Cloing And Expression Pattern Analisis Of Lemon UBE2A And UBE2I Homologous Genes

The ubiquitin proteasome pathway is closely related to gametophyte self-incompatibility,which involves in the process of self-incompatibility in many flowering plants by degrading some specific proteins.Ubiquitin binding enzyme UBE2 plays an important role in the ubiquitin proteasome pathway.The homologous gene fragments of UBE2A and CUBE2I were screened from flower transcriptional data of self-incompatibility of Xiangshui lemon in previous study.In order to understand the characteristics of the genes,we carried out this study.Xiangshui lemon(self-incompatibility)and White flower lemon(self-compatibility)were used as materials in this study.According to the homologous gene fragments of UBE2A and UBE2I obtained from lemon flower transcriptome,The total length of the open reading frame of and DNA of UBE2A and UBE2I homologous gene of Xiangshui lemon and White flower lemon were obtained by homologous cloning method.T Then the full length of cDNA and DNA of the UBE2I gene were analyzed by bioinformatics and its expression pattern was explored.The subcellular localization of protein was studied.The main results were as follows:1.The results of sequence analysis showed that,The total length of the open reading frame of UBE2A homologous gene in Xiangshui lemon was 564 bp,The total length of DNA was 1691 bp.the length of UBE2A homologous gene in White flower lemon was 315 bp,which is 887 bp shorter than that of Xiangshui lemon.The ORF length of UBE2I ubiquitin ligase of Xiangshui lemon is 483 bp.There only one nucleotide difference in the total length of open reading frame in this two cultivars.The total length of DNA of the UBE2I gene was 2267 bp.2.Bioinformatics analysis of UBE2A and UBE2I homologous genes was carried out by using biological online website and software.The homologous gene of UBE2A contained 7 exons in Xiangshui lemons,the lengths of which were respectively 44 bp,81 bp,115 bp,52 bp,129 bp,105 bp,and 38 bp.And four introns were166 bp,145 bp,261 bp,366 bp,182 bp,7 bp.The total length of the open reading frame of the UBE2A homologous gene in Xiangshui lemon is 564 bp,encoding 187 amino acids and consisting of 20 amino acids.The three kinds of amino acids with the highest content were:Ser21(21,11.2%),Asp18(18,9.6%),Glu16(16,8.6%).The theoretical molecular weight was 21200.77784 Daa and the isoelectric point was 4.29.The tertiary structure of the UBE2A ubiquitin ligase protein in Xiangshui had 6 a-helix,4 ?-folds and 10 random coils.The predicteion of ubiquitin site of the protein showed that the 149th(score 0.85)amino acid of the amino acid sequence of UBE2A in Xiangshui lemons had a high possibility of ubiquitization.,the 14th(score 0.72)and 148th site(score 0.79)amino acids had a middle possibility of ubiquitization.the 133rd(score 0.66)amino acids had a low possibility of ubiquidization.The whole peptide chain was hydrophobic and was not secretory protein with no transmembrane structure.The UBE2A homologous gene in White flower lemon contains four exons,the length of which were:44bp,81bp,115bp and 75bp.And three intons were:166bp,145bp,261bp.In the fourth intron of DNA of Xiangshui lemon,The 885th-887th base is TGA,which is the termination codon of UBE2A in the White flower lemon.The total length of the open reading frame of UBE2A homologous gene in White flower lemon was 315 bp,which encodes 104 amino acids and consists of 20 amino acids.The five kinds of amino acids with the highest content were:Ser(11,10.6%),Ile(9,8.7%),Val(8,8.7%),Lys(8,8.7%)Pro(8,8.7%)The tertiary structure of the UBE2A ubiquitin ligase protein has three a-helices,six ?-folds and seven random coils.The theoretical molecular weight is 11845.73124 and the isoelectric point is 5.13.The whole peptide chain is hydrophilic.The results of ubiquitin site prediction showed that there was no possibility of ubiquitin in the amino acid sequence of UBE2A homologous gene in White flower lemon,and the conserved domain of the homologous protein of Lemon UBE2A gene was:UBCc superfamily.The Xiangshui lemon and White flower lemon UBE2I homologous genes containing 5 introns were,the length were:70 bp,81 bp,157 bp,112 bp,63 bp.And 4 exons length were:77 bp,1145 bp,110 bp,452 bp.And there were two structures similar to the promoter TATAA frame in the intron sequence of 2st and 4st introns.Perhaps there were closely related with intron function.The total length of the open reading frame of lemon UBE2I homologous gene was 483bp.encoding 160 amino acids,There is no difference in the composition and content of amino acids,he content of three the highest amino acidswere:Pro(16,10%),Gly(14,8.75%),Leu(13,8.125%).Its molecular weight is 17981.47444 Da and isoelectric point of the conserved domain of UBE2I/homologous gene protein 7.64.And the conserved domain of the Lemon UBE2I protein was:UBCc superfamily.The three stage structure have 4 a-helixs,7 ?-folds and 10 random coils.The results of ubiquitin site prediction showed that 28th amino amino acid sequence(Score 0.70)of UBE2I had a middle possibility of ubiquitination,The 111st amino acids(Score 0.64)amino acids had a low possibility of ubiquidization.The prediction of SUMO modification site indicates the possibility of SUMO modification at the 28th and 54th amino acids of UBE2I.The peptide showed hydrophobic,without signal peptide and transmembrane structure.3.Using SYBR real time fluorescence quantitative PCR to analyze the expression of lemon UBE2A and UBE21 gene in different tissues and Xiangshui Lemon at different time intervals after self-pollination and hybridization.The results showed that UBE2A and UBE2I homologous genes were expressed in 11 tissues of Xiangshui lemon.The expression of UBE2I in pollen and pollen was higher than that in stem,receptacle and ovary,and the expression of UBE2I in pollen and filaments was higher than that in stem,receptacle and ovary.The expression of UBE2A in pollen was the highest,and the expression in receptacle and old stem was relatively low.The analysis of UBE2A expression in stigma at different time points after self-crossing of Xiangshui lemon and after crossing showed that after self-pollination 0h-10h,the expression of UBE2A was relatively low.The expression level of 10 h was about 2.5 times higher than that of cross pollination,and decreased at 10-15 h after self-pollination,and decreased to Oh at 15 h after self-pollination.The expression of 1-2d increased slightly after self-pollination,and reached the maximum at 2 days after self-pollination.The expression of UBE2I in stigma was 25 times higher than that after hybridization.The results showed that after self-pollination,the expression of UBE2I in stigma was higher than that of self-pollination 0-10 h.The expression level of 10 h was 4.5 times higher than that of cross pollination,and decreased at 10-20 h after self-pollination,and the expression level decreased to 0 h at 20 h after self-pollination.After 1 day of self-pollination,the expression level increased continuously,and at 4 days after self-pollination,the expression level was about 5.5 times of that after hybridization.4.The constructed transient expression vectors P1300-GFP-UBE2A and P10300-GFP-UBE2I were transformed into tobacco leaves for transient expression.After co-culture for 1-2 days,the expression was carried out under a multi-photon laser confocal microscope.The distribution of the target protein in the cells was determined by the fluorescence signal of the green fluorescent protein GFP.The results showed that the green fluorescent protein was mainly distributed in the nucleus of tobacco leaf cells transferred to P1300-GFP-UBE2A.The results indicated that UBE2A homologous gene expressed protein in cytoplasm and was transported to the nucleus to play its role,which may play a role of transcription factor.In tobacco leaf cells transformed with P1300-GFP-UBE2I,green fluorescent protein mainly distributed in the cell membrane.5.Yeast recombinant plasmids pGADT7-UBE2A,pGADT7-UBE2I and pGBKT7-F-box were constructed,and the self-activation and toxicity of pGBKT7-F-box were detected.It was proved that the recombinant expression vector pGBKT7-F-box had no self-activation and toxicity.