Dvide Differentiation Process Of Switchgrass Spike Into Stages And Identify Tify Gene Family In Switchgrass

Switchgrass(Panicum virgatum L.)is a perennial C4 herbaceous plant.It has been extensively studied internationally as a model energy crop in recent years,however,little research has been done on the characteristics of reproductive growth in switchgrass.Spike differentiation is a turning point of vegetative growth and reproductive growth,and it is of great significance to correctly judge the critical period of spike differentiation for crop growth and development regulation,cultivation management and breeding research.But the study on the stage of switchgrass spike differentiation has not been carried out at home and abroad.Reproductive growth is an important stage of individual development,which is jointly regulated by external environment and internal factors.TIFY as transcription factors or interacting with transcription factors play important regulatory role in plant growth and development,hormone and stress response and other processes,but there have been no reports on TIFY gene family in switchgrass.In order to explore the differentiation law of switchgrass spike and the function of TIFY gene family in reproductive process of switchgrass,this experiment have been carried out from the following aspects: we observed the changes of tissue morphology during the spike differentiation of switchgrass,and recorded the corresponding plant morphology in field for identifying the differentiation process;we identified members of the TIFY gene family in switchgrass genome and also analyzed the evolutionary relationship,structural and expression characteristics of each member;homologous cloning was performed on two members associated with inflorescence development in rice which belonged to JAZ subfamily,moreover,we analyzed its molecular characteristics and subcellular localization and also constructed overexpression vectors of the target genes.This essay has studied the inflorescence development of switchgrass through morphological observation and molecular regulation that laid a foundation for the exploration of reproductive growth in switchgrass.The main results are as follows:1.The spike differentiation in switchgrass was a slow-fast-slow process which could be divided into 9 consecutive stages: the differentiation stage of bract primordia,the differentiation stage of branch primordia,the differentiation stage of spikelet,the differentiation stage of floret primordia,the differentiation stage of stamen and pistil primordia,stamen connective formation stage,anther quarters stage,anther elongation stage,feathery stigma formation stage.What’s more,we could identify the differentiation progress by the corresponding plant morphology of each stage: leaf age/leaf age remainder and visible leaves number could be used to judge the stage of spike differentiation from the differentiation stage of bract primordia to anther quarters stage,meanwhile,the length and morphology of inflorescence and spikelet could be used to identify the process of spike differentiation from anther elongation stage to feathery stigma formation stage.Generally speaking,the spike differentiation in switchgrass took about 3 months when the plant developed from jointing stage to blooming stage.The florets of inflorescence bloomed from up to down,and the flowering stage continued about 3-4 weeks.2.A total of 48 PviTIFY were identified by genome-wide identification in switchgrass that belonged to TIFY(1),ZML(7)and JAZ(40)subfamilies.PviTIFY distributed on all chromosomes except chromosome 3 and half of them were located on chromosome 9.Moreover PviTIFY could be divided into 5 groups according to its evolutionary relationship and structural characteristics: JAZⅠ、JAZⅡ、JAZⅢ、TIFY、ZML,and the members of each group had similar genetic structure and conserved motif distribution.What’s more,there were various regulatory elements related to stress and hormone response in promoter region of TIFY genes in switchgrass and quantitative experiments confirmed that the members of PviTIFY10 responded strongly to treatment of JA and stresses of salt and drought.Moreover,some members of the JAZ subfamily,especially PviTIFY10 b,was highly expressed in the development process of inflorescence and roots.Based on the above results,it was speculated that TIFY gene family might play a role in many aspects such as hormone signal regulation,stress defense response and process of growth and development in switchgrass.3.We have successfully cloned the homologous genes of OsJAZ1 and OsTIFY10 b which was PviTIFY3 a and PviTIFY10 b respectively.What’s more,the conserved motifs,evolutionary characteristics,physicochemical properties and secondary structure components of the protein sequences which two genes ecoded were similar.Transient expression of fusion protein PviTIFY3a-GFP indicated that PviTIFY3 a could locate in nucleus and membrane simultaneously.However,the subcellular localization prediction software predicted that PviTIFY10 b was most likely located in the cytoplasm.At present,overexpression vectors of the two target genes have been successfully constructed.