柑橘TCP家族生物信息学及表达谱分析

Bioinformatics identification and expression analysis of the Citrus TCP gene family

【目的】分析柑橘TCP蛋白家族成员的序列特征和系统进化关系,探究TCP家族成员在柑橘果实发育中的功能。【方法】通过生物信息学方法分析柑橘基因组中TCP家族成员的结构、染色体位置和系统进化关系。以‘兴锦101’(WT)及其晚熟芽变(MT)为试材,利用TA方法克隆柑橘TCP家族2个成员Cs TCP1和Cs TCP3,通过q RT-PCR测定TCP家族成员在‘兴锦101’及其晚熟芽变果实不同发育时期的相对表达量。【结果】克隆的2个基因Cs TCP1和Cs TCP3与原序列比对结果相似性达到100%;柑橘全基因组共编码20个TCP转录因子成员,分布在7条染色体上,都含有非典型的BHLH结构域,系统进化关系结果将Cs TCPs分为2个亚家族:Class I和Class II;多数基因结构简单,没有内含子;同时,定量结果表明Cs TCP基因在不同组织中的相对表达量存在差异,且被聚为2类;此外,在‘兴锦101’及其晚熟芽变果实不同发育时期中同样差异表达,多数基因相对表达量在花后215 d达到峰值,且在该时期2者间差异较显著。【结论】获得20个柑橘TCP家族成员基因,表达量分析显示TCP家族成员基因可能在柑橘的生长发育过程中起到重要的作用,为今后柑橘育种工作奠定了一定的理论基础。

【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 Cs TCP1（Cs2g0808.1）and Cs TCP3（Cs2g06240.1）were designed and synthesized according to their c DNA coding frame sequences. It is necessary to obtain the sequences of Cs TCP1 and Cs TCP3 by employing a PCR amplification reaction with c DNA 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 Cs TCP1 and Cs TCP3 are 1 515 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 Cs TCP1-Cs TCP20, 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. Cs TCPs 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‘Xingjin101＇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‘Xingjin101＇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 Cs TCPs genes infer that a evolutionary relationship exists between them. Using a phylogenetic analysis, we found that there is a high homology between Cs TCP9 and At TCP6, At TCP18, which is the same as Cs TCP1 and At TCP2. 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 Cs TCP 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 Cs TCPs will be an area to focus on future research.