Cloning And Functional Analysis Of CbuATX2 Gene From Catalpa Bungei

Flowering is a complex life process regulated by both internal genetic and external environmental factors of higher planta,and it was also a symbol of the genetic and reproductive capacity when the planta enter into reproductive stage.In this paper,the CbuATX2 gene in Catalpa bungei was cloned,and analyzed by bioinformatics.The interaction of CbuATX2 with RBL and THX protein,and the biological characteristics of CbuATX2 in transgenic Arabidopsis thaliana were also studied.The main results were as follows:(1)The full-length CDS sequence of CbuATX2 gene in Catalpa bungei was 1554 bp,encoding 517 amino acid residues,and was an unstable hydrophilic protein without signal peptide and transmembrane structure.CbuATX2 had two conserved domains,which belonged to Pro-Trp-Trp-Pro(PWWP)superfamily.The two domains could bind with lysine and histone methylated on H4K20me.Therefore,the domains were proved to be a methyllysine recognition motif.The phylogenetic tree analysis showed that CbuATX2 had higher homology with other EgATX1-like proteins.CbuATX2 gene promoter contained CAAT,TATA box and other basic elements,as well as light response,low temperature response,auxin response and other elements.These indicated that CbuATX2 gene was closely involved with light response,and might also participate in the process of environmental stress response;Protein interaction prediction analysis displayed that there were 10 proteins interacting with CbuATX2.Therein,4 proteins(HTR4,AT1G13370,AT1G75600,HTR11)belonged to histone superfamily,besides WDR5a(h3k4 histone methylase),ATXR5(histone-lysine N-methyltransferase),RBL(transducing protein),TRO(protein Trauco),P1E1(chromatin remodeling protein),THX(thioredoxin x).RBL had stronger protein interaction with CbuATX2 in the prediction.(2)The yeast expression vector pGBKT7-CbuATX2 was constructed,and the results explained that CbuATX2 protein had no self-activating activity and no toxic effect on Y2HGold strain;The yeast two hybrid experiment illustrated that CbuATX2 weakly interacted with RBL and THX protein;It was speculated that CbuATX2 might coordinate RBL and THX genes to regulate plant flowering and growing development.(3)pROKⅡ-CbuATX2 plant overexpression vector was constructed and transformed into Arabidopsis thaliana by inflorescence staining.PCR results revealed that CbuATX2 gene had been integrated into Arabidopsis genome.There was no significant difference in seed germination rate between transgenic lines and wild type WT,which indicated that overexpression of CbuATX2 gene had no harm to seeds;Flowering stage of transgenic lines was later than that of WT,and overexpression of CbuATX2 gene could delay flowering.Meanwhile,the numbers of rosette leaves in transgenic lines was more than that of WT at flowering stage;Plant height of transgenic lines was significantly higher than that of WT,but pod length was no significantly difference with WT.The root length of transgenic lines was significantly shorter than that of WT,suggesting that CbuATX2 gene also had a certain effect on regulating plant height and root length of Arabidopsis;The rosette leaf area of transgenic lines increased,and had a significant difference with that of WT.The growth of transgenic and WT lines was.The rosette diameter of transgenic lines was significantly larger than that of WT through continuous observation These results indicated that CbuATX2 might significantly promote the rosette leaf growth in Arabidopsis.(4)RT-qPCR technology was used to analyze the expression level of other endogenous gene in transgenic Arabidopsis.The relevant results showed that CbuATX2 gene influenced the expression of CO,FT and CCA1 genes in Arabidopsis.CbuATX2 gene had a negative regulatory effect on the expression of CO,FT genes,but a positive regulatory effect on the expression of CCA1 genes.