Identification of C2H2 zinc finger genes through genome-wide association study and functional analyses of LkZFPs in response to stresses in Larix kaempferi
BMC Plant Biology volume 23, Article number: 298 (2023)
C2H2 zinc finger proteins (C2H2-ZFPs), one of the largest transcription factors, play a variety of roles in plant development and growth as well as stress response. While, the evolutionary history and expression profile of the C2H2-ZFP genes in Larix kaempferi (LkZFPs) have not been reported so far.
In this study, the whole genome of the LkZFPs was identified and characterized, including physicochemical properties, phylogenetic relationships, conservative motifs, the promoter cis-elements and Gene Ontology (GO) annotation. We identified 47 LkZFPs and divided them into four subfamilies based on phylogenetic analysis and conserved motifs. Subcellular localization prediction showed that most of the LkZFPs were located in the nucleus. Promoter cis-element analysis suggested that the LkZFPs may be involved in the regulation of stress responses. Moreover, Real-time quantitative PCR (RT-qPCR) results showed that Q-type LkZFP genes were involved in the response to abiotic stress, such as salt, drought and hormone stresses. Subcellular localization results showed that LkZFP7 and LkZFP37 were located in the nucleus, LkZFP32 was located in both cytoplasm and nucleus.
The identification and functional analysis of LkZFPs suggested that some LkZFP genes might play important roles in coping with both biological and abiotic stresses. These results could further increase understanding of the function of the LkZFPs, and provide some research direction and theoretical support.
Zinc finger proteins (ZFPs), one of the largest transcription factor families in eukaryotes, are known for their ability to bind Zn2+ and their finger-like structure [1, 2]. The proteins contain a highly conserved “zinc finger” (ZF) domain, which is a stable three-dimensional structure consisting of different amounts of cysteine (C) and/or histidine (H) residues bound to zinc ions . Based on the number and location of these residues, ZFPs are divided into ten types, including C2H2, C2C2, C3H, C3HC4, C2HC5, C4HC3, C2HC, C4, C6 and C8 . ZFPs play a key transcriptional regulator in a number of biological processes in plants, such as hormone signal transduction, transcriptional regulation, trichomes and root hairs development .
C2H2 zinc finger proteins (C2H2-ZFPs), also called as TFIIIA-type or classical zinc finger proteins, account for a large proportion of zinc finger proteins currently studied . The C2H2-ZFPs have two cysteines, two histidines and one Zn2+, which together form a tetrahedral structure containing an α-helix and two β-hairpins , among them Zn2+ guarantee the stability of the structure [8, 9]. The C2H2-ZFPs contain a characteristic motif composed of 25 to 30 amino acids, X2-C-X (2–4)-C-X12-H–X (3–5)-H, which has been widely found and verified in plants, animals and yeast [10, 11]. The C2H2-ZFPs include Q-type C2H2-ZFPs and C-type C2H2-ZFPs. The Q-type C2H2-ZFPs refer to C2H2 zinc finger protein containing highly conserved “QALGGH” sequence, which is unique in plants and does not exist in animals or yeast . Any amino acid mutation of the “QALGGH” sequence could affect the DNA-binding ability of C2H2-ZFPs . But the “QALGGH” sequence is not present in all C2H2-ZFPs, the C-type C2H2-ZFPs don’t have the conserved sequence. The C2H2-ZFPs can also be divided into four groups according to the form and number of zinc fingers, such as single-C2H2, triple-C2H2 (tC2H2), multiple-adjacent-c2H2 (maC2H2) and separated-paired-C2H2 (spC2H2) . In addition, C2H2-ZFPs may have other functions with EAR motif. The EAR motif is hydrophobic and is thought to keep the zinc finger domain folded . The most common types of the EAR motif are “LXLXL” and “DLNXXP” (where X represents any amino acid) [16, 17].
Since the first plant C2H2-ZFP was identified in Petunia and its expression was found to be tissue-specific and development-regulated , C2H2-ZFPs have been identified in numerous plants. As a transcription factor, the C2H2-ZFPs can effectively enhance plant tolerance to stresses such as low temperature, high salt and drought, by binding to specific promoter cis-elements of target genes . For example, AtSIZ1 enhances salt tolerance in Arabidopsis thaliana by reducing oxygen species (ROS) damage and osmotic stress and maintaining ion homeostasis through abscisic acid (ABA) signaling pathway . TaZFP1 and MpZFP1 enhance plant tolerance to salt stress through a similar mechanism [21, 22]. Moreover, MaC2H2s may be involved in controlling cold stress in bananas by inhibiting the transcription of MaICE1 . In addition, transcriptomic analysis showed that nine typical CsZFPs in Cucumis sativus were significantly correlated with drought, low temperature, heat, and salt stress .
So far, the genome-wide analysis of C2H2-ZFP genes in higher plants has been reported widely: a total of 173, 109, 79 and 98 C2H2-ZFPs have been identified in A. thaliana, poplar (Populus trichocarpa), potato (Solanum tuberosum), grapevine (Vitis vinifera) [25,26,27,28]. However, C2H2-ZFPs has not been identified in L. kaempferi, even though L. kaempferi is an important ecological and economic afforestation species in Northeast China [29, 30]. The growth and development of larch are affected by various abiotic stresses, containing drought stress and cold stress. Therefore, the genome-wide identification of the C2H2-ZFPs gene family is very important to analyze and clarify their molecular function in L. kaempferi. In this study, we identified 47 LkZFPs and analyzed their physicochemical properties, phylogenetic relationships, conservative motifs, the promoter cis-elements, Gene Ontology annotation and subcellular localization. Since “QALGGH” sequence is critical to the DNA binding activity of C2H2-ZFPs, we first analyzed the expression pattern of Q-type LkZFP genes under salt, drought stress, ABA, methyl jasmonate (MeJA) and salicylic acid (SA) treatment by RT-qPCR. Our results enriched the structural information and expression pattern of LkZFPs, and provided a basis for investigating the role of C2H2-ZFPs in response to abiotic stress and hormone treatment L. kaempferi.
Genome-wide identification of C2H2 zinc finger genes in L. kaempferi
After Blast alignment of C2H2-ZFPs in Arabidopsis and HMMER query, we screened these sequences manually based on “X2-C-X (2–4)-C-X12-H–X (3–5)-H” model and detected their structural domains. Finally, a total of 47 C2H2 zinc finger genes from L. kaempferi were identified in L. kaempferi genome and assigned from LkZFP1 to LkZFP47. For the convenience of experimental analysis, the retrieved transcript ID was converted into gene ID. We recorded their detailed physicochemical information and subcellular localization results (Table 1). The number of amino acids ranged from 104 to 896, with an average of 384.61. The molecular weight ranged from 11.19 kDa to 98.49 kDa with the average 42.53 kDa. The isoelectric point (pI) ranged from 4.65 to 9.77. The value of GRAVY is negative and the instability coefficient is greater than 40, which means that most of LkZFPs are unstable hydrophilic proteins. The subcellular localization results of WoLF PSORT showed that LkZFPs was mainly located in the nucleus, and a small portion of LkZFPs might also be located in cytosol, chloroplast and mitochondrion.
The model plant Arabidopsis thaliana has been extensively studied, and the functions of many C2H2-ZFPs have been identified. Therefore, the phylogenetic tree of the C2H2-ZFPs of L. kaempferi and A. thaliana was constructed by the maximum likelihood method (Fig. 1), and the evolutionary relationship was further analyzed. According to sequence similarity and phylogenetic tree, these genes were divided into four subfamilies, with 17, 38, 47 and 118 members in subfamilies A, B, C and D, respectively. These four subfamilies could be further divided into ten subsets. The distribution of C2H2-ZFPs in L. kaempferi and A. thaliana was relatively uniform in the four groups, indicating that the genes of the two species were closely related. The adjacent parts of the phylogenetic tree may represent high homology.
Q-type C2H2-ZFPs and EAR motif
By analyzing the identified LkZFPs, we found that there were eight Q-type zinc finger proteins in subgroup D-4. We compared these amino acid sequences and marked the positions of the C2H2-ZFP conserved motif in the figure (Fig. 2). These LkZFPs contain the common zinc finger domain "X2CX2CX3FX3QALGGHX3H". During the comparison, it was found that six of the eight Q-type LkZFPs had the EAR motif “LXLXL” at C-terminus. The EAR motif has been identified as an activity suppressor gene [31, 32]. Therefore, we speculate that these six LkZFPs may have transcriptional inhibitory effects.
Conserved motifs of LkZFPs
We drew phylogenetic tree of LkZFPs separately (Fig. 3). In order to further analyze the diversity of conserved motifs, we used the tool MEME to retrieve 10 different motifs (Fig. 3). Motif 1 is distributed in almost all proteins. We suggested that Motif 1 (Fig. 4) may be considered as a conserved Motif of the LkZFPs. However, the protein motifs in subgroup D are less than others, which may be due to the poor similarity of genes and proteins. Motif 2 and Motif 3 only existed in subfamily A, Motif 9 only existed in subfamily B, Motif 4 and Motif 8 only existed in subgroup C-1, and other motifs were scattered in various subfamilies. As can be seen from the figure, proteins in the same subfamily have similar motif composition, indicating that their functions may have the similar functions, while proteins in different subfamilies may have different functions. Combined with the results of phylogenetic analysis, the reliability of classification is supported.
Promoter cis-element analysis of LkZFPs
When plants respond to abiotic stress, such as light, temperature and water, plants can regulate gene expression by inducing transcription factors to interact with corresponding cis-elements, so promoter cis-elements play a key role in the regulation of gene transcription. In order to better and intuitively understand the possible expression functions of the LkZFPs, we used PlantCARE to predict the cis-elements of the 2 KB promoter region upstream of the genes (Fig. 5).
We mainly extracted some stress response factors that have been widely studied. The predicted results showed that these promoter sequences contained multiple cis-elements, and most of them were involved in abiotic stress and plant hormones response, such as salicylic acid (SA), jasmonic acid (JA), auxin and abscisic acid (ABA). Among them, cis-elements related to light response are most widely distributed, including MRE (MYB binding site), Box II, AE-Box, G-box, GT1. There are many types of promoter cis-components, such as MBS (MYB binding site, involved in drought induction), ABRE (abscisic acid response element), ARE (anaerobic response element), TGA (auxin response element), TCA (salicylic acid response element), GARE and TATC-Box (gibberellin response element), LTR (low temperature response element), CGTCA and TGACG (jasmonic acid response element), and TC rich areas that can participate in the stress response. The presence of these promoter cis-elements is essential for plants to acquire the ability to adapt to abiotic stresses.
Gene Ontology annotation of LkZFPs
The biological processes, molecular functions and cellular components of the LkZFPs were studied analyzed based on Gene Ontology (GO) term assumption assignment (Fig. 6). The results indicate that LkZFPs may be involved in many biological processes. Of the biological process terms, some LkZFPs are predicted to play roles in the cellular processes (~ 20%), the metabolic processes (~ 19%) and the biological regulation (~ 19%), followed by the stimulus response (~ 15%). Molecular function prediction showed that more than half of LkZFPs were labeled as small molecules or/ion binding (~ 57%), which was consistent with the molecular role of C2H2-ZFP in DNA and metal ion binding. In addition, some LkZFPs were involved in transcription factor activity (~ 32%) and catalytic activity (~ 11%). The prediction of cell composition showed that most of LkZFPs were located in the cell (~ 80%) and others were located in the organelle (~ 20%).
Expression pattern of Q-type LkZFP genes under abiotic stress and hormone treatments
Since “QALGGH” sequence plays an important role in the DNA binding activity of C2H2-ZFPs, we gave priority to the expression pattern analysis of Q-type LkZFP genes in this study. We used RT-qPCR to detect the relative expression levels of eight Q-type LkZFP genes in different tissues and different treatment times, so as to analyze their expression rules under different abiotic stress and hormone treatments. Studies have shown that these genes can effectively enhance plant tolerance to abiotic stress.
After treated with 200 mM of NaCl for 24 h, LkZFP6, LkZFP7, LkZFP24, LkZFP26, LkZFP27, LkZFP29, LkZFP36 and LkZFP41 in the leaf and root showed different expression pattens as follows. In the leaf, LkZFP6, LkZFP7, LkZFP24, LkZFP27, LkZFP29, LkZFP36 and LkZFP41 were induced by NaCl treatment. LkZFP6 and LkZFP7 was significantly up-regulated at all time points. LkZFP26 was significantly down-regulated at 3 h and 12 h. LkZFP7, LkZFP24, LkZFP26, LkZFP27 and LkZFP36 reached their highest level after 24 h of treatment. LkZFP29 and LkZFP41 reached their highest level after 6 h, and LkZFP29 was comparable to untreated control at 24 h (Fig. 7A). In the root, LkZFP6, LkZFP7, LkZFP24, LkZFP27, LkZFP29, LkZFP36 and LkZFP41 were inhibited after 3 h of treatment, reached the highest level after 6 h and then gradually decreased at the following time points. However, LkZFP6 and LkZFP26 were up-regulated at all time points (Fig. 7A).
After treated with 7% PEG6000 in the leaf, LkZFP24, LkZFP36 and LkZFP41 were significantly down-regulated at 3 h. LkZFP6, LkZFP7, LkZFP24, LkZFP27, LkZFP29, LkZFP36 and LkZFP41 were up-regulated to the maximum at 6 h, and then gradually decreased. LkZFP26 was up-regulated at all time points (Fig. 7B). In the root, LkZFP24, LkZFP26, LkZFP27, LkZFP29 and LkZFP36 were induced by drought treatment. LkZFP36 showed the highest expression among eight Q-type LkZFP genes. The expression of LkZFP7 and LkZFP41 reached their highest level after 6 h and 12 h treatment, and lower than that of the untreated control group at 3 h and 24 h (Fig. 7B).
After treated with 200 μM of ABA in the leaf, LkZFP36 reached the highest level after 24 h treatment, LkZFP6, LkZFP7, LkZFP24, LkZFP27, LkZFP29 and LkZFP41 reached the highest level at 6 h. LkZFP29 was significantly down-regulated at 24 h. The expression of LkZFP26 was significantly up-regulated and the highest at 3 h, gradually decreased after 6 h, and significantly down-regulated at 12 h and 24 h (Fig. 8A). In the root, LkZFP6, LkZFP24, LkZFP26 and LkZFP29were up-regulated at all time points. At 3 h of treatment, LkZFP7 and LkZFP41 was down-regulated. The expression level of LkZFP24, LkZFP27 and LkZFP36 was up-regulated to the maximum at 3 h, and then gradually decreased (Fig. 8A).
After treated with 200 μM of MeJA in the leaf, LkZFP6, LkZFP7, LkZFP26, LkZFP29, LkZFP36 and LkZFP41 reached the highest level at 12 h, LkZFP24 and LkZFP27 reached the highest level at 24 h. LkZFP36 showed the highest expression among Q-type LkZFP genes. LkZFP24 and LkZFP41 were significantly down-regulated after 3 h and 6 h. LkZFP26 and LkZFP29 were only significantly down-regulated after 3 h. LkZFP41 was significantly down-regulated after 3 h, 6 h and 24 h (Fig. 8B). In the root, LkZFP6, LkZFP7, LkZFP24, LkZFP26, LkZFP27, LkZFP29, LkZFP36 and LkZFP41 reached the highest level at 6 h and were inhibited at most of the time. And LkZFP6, LkZFP7, LkZFP24, LkZFP26, LkZFP27, LkZFP36 and LkZFP41 were significantly down-regulated at 24 h (Fig. 8B).
After treated with 200 μM of SA in the leaf, LkZFP6, LkZFP7, LkZFP26, LkZFP27, LkZFP29 and LkZFP36 were up-regulated at all time points, LkZFP41 were down-regulated at all time points. LkZFP24 was significantly down-regulated after 3 h and 12 h of treatment (Fig. 8C). In the root, LkZFP6, LkZFP24, LkZFP27, LkZFP29, LkZFP36 and LkZFP41 were up-regulated at 6 h. LkZFP7 and LkZFP26 were down-regulated at all time points. LkZFP6, LkZFP7, LkZFP24, LkZFP27 and LkZFP41 were significantly down-regulated after 3 h and 24 h (Fig. 8C).
To verify the prediction of subcellular localization using online tool WoLF PSORT, we randomly selected LkZFP7, LkZFP32 and LkZFP37 and we transformed the GFP fusion vector (35Spro::LkZFP7-GFP, 35Spro::LkZFP32-GFP and 35Spro::LkZFP37-GFP) into “Yinzhong” Qu 2 protoplasts. The results of confocal microscopy revealed that showed that 35Spro::GFP, a positive control, showed a green fluorescence signal in both cytoplasm and nucleus. 35Spro::LkZFP32-GFP was located in both cytoplasm and nucleus, 35Spro::LkZFP7-GFP and 35Spro::LkZFP37-GFP were only located in the nucleus (Fig. 9). The subcellular localization results of LkZFP7, LkZFP32 and LkZFP37 were consistent with the prediction.
C2H2-ZFPs are widely distributed in plants and play an important role in the regulation of various stages of plant growth and development as well as abiotic stress responses [33,34,35]. Over the past few decades, this gene family has been extensively studied and proved to have different functions in many plants [36,37,38], but no comprehensive investigation was reported in L. kaempferi which has important economic value. In this study, we identified 47 LkZFPs with the conserved domain of “X2-C-X (2–4)-C-X12-H–X (3–5)-H”. The length of these sequences varied significantly from 104 to 896 amino acid residues, indicating a high degree of complexity between LkZFPs. The structural diversity may reflect different functions in response to signaling pathways in multiple environments [39, 40].
Accurate phylogenetic trees could help us to understand the evolutionary process of genes, and members of the same group generally have the same ZFP domain number and motifs [41, 42]. By combining the analysis of phylogenetic tree and conserved motifs, we found that the type and arrangement of motifs in the same group were very consistent. It illustrated that LkZFPs in the same subgroup may have similar biological functions. Many Q-type C2H2-ZFPs play an important role in different environmental stress responses [43, 44]. Among eight Q-type C2H2-ZFPs identified, we found that six LkZFPs contain EAR motif at C-terminus. They may be involved in transcriptional inhibition but require further experimental verification [45, 46]. In addition, since the CDS sequences of the third-generation transcriptions of larch were not available, we could not analyze the exon–intron structure of these genes.
It has been widely reported that cis-elements in gene promoters play an important role in transcriptional regulation [47, 48]. Analysis of cis-elements is helpful to study expression regulation of transcription factors . The analysis results showed that each member of the LkZFP genes contained three or four cis-elements associated with hormones or environmental stresses, suggesting that they could regulate reaction. Some LkZFP genes have both drought and ABA response cis-elements. It may indicate these genes may respond to drought stress through the ABA signaling pathway , but specific functions need to be confirmed by further research. Through GO analysis, the LkZFPs may be involved in varies biological processes, such as stimulus response and biological regulation.
Previous studies have shown that C2H2-ZFP gene expression is affected by tissue differences and abiotic stresses [51, 52]. Moreover, ABA can accumulate up-regulation during drought and salt treatments and resist osmotic stress by inducing the expression of a range of resistance genes [53, 54]. For example, StZFP1 in potato and ZFP179 in rice can be induced by salt stress, drought stress and exogenous ABA . AtAZF2 may respond to stress through an ABA-dependent pathway . According to the results of RT-qPCR, we considered that LkZFP6, LkZFP7, LkZFP29, LkZFP36 and LkZFP41 could be induced by ABA treatment, salt and drought stresses, LkZFP24, LkZFP26, LkZFP27 and LkZFP36 showed similar expression patterns after salt and ABA treatments. In L. kaempferi, we found that the transcription levels of many LkZFP genes increased under different abiotic stress, but the LkZFP genes were more sensitive to salt stress, drought stress, and ABA treatment than MeJA and SA treatments. Interestingly, the relative expression levels of LkZFP24, LkZFP27 and LkZFP36 in the root were significantly higher than those in the leaves under drought stress. Differences in expression patterns suggest that these genes perform different biochemical functions to adapt to complex challenges. The expression patterns of LkZFP genes under different abiotic stress will provide many new insights into the resistance mechanism of L. kaempferi. Subcellular localization of three LkZFPs (LkZFP7, LkZFP32 and LkZFP37) demonstrated the accuracy and reliability of the prediction results.
In this study, we identified 47 LkZFP genes from three generations of larch transcription files and performed a comprehensive bioinformatic analysis. The LkZFP genes were divided into 4 subfamilies and 10 subgroups by phylogenetic analysis. By conserved motif analysis, EAR motif, transcriptional inhibition domain, was found in six of the eight Q-type C2H2-ZFPs. GO annotation predicted that LkZFPs were involved in a variety of biological processes, such as metabolic processes and biological regulation. Based on promoter cis-element and RT-qPCR analysis, some of LkZFP genes respond to salt, drought stress, ABA, MeJA, SA treatment. Subcellular localization results showed that LkZFP7 and LkZFP37 were located in the nucleus, LkZFP32 was located in both cytoplasm and nucleus. The results of this study provide a solid foundation for further functional studies of the LkZFP gene family.
Data collection and identification of LkZFPs
Due to the large size of larch genome files in NCBI, we turned to three generations of larch transcription files as the base database. All 173 C2H2 zinc finger gene sequences of Arabidopsis thaliana were downloaded from the Arabidopsis Information Resource (TAIR) (https://www.arabidopsis.org/), regarded as reference sequences and compared in BioEdit 7.0  to acquire similar genes. The Hidden Markov Model (HMM) of C2H2-ZFPs (Pfam ID: PF00096) downloaded from the Pfam database (http://pfam.xfam.org/) , and were used to extract the sequences containing conservative domain by the HMMER 3.3.2 (http://hmmer.org/). Then, we detected their structural domains by Batch SMART program of TBtools 1.09  and deleted redundant sequences. Finally, we summarized and gained the LkZFPs. The ProtParam tool of ExPASy (https://web.expasy.org/protparam/)  was used to predict the physicochemical properties, such as amino acid (aa) length, molecular weight (MW), theoretical isoelectric point (pI), GRAVY, aliphatic index and instability index. WoLF PSORT (https://wolfpsort.hgc.jp/)  was used to predict the subcellular localization.
The protein sequences of L. kaempferi and Arabidopsis thaliana were compared by The ClustalW function of Mega-X 10.0.5 . Then the phylogenetic evolutionary tree was constructed by maximum likelihood estimation (MLE) with 1000 times bootstraps . Furthermore, we used Evolview (https://www.evolgenius.info/evolview-v2)  to beautify the phylogenetic tree. The Q-type zinc finger proteins in L. kaempferi were aligned by ClustalX 2.0 . The alignment results were mapped and marked with GeneDoc 2.7 to analyze the homologous parts of C2H2-ZFPs.
Identification of conserved motifs
In order to further explain the evolutionary relationship between the further, the phylogenetic tree of the LkZFPs was drawn separately. The protein conserved motifs were searched by MEME (http://meme-suite.org/tools/meme) , and the maximum number was set to 10. Then, the evolution tree and conserved motifs of the LkZFPs were visualized using TBtools 1.09.
Promoter cis-element analysis and Gene Ontology Annotation analysis
NCBI BLAST was used to find the 2000 bp promoter sequence of the LkZFPs, and it was submitted to PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) for prediction and analysis of cis-elements. The results were then visualized with TBtools software. Sequence alignment was plotted using GeneDoc 2.7. EggNOg-Mapper (http://eggNOG-mapper.embl.de) can associate proteins with GO annotations (parameter default), in the terms of biological process, molecular function and cellular component. Then we used TBtools to collate the data and draw.
Plant materials and stress treatments
The wild-type L. kaempferi was grown in pots containing vermiculite and a soil mix of humus in a ratio of 1:1. The seedlings were grown at 23–25℃ culture room with a 16-h photoperiod. After three months of cultivation, we conducted the following treatments. The seedlings were immersed in 1/2 MS liquid medium containing 200 mM NaCl, 7% polyethylene glycol (PEG) 6000, 200 μM ABA, 200 μM MeJA and 200 μM SA for salinity, drought stress and hormone treatments, respectively. The leaves and roots from different seedlings were collected after 3, 6, 12 and 24 h of treatment respectively and the samples without treatment (0 h) were used as the control. All samples were frozen in liquid nitrogen after immediately collected, and then stored at − 80 °C until analysis.
RNA isolation and RT-qPCR
Total RNA was extracted from L. kaempferi leaves and roots using the Plant RNA Reagent Kit (Bioteke, Wuxi, China). Total 1 μg of total RNA was used for the cDNA synthesis by using the MonScript™ RTIII All-in-One Mix with the dsDNase Kit (Monad, Wuhan, China). The synthesized cDNA was diluted ten times for RT-qPCR template and three replicate PCR amplifications were performed for each sample. The α-tubulin gene and actin gene were selected as internal references and Primer Premier 5 was used to design primers with amplicon lengths of 175–221 bp. The primer sequences of LkZFP genes are listed in the supplementary material (Table S1). The RT-qPCR used ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The reaction system consisted of 10µL of 2 × ChamQ Universal SYBR qPCR Master Mix, 0.4µL (10 µM) of forward primer, 0.4µL (10 µM) of reverse primer, 1µL (100 ng) of cDNA, and 8.2µL of ddH2O. The reaction process was performed with the following steps: 95℃ for 30 s; forty cycles were performed with 95℃ for 10 s and 60℃ for 30 s. Relative expression levels of LkZFPs were determined using the 2−ΔΔCt method .
Subcellular localization analysis
The full-length CDS of randomly selected three genes LkZFP7, LkZFP32 and LkZFP37 were amplified using specific primers (Table S2) and KOD FX DNA Polymerase (TOYOBO, Osaka, Japan), respectively, and then cloned into plasmids 35Spro::GFP. Protoplasts were extracted following the procedure described previously . The constructed GFP fusion vector (35Spro::LkZFP7-GFP, 35Spro::LkZFP32-GFP and 35Spro::LkZFP37-GFP) were transfected into “Yinzhong” Qu 2 protoplasts  and cultured in dark at 25℃ for 16 h. The 35Spro::GFP transfected into the protoplasts as control. The fluorescence signals were observed and collected by a laser scanning confocal microscopy (LSM880, ZEISS, Jena, Germany) [69, 70].
Statistical testing was performed with IBM SPSS statistical software (version 23). Three biological replicates were set for each sample of experiments. The data were tested by Student’s t-test (*P < 0.05 or **P < 0.01).
Availability of data and materials
The sequences of LkZFP7 (OQ630901), LkZFP32 (OQ630902) and LkZFP37 (OQ630903) are available in NCBI (https://submit.ncbi.nlm.nih.gov/).
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This work was supported by the Major projects of agricultural biological breeding (2022ZD0401602).
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Shao, L., Li, L., Huang, X. et al. Identification of C2H2 zinc finger genes through genome-wide association study and functional analyses of LkZFPs in response to stresses in Larix kaempferi. BMC Plant Biol 23, 298 (2023). https://doi.org/10.1186/s12870-023-04298-5