰinvolved in the differentiation of the mesophyll cells in palisade tissues.The promoter region of OsSPE11 contained cis-acting elements that regulate meristem.The analysis of signal peptide,transmembrane domain and subcellular localization indicated that the OsSPE01,OsSPE06,OsSPE07 and OsSPE13 all contained signal peptides and transmembrane domains,suggesting that these proteins may be secreted proteins.Both OsSPE10 and OsSPE11 were located in mitochondria and contained a signal peptide and a transmembrane domain,speculating that it may play a role in the mitochondrial membrane.There were no signal peptide and transmembrane domain of OsSPE02 and OsSPE16,and their subcellular localization was unclear.OsSPE05 had no signal peptide but contained a transmembrane domain,which may be secreted protein.OsSPE08 and OsSPE15 contained a signal peptide but not contain a transmembrane domain,possibly be secreted to the extracellular for its function.OsSPE18 had no signal peptide but contained a transmembrane domain,and its subcellular localization was unclear.(2)The promoter sequences of the 12 genes,including OsSPE01,OsSPE02,OsSPE05,OsSPE06,OsSPE07,OsSPE08,OsSPE10,OsSPE11,OsSPE13,OsSPE15,OsSPE16 and OsSPE18,were cloned and inserted into the MCS(Multiple cloning site)of pCAMBIAI1300-GFP vector for obtaining recombinant vectors,respectively.Thereafter,the recombinant vectors was transferred to E.coli,Agrobacterium,and rice callus,respectively.According to the identified results of PCR,the positive transgenic plants of rice were screened and obtained.(3)The transgenic plants were obtained from callus induced from rice Nipponbare,which were infected by Agrobacterium(EHA105)-mediated transformation,Then,the positive transgenic plants of 12 genes were identified by PCR using the genome extracted from transgenic plants as template.The GFP fluorescence of the ovary,ovule,filament,14-day-seedling roots and leaves in the positive transgenic plants were observed under confocal microscopy.The results showed that OsSPE01 waspreferentially expressed on the stigma after pollination,also detected on the ovary wall,style and integument before and after pollination in pistil.Besides,it was also expressed in the stomatal apparatus of the leaves and the epidermis and cortex of coronal,and lateral roots.OsSPE02 was dominantly expressed in the stigma,style,ovary wall and integument in the pistil before and after pollination,meanwhile expressed in the primordium of lateral root continuously until the lateral root mature.In the pistil before pollination,OsSPE05 was expressed in the style,ovary wall,integument and filaments.In addation,it was specifically expressed in the stomatal apparatus of leaves and specifically expressed in the pericycle of the crown root,speculated that it may participate in the formation of the stele tissues.OsSPE06 was specifically expressed in the wall of the embryo sac in the pistil.After pollination,the GFP fluorescence signal extended outward with the elongation of the embryo sac.The gene was specifically expressed in the root cap,suggesting that it may participate in the morphogenesis and developmental process of the root cap.OsSPE10 was weakly expressed in the style,ovary wall,the integument of the pistil,however there was no significant difference compared with wild type plants.OsSPE11 was specifically expressed in filaments and lodicules.The expression pattern of OsSPE13 in the pistil was uncertain,but expressed in the stomatal apparatus,cork cells and the epidermal cells between adjacent stomata of the leaves.In the pistils before and after pollination,OsSPE16 was predominantly expressed on the style,ovary wall,integument and it was specifically expressed on the guard cells of the leaves.The expression of OsSPE07,OsSPE08 and OsSPE15 at the organizational level was uncertain.In summary,this research revealed that the first gene(OsSPE05)specially expressed in the stomatal apparatus of the leaves and in the pericycle of the crown root,the second gene(OsSPE06)specially expressed in the wall of the embryo sac and root cap,the third gene(OsSPE11)specially expressed in filaments and lodicules.