Genome-wide identification of TALE family members in walnut
Sixty-nine TALE gene family members were identified from the walnut genome sequence, which were encoded by twenty-seven genes and located on twenty-five scaffolds. As can be seen from Table 1, the length of the encoded amino acid sequence is between 251-550aa, of which JrTALE55 encodes the shortest amino acid, and JrTALE16-JrTALE22 has the longest; the molecular weight of the protein is 28666.7-93611.11 Da, and the isoelectric point is 4.87-9.77. ProtComp 9.0 online software was used to perform subcellular localization analysis on the predicted protein. The results showed that JrTALE was located in the nucleus. As shown in Additional file 2: Table S1.
Phylogenetic analysis of TALE gene family
Based on MUSCLE multiple sequence alignment, the MEGA6.0 software was used to construct a neighbor-joining (NJ) phylogenetic tree for the amino acid sequences of 69 walnuts TALE proteins, 33 Arabidopsis TALE proteins, and 21 grape TALE proteins (Fig. 1). Based on the phylogenetic tree and previous studies, all members of the TALE gene family were divided into two subfamilies KNOX and BELL according to different domains. The distribution of JrTALE proteins among different subfamilies was as following: KNOX (14) and BELL (55), In each evolutionary branch, there were multiple branches from the same species, which may be caused by gene replication events, and the lack of representatives of certain species may be related to gene loss events in the family. In the KNOX subfamily, JrTALE35 and grape GSVIVT01007715001 were orthologous genes; In the BELL subfamily, GSVIVT01009779001 and JrTALE40 were orthologous genes.
Exon-intro Structure Analysis of TALE family members in walnut
The gene structure of JrTALE family members was analyzed online by GSDS web server (Fig. 2). The results showed that there were evident differences in the number of exons among all family members, with a maximum of 6 exons. Only JrTALE55 contains 3 exons; most family members contained four exons. JrTALE55, JrTALE26, JrTALE27, JrTALE36, and JrTALE37 contained 3’ UTR (untranslated regions), which helped to bind microRNAs and regulate the expression of transcription factor proteins. the gene structure was combined with the clustering of the phylogenetic tree. the differences in gene structure between each subfamily were small, the result indicated that the structural similarity of the same subfamily was high.
Conserved motifs analyses
Conservative motif analysis can reveal the diversity of TALE protein and its functional differentiation in walnuts. family members containing the same motif may have similar functions; family members contained different motif elements suggesting that may have undergone functional differentiation and have other special functions. the MEME online tool was used to predict the conserved motifs of walnut TALE family members (Fig. 3). All JrTALE members contained motif 1, the results showed that it was highly conserved in the TALE gene family; motif 9 was highly conserved at the N-terminus in the KNOX subfamily. Except JrTALE55, all BELL subfamily members had seven common conserved motifs were motif8, motif5, motif2, motif4, motif3, motif1, motif7, respectively. The phylogenetic tree shows that closely related members of the TALE family contain the same conserved elements, however, the same subfamily has a proprietary conserved motif, indicating that the same subfamily has similar functions.
Cis-elements prediction in the promoter of JrTALE gene
In response to plant hormones, stress factors and light response, the cis element in the promoter region affects gene expression to regulate plant development and adaptability to environmental changes. In order to obtain more information about the function of the TALE gene in walnuts, 26 JrTALE gene promoters were analyzed using the online software PlantCARE (of which the JrTALE55sequence was too short to be analyzed). The cis-elements were divided into four main subgroups: stress-responsive, hormone-responsive, light-responsive, and MYB binding sites (Fig.4). Most genes were involved in the light-response type promoter (52.51%), followed by hormones (27.41%) and other stress-related genes (13.51%). Among 26 JrTALEs, eleven of them possesed MYB binding sites. In addition, we found that there were abundant light-responsive cis-elements in the JrTALEs, especially in the JrTALE41 gene with a number of 19.
Gene Expression Analysis
Based on RNA-seq data, we used the FPKM values of 27 JrTALE genes to investigate the expression profile of JrTALE family genes. (i) Comparison of JrTALE gene expression levels in female flower buds and leaf buds (Fig. 5A). JrTALE36-37 and JrTALE46-53 were highly expressed in female and male flower buds, JrTALE29-31, JrTALE55, JrTALE32-33, and JrTALE54 were lowly expressed in female and male flower buds, and the expression levels of JrTALE41 and JrTALE42 in female flower buds were higher than male flower buds. (ii) The expression levels of JrTALE gene before, during, and after transformation of female flower buds (FB-1 / FB-2 / FB-3) and before, during, and after transformation of male flower buds (MB-1/ MB-2 / MB-3) were compared (Fig. 5B). JrTALE55 was not expressed in FB; JrTALE8-12, JrTALE46-53 and JrTALE14-15 were highly expressed in FB-1 and FB-3; JrTALE23-25, JrTALE55, JrTALE29-31 were lowly expressed in female and male flower buds; JrTALE14-15, JrTALE41 and JrTALE42 were differentially expressed in FB-1, FB-2, FB-3, MB-1, MB-2, MB-3.
Verification of JrTALE expression pattern by qRT-PCR
Based on this data comes from the analysis results of JrTALE expression pattern, six genes (JrTALE4-5, JrTALE8-12, JrTALE14-15, JrTALE23-25, JrTALE36-37, JrTALE46-53) were further analyzed to detect their expression patterns in different tissues and different development stages using qRT-PCR (Fig. 6). Of these, the expression levels of JrTALE4-5, JrTALE23-25, JrTALE36-37 and JrTALE46-53 were the highest in the male flower buds, consistent with those of the RNA-seq data; As for flower bud transition periods, three genes (JrTALE4-5, JrTALE36-37 and JrTALE46-53) were up-regulated in FB / MB-3 than that in FB / MB-2; Moreover, At different developmental stages, the expression of JrTALE14-15 in female flower buds was higher than that of male flower buds; The suggest that this gene should work as the candidate gene for female and male flower buds difference expression in walnut (Fig. 6).
Protein interaction network of the JrTALE family
The function of Arabidopsis TALE has been studied in depth, but the relationship of walnut TALE protein interaction has not been reported. Therefore, we can construct a walnut TALE protein interaction network through the interaction of Arabidopsis TALE protein. twenty-one homologous proteins and five predicted functional proteins were obtained, and the walnut TALE protein corresponding to the Arabidopsis TALE protein was listed below it (Fig. 7). It can be seen from the figure that several JrTALE genes (For example, the homologous Arabidopsis BLH2 and KNAT6 corresponding to the walnut TALE proteins) were the core nodes for predicting protein interactions. this result suggests that they may participate in multiple functions by interacting with other proteins.