Screening And Functiaonal Identification Of Switchgrass Dedifferentiation Gene,PvCAL1

Switchgrass(Panicum virgatum L.)is a C4 grass energy plant with a high bioenergy value.It can be used to produce bioethanol replacing petroleum and other fossil fuels,and so it has high bioenergy potential.However,due to the poor dedifferentiation ability and genotype restriction,switchgrass has seriously restricted the progress of genetic transformation research.And hindered the development and utilization of switchgrass.Establishing an efficient genetic transformation system and cultivating the excellent varieties of switchgrass are conducive to expand the bioenergy value of switchgrass and improve its economic benefits.Previously,we obtained the FOX Arabidopsis library by FOX hunting system(Full-length cDNA Over-expression gene hunting system).Screening the switchgrass callus-inducing related genes and study their functions.A detailed information for this research was listed as follow:1 PvCallus1(PvCAL1)is a dedifferentiation gene of switchgrass,it was obtained by phenotypic screening.PvCAL1 was cloned into inducible vector.Subsequently,the resulting construct was transformed into Arabidopsis thaliana.Two inducible homozygous lines,PvCAL1-IOE1 and PvCAL1-IOE3 were obtained.2 Functional domains prediction showed that PvCAL1 contains several domains including Alanine-rich region,WRKY domain and WRKY DNA-binding domain.It is homologous with WRKY,and subcellular localization analysis revealed that PvCAL1 was localized to the nucleus.These data indicated that PvCAL1 is a transcription factor.Tobacco light energy conversion efficiency experiment demonstrated that these three domains are the key part to result in withered leaves,photosystem Ⅱ’s maximum photosynthetic efficiency is reduced.3 The inducible homozygous line PvCAL1-IOE1 was used for transcriptome analysis.Transcriptome analysis showed that the numbers of differentially expressed genes(DEGs)in 8 h vs CK were more than that of 16 h vs CK,which were 8685 and 4586.Respectively,the up-regulated genes from DEGs were 3959 and 2306 and the down-regulated genes were 4726 and 2280.The DEGs mainly includes:(1)3-ketoacyl-CoA synthetase family proteins(2)transcription factors(3)auxin response factors(4)zinc finger proteins(5)dormancy/auxin-related family proteins(6)response stress related genes.4 GO enrichment analysis of DEGs:For biological processes,the DEGs of 8 h vs CK were involved in metabolic process、biological regulation and response to stimulus.For cell component,mainly included cell、cell part and organelle.For molecular function,they mainly included structural molecular activity、molecular function and the structural composition of ribosome.For biological processes,the DEGs of 16 h vs CK were mainly involved in the response to stimulus.For cell component,the DEGs of 16 h vs CK was similar as that of 8 h vs CK.For molecular function,they were enriched in DNA-binding transcription factor activity.The KEGG pathway analysis indicated that the DEGs were mainly involved in metabolic pathways、biosynthesis of secondary metabolites pathway and plant hormone signal transduction.The LATERAL ORGAN BOUNDARIES DOMAIN(LBD)gene family was showed a significant changes in PvCAL1 overproducing plants,which indicated that LBDs may be involved in the dedifferentiation at the downstream of PvCALl.This is verifired by qRT-PCR.Furthermore,we employed ChIP approache to confirmed that PvCAL1 could activate LBDs expression through the direct binding to the promoter w-box region of the LBD genes.5 Using yeast two-hybrid technique to screen PvCAL1 interaction proteins,36 positive clones were obtained.Three genes were confirmed by the following experimants,which are AT3G12050、AT3G26900 and AT5G47430.Using immunoprecipitation-mass spectrometry to screen PvCAL1 interaction proteins,234 proteins were identified.Through functional annotation,around 30 proteins might be the PvCAL1 interactors.