Structure Feature And Expression Analysis Of The LEAFY Gene From Hyophila Javanica

The FLORICAULA/LEAFY(FLO/LFY)homologous gene controls flower development,which involved in the flowering process of plants.It plays an important role in flower development regulation networks.At present,most studies on LFY gene are focused on flowering plants,but LFY gene is not unique to flowering plants,and it also exists in non-flowering plants such as mosses.In this paper,Hyophila javanica as materials to clone the homologue of FLORICAULA/LEAFY of cDNA sequence,DNA sequence and promoter were isolated from Hyophila javanica by polymerase chain reaction(PCR),RT-PCR and hermal asymmetric interlaced polymerase chain reaction(Tail-PCR).Based on bioinformatics analysis,the genetic structure,protein sequence,and the analysis of the physical and chemical properties were studied,the construction of the evolutionary tree determines the evolution position of Hyophila javanica.The pThioHis A/LFY prokaryotic expression vectors was successfully constructed and the prokaryotic expression conditions were optimized.Expression Analysis of the LEAFY Gene from Hyophila javanica were studied by real-time quantitative PCR.This study has some scientific significances in elucidating the function of LFY in mosses.The main results of this study are below:(1)Via RT-PCR and RACE-PCR to amplify full sequence of LFY from Hyophila javanica.DNA sequence of LFY includes four exons and three introns,There has a whole length of 2633 bp.Also an open reading frame 1050 bp and 349 coding amino acid sequence are involved in this gene.(2)By analyzing amino acid sequence through the Protparam software,its theoretical relative molecular mass is surmised to be 40389.07 Da and the theoretical isoelectric point is 6.59.As for secondary structure,Hyophila javanica LFY protein has Alpha helix regions,random coil regions and Beta folding regions.Alpha helix regions take up 41.26 % in secondary structure,Beta folding regions take up 3.72 % in secondary structure,and random coil regions take up 55.01 % in secondary structure.And the folded structure of the alpha helical and beta sheet are convinced to be the main composition.(3)The 821 bp of gene upstream regulatory sequences from Hyophila javanica are gained through the method that adopted Tail-PCR technology to clone the promoter sequence of Hyophila javanica genes.The analyzed by Plant CARE showed that it hadTATA-box,CAAT-bos,MYB binding site,light-induced responsive element Sp1 and other cis-acting elements.(4)The pThioHis A/LFY prokaryotic expression vectors was successfully constructed in E.coli BL 21(DE 3)strain.The analysis of SDS-PAGE indicated the presence of recombinant proteins,the prokaryotic expression protein pThioHis A/LFY was a soluble protein.And the pThioHis A/LFY was optimized for expression conditions in E.coli BL 21(DE 3)strain and was determined to be 27 ?,1 mM IPTG induced for 6 h.(5)Detecting LFY gene from Hyophila javanica in sporophyte,gametophyte through method of real-time quantitative PCR,they are all have expression,but its expression level have obvious difference,LFY had the highest amount of expression in the sporophyte.(6)Detection by using the method of real-time quantitative PCR,LFY gene from Hyophila javanica expression level showed that a rise under the condition of drought treatment,at the time of 3.5 h reached the highest expression level,achieving 1.5 times than in control group.