Bioinformatics Identification Of TCP Gene Family In Citrus And Functional Verification Of CsTCP1 Transcription Factor Involved In Fruit Ripening

Fruit ripening is a complex process that involves in many metabolic pathways. Currently, the study onripening mechanismfor non-climacteric fruit is less. So it has an important significance for citrus industry to research fruit ripening-related genes, explore the relationship between themand regulatory mechanism of fruit ripening, and then breed new cultivars with earlier or later ripening time. TCP(TEOSINTEBRANCHEDI/ CYCLOIDEA/PROLIFERATING CELL FACTOR) protein family is a plant-specific transcription factor that involves in the development of plant leaf, flower symmetry, branching of stems and other processes, but few studies on fruit. Previous study indicated that CsTCP1 may participate in the process of fruit ripening by transcriptome sequencing and SSR analysis.In this rearch, we studied TCP family members of citrus both bioinformatics and gene expression level. ThenCsTCP1 was cloned andits functions were analyzed by genetic transformation of tomato. The main results are as follows:1. A total of 20 TCP members were identified in the whole genome database, named CsTCP1~CsTCP20, which weredivided into 2 groups of Class I and Class II. They are distributed on 7 chromosomes except for the first and the fourth chromosome. Sequence alignment found that each of TCP family members has a non-canonical BHLH domain. Most of genes are simple, and have no introns.2. The expression patterns of TCP members in citrus different tissues can be divided into 2 categories, the expression level in group 1 was higher than group 2, and most of genes were highly expressed in leaves, flowers and fruits, while the expression level was low in callus. In addition, the expression levels during the six periods after flowering in ‘Xingjin101’(WT) and its spontaneous late-ripening mutant(MT) were detected by fluorescence quantitative polymerase chain reaction. The relative expression levels of most genes peaked in 215 days after flowering, and there were significant differences between WT and MT. So we speculated that TCP family members may play an important role in the regulation of fruit ripening.3. The primers of CsTCP1-a/CsTCP1-bwere designed and synthesized according its cDNA coding frame sequences. With cDNA of ‘Xingjin 101’ as a template, and the sequence of CsTCP1 was amplified by PCR amplification reaction, then CsTCP1 was cloned through the method of TA technology. We made a sequence alignment between cloned sequence and original sequence to ensure the accuracy of the sequences, which can match completely.Cs TCP1 is located in the nucleus by Subcellular localization. We got transgenic tomato by resistance screening, PCR and qRT-PCR. The results of phenotype observation showed that transgenic plants changed dwarf. In addition, the shape of overexpression fruits was longer than wild fruits, and the veraisionwas earlier in overexpression fruits. The results of qRT-PCR showed that the fruit ripening-related genes were upregulated in transgenic fruits, compared to those in wild fruits, which indicated that CsTCP1 may positively regulate those genes.The TCP gene family members of citrus may be involved in fruit ripening process by analyzing the relative expression levels of genes. Meanwhile, a preliminary study reveal thatCsTCP1 gene may regulate growth of stem, and inhibit to elongation of stem, as well as accelerate fruit ripening, which lay a certain foundation for further researching the gene function.