Bioinformatics identification and expression analysis of the Citrus TCP gene family
[Objective] To search TCP (TEOSINTE BRANCHEDI/CYCLOIDEA/PROLIFERATING CELL FACTORI) family members from the citrus genome,analyze their sequence characteristics and phylogenetic relationships,then explore their expression levels in different tissues of citrus and other functions of the TCP family genes,which occur during fruit ripening,and are very important for breeding new citrus cultivars with earlier or later ripening times.The TCP protein family is a specific transcriptional factor family in plants.The studies on A.thaliana,Antirrhinum and maize show that the TCP family members are involved in many physiological and biochemical processes,such as the development of leaves,flowers bilateral symmetry and stem branching,which are rarely reported in citrus,especially in fruit ripening.[Methods] Search citrus TCP family members and their expression levels in four different citrus tissues (callus,flower,leaf and fruit),using the Sweet Orange Database and a local Blast.The structure,chromosome location,phylogenetic relationship and conserved domains were predicted through using the bioinformatics method.The primers of CsTCP1 (Cs2g0808.1) and CsTCP3 (Cs2g06240.1) were designed and synthesized according to their cDNA coding frame sequences.It is necessary to obtain the sequences of CsTCP1 and CsTCP3 by employing a PCR amplification reaction with cDNA of ‘Xingjin101 ’s pulp as a template,and making a sequence alignment between the cloned sequence and original sequence in order to ensure the accuracy of the sequences;with ‘Xingjin101 ’ (WT) and its spontaneous late-ripening mutant (MT) as test materials,fruit samples were harvested at six development stages (139,166,182,199,215 and 232 days after flowering,respectively) from September to December,and total RNAs were extracted,and reverse transcribed.The expression levels of TCP family members in the six periods were determined by means of fluorescence quantitative PCR,and the genes which were differentially expressed between WT and MT and differentially expressed in six different developmental stages in ‘Xingjin101’and its spontaneous late-ripening mutant were analyzed.[Results]The sequence lengths of CsTCP1 and CsTCP3 are 1515 and 954 bp respectively,and they can be matched completely with the original sequence,which shows the accuracy of their sequences.A total of 20 members were identified in the whole genome database,named CsTCP1-CsTCP20,which were divided into 2 groups of Class I and Class Ⅱ.The length ranges for their amino acids are from 174 aa to 577 aa,and the ranges of their isoelectric points are 4.44-9.47.CsTCPs are distributed on 7 chromosomes,except for the first and the fourth chromosomes,and each of the TCP family members have a conserved domain which contains about 60 amino acids,that is a non-canonical BHLH (basic-helix-loop-helix) domain;and most of the genes had simple structures with no introns.The expression patterns of the TCP members in the different citrus tissues can be divided into 2 categories,and we find that the expression level in group 1 is higher than group 2,and most of the genes are highly expressed in leaves,flowers and fruits,while the expression level is low in callus.Meanwhile,the expression levels are different during fruit ripening between the ‘Xingjin 101’ sweet orange and its spontaneous late-ripening mutant.The relative expression levels of most genes peak in 215 days after flowering,and there are significant differences between WT and MT.The expression trends of the genes in ‘Xingjin101 ’ sweet orange and its spontaneous late-ripening mutant are divided into five categories,A,B,C,D,and E.Few differences and lower expression levels between WT and MT are found in category A.The expression levels of genes in category B show significant differences between WT and MT,which reach their maximum at the fifth stage in WT,while they remain steady during the whole ripening stages in MT.The expression levels of genes in category C are higher in the spontaneous late-ripening mutant than the Wide type in the early stages,while they are contrary in the late stages.The expressions of genes in category D are higher in ‘Xingjin 101’ than its spontaneous late-ripening mutant for the entire period.The expression levels of genes in category E are the reverse,showing a higher expression level in MT than WT during the whole period,and there are also great differences between them.[Conclusion]We identified 20 citrus TCP family members,which contain a TCP conserved domain.The structural similarities of the CsTCPs genes infer that a evolutionary relationship exists between them.Using a phylogenetic analysis,we found that there is a high homology between CsTCP9 and AtTCP6,AtTCP18,which is the same as CsTCP1 and AtTCP2.Combining these facts with previous studies,it becomes evident that these genes may be involved in the development of leaves.Meanwhile,the results of applying qRT-PCR show that CsTCP genes may participate in the development of leaves,flowers,fruits,and play an important role in the regulation of fruit ripening.According to the above research results,the functional verification of CsTCPs will be an area to focus on future research.
国家科技期刊平台
NSTL
万方数据
维普
