Cloning And Expression Analysis Of MYB-related Transcription Factor Gene In Longan

Longan(Dimocarpus longan Lour.)is a typical subtropical fruit.In recent years,due to the influence of warm winter climate,the problem of flower formation or bud differentiation of Longan has become increasingly prominent.MYB-related gene family sequences were obtained based on transcriptome third-generation sequencing,longan database and bioinformatics analysis.In this study,the MYB-related homologous gene of longan was cloned and its physical and chemical properties were further analyzed.The expression of MYB-related family members in different tissues and annual cycles was explored by using real-time fluorescence quantitative q RT-PCR technology,and the genes related to longan flowering were preliminarily screened.In order to further explore the function of this gene,it lays a foundation for improving the gene function of MYB-related transcriptome gene family expressed in longan,and provides a molecular basis for the regulation of flower formation in longan.The specific experimental results are as follows:(1)The laboratory obtained the transcriptional sequencing data of the thirdgeneration of Longan by testing the longan ‘Sijimi' material,and obtained the full length of longan transcriptional gene sequence through bioinformatics analysis combined with longan database.Using the leaves of ‘Sijimi' longan as experimental materials,10 homologous genes of MYB-related gene family of Longan were cloned,providing sequence information of MYB-related gene of Longan for identifying the biological process in which members of this family participated in the next step.(2)Through further comparative analysis,it was found that the sequences of MYB-related family members were quite different from each other,among which genes MYB6,MYB14 and MYB23 all contained LKDKWRN motif,belonging to the TBP-like subfamily of MYB-related gene family.Genes MYB133,MYBS3,MYB1R1 and REVEILLE-8 all contain SHAQKYF motif,and these four genes can be classified as CAA1-like R-R subfamily in the MYB-related gene family.No conserved amino acid sequence of MYB duplication region in genes SWR1,ADA2 b and MAMYB of the MYB-related gene family has been found.Through online analysis software MYB-related transcription factor family,bioinformatics analysis found that MYB-related ORF length difference between the members,most of the sequence length between 830 bp to 1050 bp,SWR1 and ADA2 b is bigger,its molecular weight is higher,also the highest molecular weight ADA2 b,its value is 59390.23 D,the rest of the sequence of the molecular weight size difference is not obvious,both up and down in the 33000 D.Theisoelectric point of gene MYB1R1 was 6.99,which was a neutral protein;the isoelectric point of gene ADA2 b was 6.76,which was an acidic protein;the isoelectric points of other members were all higher than 7.0,indicating that most of them were alkaline proteins.Except for the genes REVEILLE-8,MYB6 and MYB133,most of the MYB-related genes were unstable proteins.Members of the MYB-related family are all hydrophilic proteins.The physical and chemical properties of most members are similar except for a few.Bioinformatics analysis showed that the proteins encoded by MYB-related family members were all non-transmembrane proteins.The results of subcellular localization prediction showed that the gene MYB14 was located in the cytoplasm,the gene MYBS3 and the gene REVEILLE-8 were located in the mitochondria,and the other members were all located in the nucleus.Phylogenetic tree shows that the phylogenetic tree of proteins MYB6,MYB14 and MYB23 of the TBP-like subfamily belongs to the same branch,which is closely related to citrus.It is known that the sequences of the members of the TBP-like subfamily are extremely conservative.CAA1-like R-R subfamily gene MYB133,REVEILLE-8,MYB1R1 and MYBS3 were different from other species,among which gene MYBS3 belonged to CAA1-like R-R subfamily,but in the analysis of phylogenetic tree species,it was in the same branch as the members of the TBP-like subfamily,and was most closely related to citrus.It was speculated that among the five subfamilies of MYB-related transcription factors,the CAA1-like R-R subfamily and the TBP-like subfamily functionswere the closest.(3)Through real-time fluorescence quantitative analysis of the expression of MYB-related members in different tissues of longan,it was found that genes MYB14,SWR1 and MAMYB were mainly expressed in flower buds and leaves,and it was preliminarily speculated that the function of this gene might be related to the formation of longan into flowers.Both UK and MYBS3 were highly expressed in longan flower buds,but both genes were highly expressed in the leaves of ‘Shixia'.The difference of expression of REVEILLE-8 was found in the same tissue of longan of ‘Sijimi' and ‘Shixia'.At the same time,through real-time fluorescence quantitative analysis of the expression of MYB-related members in a natural annual cycle of longan,it was found that the expression levels of genes MYB14,SWR1,MAMYB and MYBS3 in January to April were significantly higher than those in other months,and this month was the stage of longan's growth and bud differentiation,suggesting that the gene was related to the process of longan's flowering.The study on the different tissues and annual cycles of the two longan varieties of ‘Sijimi' and ‘Shixia' through spatio-temporal expression mode found that the REVEILLE-8 was highly expressed in January,may,July and December of ‘Sijimi' longan,but this gene had no specific expression of the varieties.Gene UK was highly expressed in February,April,June and October in longan of‘Sijimi',and the expression levels were significantly higher than those in‘Shixia' longan.Considering that ‘Sijimi' longan flowers all the year round,itwas preliminarily speculated that this gene might be related to ‘Sijimi' flowering all the year round.