Genome-wide identification and analysis of the Q-type C2H2 gene family in walnut (Juglans regia L.)

Abstract:【Objective】Q-type C2H2 zinc finger protein plays an important role in plant resistance to abiotic stress. As an important species of economic forest, walnuts are often exposed to adverse conditions. In order to further understand the characteristics of Q-type C2H2 zinc finger proteins in walnut genome, the whole genome of Q-type C2H2 zinc finger proteins (JrZFPs) in walnut was identified and analyzed, and their expression patterns in response to drought stress and salt stress were studied. We carried out this research to provide a reference for the study of walnut resistance to stress.【Methods】4-week- old Liaohe No. 4 tissue cultured seedlings were used as materials. These plantlets were treated with NaCl (100 mmol·L-1 ) and PEG (15.0%, ρ) for 0, 6, 12 and 24 h, then frozen in liquid nitrogen immediately and stored at −80 ℃ for RNA extraction and gene expression analysis. Each treatment had 3 replicates, with nine plants per replicate. Additionally, through BLAST Arabidopsis thaliana Q- type C2H2 protein sequences, 82 JrZFPs were screened from the walnut genome database. With the help of ExPASy, MEGA X, TBtools, MEME and Plant CARE online website, bioinformatics analysis of the gene families was performed, including the phylogenetic analysis, gene structure and protein sequence analysis, chromosome location, series replication analysis, and inter species collinear analysis. Finally, the expression patterns of 15 Q-type C2H2 genes were analyzed under drought and salt stress through qRT-PCR.【Results】A total of eighty-two members of JrZFP were identified from the walnut genome database (named as JrZFP1-JrZFP82). They all contained invariant“QALGGH”motif in the zinc finger domains (CX2–4CX3FX3QALGGHX3–5H). In addition, the number of amino acids, relative molecular weight, isoelectric point and subcellular localization of JrZFP family genes were analyzed. The number of amino acids of the 82 JrZFP gene family members ranged from 59 (JrZFP26) aa to 636 (JrZFP63) aa, with a molecular weight ranging from 6.72 (JrZFP26) to 70.33 (JrZFP63), and an isoelectric point from 5.33 (JrZFP80) to 10.23 (JrZFP47). Prediction of subcellular location showed that JrZFPs were mainly located in the nucleus, with a small amount distributed in the cytoplasm, peroxisomes and chloroplasts. Chromosome location analysis showed that these JrZFPs were unevenly distributed in all the walnut except for chromosome 16. In the present study, 11 pairs of JrZFP genes (22/82, 26.83%) were identified as tandemly duplicated genes, of which chromosomes 1, 2, 3, 4, 6, 10 and 14 had one pair of tandem duplication genes. Chromosome 9 and 13 had two pairs of genes. Besides the tandem duplication, 63 (63/82, 76.83% ) segmental duplication genes were also identified using MCScanX methods. In order to study the evolutionary relationship between walnut and Arabidopsis Q-type C2H2 genes, a phylogenetic tree analysis was performed on the amino acid sequences of 140 Q-type C2H2 (including 58 Arabidopsis genes and 82 walnut genes). According to sequence similarity and topological structure, the phylogenetic tree could be divided into 7 ZFP subfamilies, namely C1, C2-Ⅰ, C2- Ⅱ, C2-Ⅲ, C2-Ⅳ, C3-Ⅰ and C3-Ⅱ. In order to understand the gene structure of JrZFPs, their introns, exons and conservative motifs were analyzed. The results showed that 75 JrZFP genes (91.46%) had no introns, and a total of 10 Motifs were identified in the conservative motif distribution. Motif 1 and Motif 2 both contained Q-type C2H2 core sequences, and Motif 5 was an EAR-motif. The JrZFPs in the C1 and C2 subfamily contained two zinc finger domains, while the members of the C3 subfamily contained only one zinc finger domain. Motif 1 and Motif 2 were Q-type zinc finger domains, which are widely distributed in 82 JrZFPs. All 76 JrZFPs contained EAR-motif, and the remaining members did not have EAR-motif. Ninety-eight percent of the members of this family had light-responsive elements, 93% of members contained abscisic acid-responsive elements and 50% of members had low-temperature response elements. Additionally, 44% of members had drought-responsive elements, indicating that the JrZFPs family plays an important role in response to drought environment. At last, the 15 JrZFP genes showed different response patterns under salt stress, all of which were significantly up-regulated. Especially, the relative expression of JrZFP55 was the highest compared with S0, indicating that JrZFP55 played a positive regulatory role in walnut NaCl stress. On the other hand, the 15 JrZFP genes showed different response patterns under drought stress, which were either significantly up-regulated or down-regulated. Among them, JrZFP12 was up-regulated the most, which was 13.97 times that of S0. At the same time, JrZFP4, JrZFP55 and JrZFP57 were down- regulated most significantly under drought stress, suggesting that these three genes play a negative regulatory role in walnut drought stress.【Conclusion】There are 82 JrZFP family members in the whole genome of walnut, all of which contain the typical Q-type C2H2 domain. JrZFP genes may be involved in drought and salt stress based on real-time fluorescence quantitative analysis.