Function Analysis Of Three Key CesAs In Sugarcane

Sugarcane(Saccharum spp.)is the main raw material for the production of sucrose worldwide.It is also known as the C4 crop with the highest acclimated biomass.Sugarcane contains rich carbohydrates such as cellulose and is an ideal energy crop for biofuels production.Cellulose synthase(CesA)is the key enzyme for plant cellulose synthesis.Therefore,it is of great theoretical significance and application value to study the molecular mechanism and the function of SsCesAs.In this stduy,we used comparetive genomics and the model plant transgeneitc system to study the funcion of the sugarcane primary cell walls and secondary cell wall synthesis related genes,includingSsCesA3,SsCesA9 and SsCesAll.The main results are as follows:(1)Used comparative genomics analysis,we identifiedll members ofcellulose synthase A gene family in Saccharum spontaneum.A phylogenetic tree contained the CesA genes from six plant speices including Arabidopsis thaliana,Vitis vinifera,Oryza sativa,Zea mays,Sorghum bicolor andSaccharum spontaneum,were constucted for studying the gene evolution of CesA in plants.Based on the RNA-seq expressional analysis and combined with previous studies in Arabidopsis for the CesA family,we assumed that ScCesA3 and ScCesA9may play important roles in primary cell wall formation,and ScCesAll may be indispensable for secondary cell wall formation.(2)At the stage of preliminary exploration for Saccharum spontaneum tissue culture conditions,by adjusting the concentration of 2,4-Dichlorophenoxyacetic we promoted the differentiation of callus,and effectively slowing down the browning of the callus by adding PVP(polyvinyl pyrrolidone).On the basis of improving the culture system,we finally obtained the aseptic seedling of Saccharum spontaneum.(3)To explore the expression of three key SsCesAs,we constructed the promoter expression vector:pSsCesA3::GUS,pSsCesA9::GUS and pSsCesAll::GUS to transform the tissue culture of Saccharum spontaneum.GUS staining results showed that:the expression signals were detected in the tissue culture of Saccharum spontaneum,which means the promoter of these three key SsCesAs can express normally.However,their specific expression in different tissues needs further experimece.(4)To explore the function of these key SsCesAs,we constructed overexpression vector p35S::SsCesA3,p35S::SsCesA9,p35S::SsCesAll and then transformed to Arabidopsis thaliana(Dicotyledonous).The expression levels of SsCesAs in transgenic plants and wild-type plants were detected by Q-PCR,and the results indicated that SsCesAs expressional levels have a significant increases for transgenic plants.And through the observation of fluorescence intensity in stem by frozen section staining,proved that ScCesA9 and ScCesAll were related to the synthesis of cellulose.(5)For the further study of these key SsCesAs,we constructed two overexpression vector and then transformed to Japonica(Monocotyledons).Thirty eight and forty transgenic plants were obtained for target genes ScCesA9 and ScCesA11.The expression levels of SsCesAs,fluorescence intensity of cellulose in cell wall and the cellulose content confirmed that SsCesA9 and SsCesAll were related to cellulose synthesis for primary cell wall and secondary cell wall respectively.