Identification of CDPK Gene Family in Solanum habrochaites and Its Function Analysis under Stress

Tomato is an important vegetable crop. In the process of tomato production, it will encounter abiotic stress, such as low temperature, drought, and high salt, and biotic stress, such as pathogen infection, which will seriously affect the yield of tomato. Calcium-dependent protein kinase (CDPK) is a class of major calcium signal receptor which has an important regulatory effect on the perception and decoding of calcium signals. CDPK plays a key role in many aspects of plant growth, such as the elongation of pollen tubes, plant growth, and response to biotic and abiotic stress. While some studies have concentrated on Arabidopsis and pepper, Solanum habrochaites is a wild species relative of cultivated tomato and there is no report on CDPK in Solanum habrochaites to date. Using tomato genomic data, this study identified 33 members of the CDPK gene family. Evolutionary analysis divides family members into four Asian groups, of which the CDPK family members have 11 gene replication pairs. Subcellular location analysis showed that most proteins were predicted to be located in the cytoplasm, and less protein existed on the cell membrane. Not all CDPK family members have a transmembrane domain. Cis regulatory elements relating to light, hormones, and drought stress are overrepresented in the promoter region of the CDPK genes in Solanum habrochaites. The expression levels of each gene under biotic stress and abiotic stress were quantified by qRT-PCR. The results showed that members of the CDPK family in Solanum habrochaites respond to different biotic and abiotic stresses. Among them, the expression of ShCDPK6 and ShCDPK26 genes change significantly. ShCDPK6 and ShCDPK26 genes were silenced using VIGS (virus-induced gene silencing), and the silenced plants illustrated reduced stress resistance to Botrytis cinerea, cold, and drought stress. The results of this study will provide a basis for the in-depth study of the CDPK gene family in Solanum habrochaites, laying the foundation for further analysis of the function of the gene family.


Introduction
Plants often encounter a wide variety of abiotic and biotic stresses in nature and thus they have evolved a complex defense system to resists external pressure. This system facilitates the recognition and perception of external attack, which in turn will be converted to defense mode. Ca 2+ is an important second messenger in the transduction of plant cell signaling [1]. When plants are subjected to an external adverse stress, calcium signals in cells are transmitted to downstream components through calcium sensing proteins, such as calmodulin, Calcinerium-B Like, and the Calcium-dependent protein kinase (CDPK) proteins, thereby modulating the expression of associated genes [2][3][4]. In plants, cytoplasmic regulation of Ca 2+ concentration can be a response to various endogenous and exogenous signals, including changes in hormone level, abiotic stress (such as drought, high and low temperature, or light), and biotic varieties. Therefore, as superior stress resistant materials, Solanum habrochaites often play an important role in improving the quality characteristics of tomatoes.
While CDPKs' important role in calcium signal transduction has been studied in Arabidopsis and corn, among others, it has not been studied in Solanum habrochaites. We suspect that the CDPK gene family plays an important role in affecting the strong low temperature and disease resistance of Solanum habrochaites. In order to analyze the function and genetic evolution of the CDPK gene family in Solanum habrochaites, the genomic data of Solanum habrochaites were first studied by bioinformatics, where the gene family members of CDPKs were identified and their gene and protein sequences, conservative motif, and cis acting elements were analyzed in this study. The expression of the CDPK gene family under biotic stress (Botrytis cinerea) and abiotic stress, such as low temperature and drought, was investigated by qRT-PCR. The functions of ShCDPK6 and ShCDPK26 genes were further verified by virus-induced gene silencing. This study provides a theoretical basis for clarifying the biological function of CDPK genes in response to stress, and further lays a foundation for tomato crop improvement.

Identification of CDPK Family Members in Solanum habrochaites
In order to identify the members of the CDPK family in Solanum habrochaites, we conducted local tBLASTn comparison between the nucleotide sequences of the existing tomato annotated genes and the CDPK amino acid sequences of Arabidopsis thaliana (E-value < 1 × 10 −7 , identity >50%), removing duplicates to obtain tomato candidate CDPK. We used the Prosite (http://prosite.expasy.org/ accessed on 24 July 2020) website to identify candidate CDPK protein domains, screening CDPKs with NAF domain structure, and eventually received 33 members of the CDPK family (Table 1). These genes were named ShCDPK1-ShCDPK33 according to chromosome location information. The CDPK gene family is distributed on 12 chromosomes, of which six family members are distributed on chromosome 1. We analyzed the physical and chemical properties of CDPK family gene length, amino acid length, isoelectric point, and molecular weight of Solanum habrochaites. It was found that the gene length of CDPK family members varied greatly, ranging from 1506 to 3204 bp, of which ShCDPK19 gene was the shortest and ShCDPK31 gene was the longest. The range of amino acids encoded by CDPK family is 501~1067 aa. The minimum isoelectric point (ShCDPK17) was 4.77 and the maximum isoelectric point (ShCDPK8) was 9.77. The members of the gene family were acid protein and basic protein. Subcellular localization prediction analysis showed that most genes were localized in the cytoplasm and two family members existed not only in the cytoplasm, but also on the cell membrane. All family members predicted that there was no transmembrane domain (Table 1).

Phylogenetic Analysis of CDPK Gene in Solanum habrochaites
In order to further understand the evolutionary relationship of CDPK gene in Solanum habrochaites, we constructed a phylogenetic tree with Arabidopsis, tomato and pepper CDPK family members. It can be seen from Figure 1

Motif Analysis of CDPK Gene Family in Solanum habrochaites
Ten motifs were predicted in the tomato CDPK family by meme software, and their motif types and sequence were basically the same. The higher the homology, the stronger the similarity of gene motif arrangement ( Figure 3). Hence, 33 ShCDPK genes have the same conservative motif and order. ShCDPK4, ShCDPK10, ShCDPK11, ShCDPK18, ShCDPK22, ShCDPK25, and ShCDPK28 do not have motif 9. Compared with other family members, they contain a unique motif 10. ShCDPK4, ShCDPK10, ShCDPK11, ShCDPK18, and ShCDPK22 do not have motif 4 or motif 10, but have two motif 6.

Motif Analysis of CDPK Gene Family in Solanum habrochaites
Ten motifs were predicted in the tomato CDPK family by meme software motif types and sequence were basically the same. The higher the homology, th the similarity of gene motif arrangement ( Figure 3). Hence, 33 ShCDPK gene same conservative motif and order. ShCDPK4, ShCDPK10, ShCDPK11, S ShCDPK22, ShCDPK25, and ShCDPK28 do not have motif 9. Compared with other f members, they contain a unique motif 10. ShCDPK4, ShCDPK10, ShCDPK11, ShCD and ShCDPK22 do not have motif 4 or motif 10, but have two motif 6.

Gene Replication and Collinearity Analysis of CDPK Gene Family in Solanum habroch
The gene replication of CDPK gene family in Solanum habrochaites is analyzed in S2.

Analysis of Cis Acting Elements of CDPK Gene Family in Solanum habrochaites
The structure of the promoter affects the affinity between promoter and RNA polymerase, thus affecting the level of gene expression. By analyzing the 1500 bp upstream of CDPK gene family members in Solanum habrochaites, many cis acting elements associated with plant stress were determined (Table S2). In the analysis of cis acting elements of ShCDPKs, in addition to the common CAAT box and TATA box originals, they can be divided into three categories. The first category comprises hormone related components, such as ABRE and CGTCA motif. The second category is the original related to regulation, such as light regulation. The third category is the original related to biological stress, such as TC rich repeats, MBS, and LTR. All members have photoregulated cis acting elements, 23 family members have abscisic acid (ABRE), 19 family members contain methyl jasmonate (CGTCA motif), 19 family members have cis acting elements of defense and adversity responses (TC rich repeats), and 18 family members contain anaerobic induction cis acting elements (ARE), 13 family members have salicylic acid cis acting elements (TCA), 11 family members have cold stress cis acting elements (LTR), nine family members have drought stress cis acting elements (MBS), 10 family members have auxin related cis acting elements (TGA element), and six family members have gibberellin related cis acting elements(P-Box).

Expression Analysis of CDPK Gene Family in Solanum habrochaites under Various Stress
Bioinformatics analysis revealed that members of the CDPK gene family in Solanum habrochaites were associated with multiple stresses (biotic and abiotic). Therefore, in order to further analyze the function of the CDPK gene family, we treated tomato seedlings with low temperature (4 • C), drought, and Botrytis cinerea and detected the gene expression under stress by qRT-PCR.
The expression of the CDPK gene family in Solanum habrochaites changed to a certain extent under cold stress. The expression of ShCDPK5, ShCDPK6, ShCDPK12, ShCDPK15, ShCDPK26, ShCDPK29, and ShCDPK33 changed significantly under cold stress. The expressions of ShCDPK5 and ShCDPK6 were significantly up-regulated after cold induction for 0.5, 12, and 24 h. The expression of ShCDPK5 at 12 h was about 11.0 times that of untreated leaves (the expression at 0 h, CK). The expression of ShCDPK6 was the highest at 12 h, about 31.8 times that of the control group (CK). ShCDPK19, ShCDPK29, and ShCDPK33 showed an upward trend as a whole. The highest value of ShCDPK19 appeared at 24 h, about 18.1 times that of the control group. The highest value of the ShCDPK29 occurred at 12 h and was about 12.7 times higher than the control group. The highest value of the ShCDPK33 occurred at 12 h and was about 26.1 times higher than the control group. The expression of ShCDPK26 increased significantly at the beginning of stress, and the highest expression was found at 12 h, about 38.9 times that of the control group. In conclusion, ShCDPK6 and ShCDPK26 were the most affected by cold stress in the CDPK gene family of Solanum habrochaites, followed by ShCDPK19 (Figure 4).
Under drought induction, the expression of CDPK gene family in Solanum habrochaites changed visibly, and the most significant appeared at 0.5 h. At this time, those with high relative expression include ShCDPK6, ShCDPK23, and ShCDPK26, and the expression of these genes can reach levels higher than 30 times. Most genes showed a wave expression trend and peaked at different time points. The expression of ShCDPK6 gene was 32.5 times that of the control group at 0.5 h and 16.4 times that of the control group at 12 h. The expression of ShCDPK23 gene was 31.0 times that of the control group at 0.5 h and 17.9 times that of the control group at 12 h. The expression of ShCDPK26 gene was 32.9 times that of the control group at 0.5 h and 20.7 times that of the control group at 12 h. ShCDPK6, ShCDPK8, ShCDPK10, ShCDPK16, ShCDPK18, ShCDPK20, ShCDPK21, ShCDPK23, ShCDPK26, ShCDPK29, ShCDPK30, and ShCDPK33 genes was higher than that of 0 h at all time points, with the most pronounced expression at 0.5 h and 24 h. In conclusion, after drought treatment, several genes with significant gene changes are ShCDPK6, ShCDPK16, ShCDPK18, ShCDPK23, ShCDPK26, ShCDPK29, ShCDPK30, and ShCDPK33 ( Figure 5).
We detected the expression changes of the CDPK gene family under Botrytis cinerea stress. Through qRT-PCR detection, we found that the expression of the CDPK gene family in Solanum habrochaites changed in varying degrees under Botrytis cinerea stress, and most of them changed significantly in 0.5 h. ShCDPK4, ShCDPK12, ShCDPK20, and ShCDPK22 genes had the same expression change trend, and showed high expression at 0.5 h, with little change at other points in time. The expression of ShCDPK6, ShCDPK18, ShCDPK19, ShCDPK25, ShCDPK26, ShCDPK29, ShCDPK30, ShCDPK31, and ShCDPK33 decreased at 0.5-9 h and increased at the later stage. The expression of ShCDPK6 and ShCDPK33 was overtly raised in the later stage of stress, and at 36 h, the expression of both genes was 43.9 times and 32.8 times higher than that of the control. The peak values of ShCDPK26, ShCDPK29, and ShCDPK31 were 25.6 times, 15.5 times, and 14.9 times that of the control at 36 h, respectively. In conclusion, ShCDPK6 and ShCDPK33 were the most affected by Botrytis cinerea stress in the CDPK gene family of Solanum habrochaites, followed by ShCDPK26 ( Figure 6). Under drought induction, the expression of CDPK gene family in Solanum habrochaites changed visibly, and the most significant appeared at 0.5 h. At this time, those with high relative expression include ShCDPK6, ShCDPK23, and ShCDPK26, and the 12 h. ShCDPK6, ShCDPK8, ShCDPK10, ShCDPK16, ShCDPK18, ShCDPK20, ShCDPK21, ShCDPK23, ShCDPK26, ShCDPK29, ShCDPK30, and ShCDPK33 genes was higher than that of 0 h at all time points, with the most pronounced expression at 0.5 h and 24 h. In conclusion, after drought treatment, several genes with significant gene changes are ShCDPK6, ShCDPK16, ShCDPK18, ShCDPK23, ShCDPK26, ShCDPK29, ShCDPK30, and ShCDPK33 ( Figure 5).  ShCDPK33 was overtly raised in the later stage of stress, and at 36 h, the expression of both genes was 43.9 times and 32.8 times higher than that of the control. The peak values of ShCDPK26, ShCDPK29, and ShCDPK31 were 25.6 times, 15.5 times, and 14.9 times that of the control at 36 h, respectively. In conclusion, ShCDPK6 and ShCDPK33 were the most affected by Botrytis cinerea stress in the CDPK gene family of Solanum habrochaites, followed by ShCDPK26 (Figure 6). Quantitative PCR data are represented as mean ± SEM. ** p < 0.01, * p < 0.05. All date represent mean values for three independent biological replicates.

Functional Analysis of Silencing ShCDPK6 and ShCDPK26 in Solanum habrochaites
Gene expression differences between ShCDPK6 and ShCDPK26 were the most obvious after three stress treatments. It is speculated that they may be involved in the response of Solanum habrochaites to stress. We will take ShCDPK6 and ShCDPK26 as the research objects to further verify the function of the gene through VIGS. VIGS vectors PTRV2-ShCDPK6 and PTRV2-ShCDPK26 were successfully constructed and transformed into Agrobacterium tumefaciens. Tomato seedlings were injected with agrobacterium tumefaciens containing the target vector and PDS (Phytoene dehydrogenase) silenced

Functional Analysis of Silencing ShCDPK6 and ShCDPK26 in Solanum habrochaites
Gene expression differences between ShCDPK6 and ShCDPK26 were the most obvious after three stress treatments. It is speculated that they may be involved in the response of Solanum habrochaites to stress. We will take ShCDPK6 and ShCDPK26 as the research objects to further verify the function of the gene through VIGS. VIGS vectors PTRV2-ShCDPK6 and PTRV2-ShCDPK26 were successfully constructed and transformed into Agrobacterium tumefaciens. Tomato seedlings were injected with agrobacterium tumefaciens containing the target vector and PDS (Phytoene dehydrogenase) silenced plants were used as indicators of the VIGS silencing effect. The expression levels of target genes in ShCDPK6 and ShCDPK26 silent plants and control plants were detected by fluorescence quantitative PCR, and the silencing efficiency of ShCDPK6 and ShCDPK26 is 69.4% and 48.5% respectively ( Figure S1). Finally, we obtained 15 ShCDPK6 and ShCDPK26 silencing plants respectively. In terms of bioinformatics analysis, ShCDPK6, ShCDPK24, and ShCDPK26 genes were found to have a close evolutionary relationship, and the expression of ShCDPK24 gene was analyzed separately for the silenced plants, as shown in Figure S2. The results revealed that the expression of ShCDPK24 gene in ShCDPK6 and ShCDPK26-silenced plants did not show significant changes in expression.
Plants will produce some reactive oxygen species after encountering adversity, which will have a toxic effect on cells, leading to plant cell and tissue damage and death. Therefore, under Botrytis cinerea, drought, and cold stress, various physiological indexes of ShCDPK6 and ShCDPK26 silenced plants and control plants were measured respectively to evaluate the degree of oxidative damage between them. The contents of superoxide dismutase (SOD), catalase (CAT), ascorbate oxidase (AAO), peroxidase (POD), and polyphenol oxidase (PPO) in silenced and control plants were determined under low temperature (4 • C), drought, and biotic stress (Botrytis cinerea). Under cold stress, SOD, AAO, POD, and PPO values of silenced plants were lower than those of control plants, indicating that the cold resistance of the silenced plants was reduced (Figure 7). Under drought treatment, the contents of CAT, POD, and PPO in silenced plant were significantly lower than that of control group. The change in the dynamic of PPO content was in keeping with that of the control plant in the early stage of stress. ShCDPK6 and ShCDPK26 decreased in varying degrees after 9 h (Figure 8). Under Botrytis cinerea treatment, changes of SOD, POD, PPO, and AAO contents in the ShCDPK6 silenced plants was distinctly lower than that of the control group, while the changes of CAT, POD, and PPO in silent plant ShDPK26 were smaller than those in control group, indicating that the Botrytis cinerea resistance of the silenced plants was reduced ( Figure 9). The phenotype of the control and silenced plants under three stresses is shown in Figure S3. Compared with the control plants, the silent plants showed more wilting under low temperature and drought stress, but the phenotypic difference was not very obvious under Botrytis cinerea stress.
PPO in silent plant ShDPK26 were smaller than those in control group, indicating that the Botrytis cinerea resistance of the silenced plants was reduced (Figure 9). The phenotype of the control and silenced plants under three stresses is shown in Figure S3. Compared with the control plants, the silent plants showed more wilting under low temperature and drought stress, but the phenotypic difference was not very obvious under Botrytis cinerea stress.

Discussion
The CDPK gene family widely exists in all parts of plants, such as roots, stems, leaves, flowers, fruits, and seeds, and plays an important role in plant state construction [17,34]. Arabidopsis CPK11 positively regulates the ABA signaling pathway in seed germination

Discussion
The CDPK gene family widely exists in all parts of plants, such as roots, stems, leaves, flowers, fruits, and seeds, and plays an important role in plant state construction [17,34]. Arabidopsis CPK11 positively regulates the ABA signaling pathway in seed germination and stomatal movement [35]. Later, it was found that Arabidopsis CPK12, as a homologue of CPK4 and CPK11, negatively regulates the process of plant seed germination and growth after germination and antagonizes CPK4 and CPK11 in the ABA signaling pathway [34]. CDPKs also play an important role in plant growth and development and response to biotic and abiotic stresses. This has been identified and studied in many species [36]. For example, the first purification and identification were carried out in soybean. Cucumber CsCDPK5 is involved in the formation of adventitious roots of hypocotyl [37]. Arabidopsis contains 34 CDPK genes [26]. There are great differences in the number of CDPK genes in different species, including 21 in potato and 18 in melon [38,39]. In this study, 33 ShCDPK genes of Solanum habrochaites were identified by BLAST analysis. The physical and chemical properties of ShCDPK gene family members, such as gene length, amino acid, molecular weight, isoelectric point, and molecular weight, were determined by bioinformatics analysis. Through phylogenetic analysis of the gene family in Arabidopsis and pepper, 33 ShCDPK gene family members were divided into four subfamilies, which is consistent with the studies conducted in cucumber [40], pineapple [41], and grape [42], indicating that there are still some commonalities among different species.
CDPK was identified in pea and soybean for the first time. However, a subsequent study showed that CDPK can be found in green algae, oomycetes, and some protozoa, such as ciliates and capsicum. The CDPK gene family is more distributed on chromosomes 1, 10, and 11 and unevenly distributed on other chromosomes. The unbalanced distribution of genes may be related to species evolution and genetic variation. The phylogenetic tree constructed from Solanum habrochaites, Arabidopsis, and Pepper is divided into four subfamilies, and each subfamily has ShCDPK distribution. Group 1 included three pairs of vertical homologous pairs of Solanum habrochaites and Pepper. Groups 2 and 3 included two pairs of vertical homologous pairs between Solanum habrochaites and Arabidopsis. Group 4 did not contain vertical homologous pairs between Solanum habrochaites and Arabidopsis. These results showed that the homologous genes related to Solanum habrochaites and Arabidopsis had a conserved structure and function. In contrast, Arabidopsis and Solanum habrochaites have vertical homologous pairs, indicating that the CDPK gene family of Solanum habrochaites is relative to Arabidopsis in functional evolution. The CDPK gene family is mainly rich in introns, and the composition of intron exons can reflect the evolutionary relationship of the gene family. ShCDPK8 and ShCDPK12 present the most intron enrichment, containing 11 introns. ShCDPK2, ShCDPK7, and ShCDPK9 have 10 introns. They are located in group 4 in the phylogenetic tree. Other introns were enriched in group 1, group 2, and group 3 of the phylogenetic tree. The number of introns in all gene family members was greater than 6, and there were no genes with intron deletion. The rate of intron acquisition is slower than that of intron deletion. The phylogeny of intron deleted CDPK gene may be the branch of intron enriched gene mRNA re inserted into the genome during the reverse transcription of intron enriched gene mRNA. There are 11 pairs of tandem repeat genes in ShCDPKs. The results showed that fragment duplication originated from stress. These 11 pairs of genes are highly consistent on the conserved motif, indicating that tandem replication plays a key role in the amplification of ShCDPKs. The KS of tandem repeat gene pairs is 0.0932819~1.30348, the separation time is 82~115.2 Mya, and it is 200~205 Mya during single dicotyledon differentiation. It is speculated that the fragment replication of ShCDPKs occurs after single dicotyledon differentiation [43].
It is well known that Ca 2+ as a ubiquitous second messenger in the plant signal system plays a great role in plant growth and development. When plants are stimulated by environment and development, the stimulation triggered by external factors, such as temperature, light, salt, and osmotic stress, can produce different calcium ion changes. Specific calcium receptors can recognize and perceive these changes, and then play a role in regulating gene expression through a series of cascade reactions [44]. Through research, the CDPK gene senses the change of Ca 2+ concentration through EF hand structure, relieves self-inhibition, activates kinase domain, and then transmits information to regulate the physiological changes of plants, widely participating in plant growth, development, and morphological construction [45]. In this study, the expression of ShCDPK family members changed significantly after cold, drought stress, and Botrytis cinerea stress. The expressions of ShCDPK6, ShCDPK19, and ShCDPK26 were significantly up-regulated under low temperature stress, suggesting that these genes may be involved in cold response. Under cold stress, the expression of ShCDPK12 and ShCDPK30 decreased significantly, indicating that different members of CDPK may play different roles in response to cold stress. At the beginning of the drought and Botrytis cinerea treatments, the expression level of most genes increased significantly, the expression of ShCDPK5, ShCDPK6, ShCDPK23, ShCDPK26, ShCDPK29, ShCDPK30, and ShCDPK33 genes increased significantly at 0.5 h of drought stress, and the expression of ShCDPK6, ShCDPK16, ShCDPK23, ShCDPK26, and ShCDPK29 peaked at 0.5 h and 12 h. This may be because of the overrepresentation of cis acting elements relating to defense and drought stress in their promoter. The results show that CPK10 and HSP1 participate in the regulation of stomatal movement through ABA and Ca 2+ signaling pathways, which may play an important role in the response of plants to drought stress [39]. It is also reported that guard cell anion channel SLAC1 is regulated by CPK21 and CPK23 and has different Ca 2+ affinity [46]. AtCDPK4 and AtCDPK11 in Arabidopsis are responsive to salt stress. Grape VpCDPK16 was similar to AtCDPK4 and AtCDPK11, while they could phosphorylate ABA response transcription factors and reduce the tolerance to salt stress in seed germination and seedling growth. VpCDPK16 also had similar expression [35]. In this study, under abiotic stress, such as low temperature and drought, and biotic stress of Botrytis cinerea infection, the expression of ShCDPK6 and ShCDPK26 changed significantly, indicating that these genes play an important role in the environmental adaptability of Solanum habrochaites. VIGS experiments were performed on ShCDPK6 and ShCDPK26.The level of gene silencing is influenced by a variety of factors, including the size of the inserted silencing fragment, the direction of insertion, and the environment in which it is grown [47], and of course, genetic differences exist in plants of different species. All these factors may make the level of gene silencing different. This silencing system mediated by TRV (tobacco rattle virus) vector has been widely used in tomato varieties [48,49], but the presence of multiple factors leads to an average level of silencing. During biotic stress, the activities of SOD, POD, AAO, PPO, and CAT in silenced plants increased to varying degrees with stress stimulation, indicating that after the silencing of ShCDPK6 and ShCDPK26 genes, the enzyme activity response is more sensitive, so as to balance the metabolic system in order to eliminate the active oxygen produced by plant stress. From the expression of various enzyme activities, the enzyme activities of silenced plants are less than those of control plants, which may be caused by the silencing of CDPK family genes. In abiotic stress, we explored the changes of physiological indexes of silent plants under drought stress and cold stress. Under low temperature and drought stress, plants will produce a large amount of reactive oxygen species which can damage the plant cell membrane. In order to effectively remove reactive oxygen species from plant cells, plants activate a series of antioxidant systems, such as SOD and POD [50]. The results showed that there were some changes between the silent plants and the control plants under the two stresses. The change of POD in silent plants increased first and then decreased, but it was less than that in CK group, which may be because POD needs to remove hydrogen peroxide produced under stress and reduce the damage caused by stress. The change of SOD value increases first and then decreases as time goes on. However, compared with ShCDPK6, ShCDPK26 gene may play a regulatory role under abiotic stress. These results further indicate that the lack of ShCDPKs family member genes make plant more sensitive to low temperature, drought, and Botrytis cinerea. However, how ShCDPK6 and ShCDPK26 respond to various stress conditions and functions needs to be further studied.

Plant Materials and Treatments
Solanum habrochaites (LA1777, https://tgrc.ucdavis.edu/, the National Plant Germplasm System (NPGS), California (for USA) accessed on 5 April 2022) was conserved in our lab supplying by the Tomato Genetics Research Center (University of California, Davis, CA, USA). The seeds were first soaked in 2% sodium hypochlorite solution for 16 min (slowly shaken), then rinsed with sterile water 3-4 times, then soaked in 75% alcohol for 30 s, and rinsed with sterile water. Sterilized seeds were pre-germinated on wet filter paper and subsequently transferred to plastic basin of the soil mixture (1 V:1 V). They were planted in an artificial climate chamber at 21-26 • C, 16 h/8 h (day/night). Tomato seedlings at five leaves and one heart were used for subsequent experimental treatment. Three plants with the same growth were taken from each treatment for three biological replicates. Three technical replicates were performed for each treatment.

Cold Stress
Plant roots were dipped in half-strength Hoagland culture solution for 2-3 days. Cold stress was exerted by exposing a subset of the plants to low temperature (4 • C). Samples were obtained at time points 0, 0.5, 3, 9, 12, 24, and 36 h.

Drought Stress
Roots of experimental and control plants were dipped in half-strength Hoagland culture solution for 2-3 days. The treatment group and the control group were sampled simultaneously. Treatment group: The roots of plants were soaked with 20% PEG6000 (1/4 Hoagland nutrient solution). They were allowed to continue growing in this medium until samples were obtained. Samples were obtained at time points 0, 0.5, 3, 9, 12, and 24 h.

Botrytis Cinerea Stress
B. cinerea spores were diluted with sterile water to make the concentration of spore suspension 10 7 CFU/mL. Treatment group: The fungus solution was sprayed on the tomato leaves and cultured in a light incubator with 90% humidity at 20-25 • C, while in the control group the tomato blade was sprayed with water, and other conditions remained constant. Samples were obtained at time points 0, 0.5, 3,9,12,24, and 36 h.

Identification and Basic Information Analysis of CDPK Gene Family Members in Solanum habrochaites
Firstly, the hidden Markov model (HMM) files corresponding to protease (PF00069) and NaF domain (PF03822) were downloaded from Pfam protein family database (https: //pfam.xfam.org/ accessed on 24 July 2020). Then, the whole genome data of Solanum habrochaites were obtained by hmmsearch (https://www.ncbi.nlm.nih.gov/data-hub/ taxonomy/62890/ accessed on 24 July 2020). CDPK gene (E-value < 1 × 10 −7 , similarity >50%), and the members and number of CDPK gene family in Solanum habrochaites were obtained. The length, molecular weight, and isoelectric point of CDPK protein in Solanum habrochaites were predicted by bio-linux software. In order to understand the chromosome situation of the gene family, the chromosome mapping map was drawn by MapChart software. At the same time, SOSUI (http://harrier.nagahama-i-bio.Ac.jp/sosui/ accessed on 24 July 2020) online software was used to predict the protein transmembrane domain. CELLO (http//cello.life.nctu.edu.tw accessed on 24 July 2020) was used to predict subcellular localization and identify the functional sites of the CDPK gene family.

Evolutionary Analysis of CDPK Gene Family in Solanum habrochaites
In order to explore the evolutionary relationship between the CDPK gene family, comparative analysis of the CDPK gene family in Solanum habrochaites, Arabidopsis, and peppers was conducted using MEGA software with bootstrap repeated 1000 times.

Analysis of Genetic Structure of CDPK Gene in Solanum habrochaites
Genomic sequence of CDPK genes consisting of the coding sequence and the noncoding regions were obtained for Solanum habrochaites. The GSDS tool (http://gsds.cbi.Pku. Edu.cn/ accessed on 24 July 2020) was used to perform gene structure analysis and draw the exon-intron structure diagram.

Analysis of CDPK Gene Family Conservative Motif in Solanum habrochaites
In order to study the evolutionary relationship of CDPK gene family in Solanum habrochaites, 10 conserved motifs of CDPK genes were analyzed online with MEME (https: //meme-suite.org/meme/ accessed on 24 July 2020).

Gene Replication of CDPK Gene Family in Solanum habrochaites
The full-length amino acid sequence is compared using Clustal. Ka is the nonsynonymous substitution rate, and Ks is the synonymous substitution rate. Ks and Ka programs were calculated using Kaks_Calculator 2.0 and the discrete time of genetic pairs was estimated using mutation rate. The substitution of each synonymation in each year is T = Ks/2X (x = 6.56 × 10 −9 ). If Ka/Ks > 1, a positive selection effect is considered. If Ka/Ks = 1, neutral selection is considered to exist. If Ka/Ks < 1, a purifying selection effect is considered.

Real-Time Quantitative PCR (qRT-PCR) Analysis
The plants were grown at 25 • C for 4 weeks. The total RNA of samples were extracted using Trizol reagent (Invitrogen, California (for USA)) and used for mRNA synthesis with High-Capacity cDNA Reverse Transcription Kits (ThermoFisher Scientific, Waltham, MA, USA) following the manufacturer's recommendations. qRT-PCR was performed with the SYBR Green qPCR Mix Kit (Biosharp, Hefei, China). NCBI (https://www.ncbi.nlm.nih.gov/ accessed on 15 July 2021) Primer Blast was used to design fluorescent quantitative primers (MAX value to 200 base pairs), and the primer sequence is shown in Table S1. The product cDNA was diluted 10 times and used as template for qRT-PCR. Each reaction contained 10 µL Chamq SYBR qPCR Master Mix (Without Rox) and 0.4 µL forward primer (10 µM), 0.4 µL reverse primer (10 µM), and cDNA samples diluted 10 times with 1.0 µL. PCR amplification was performed using a Quant Studio real-time fluorescent quantitative PCR instrument. Reaction conditions: predeformation, 95 • C for 5 min, followed by 40 cycles, 95 • C for 10 s, 60 • C for 30 s. Data represent means of three replicates ± standard deviation (SD). Analysis was performed using the Data Processing System, and Tukey's multiple range test was conducted to determine significant differences. p < 0.05 was considered to indicate statistical significance.

Virus-Induced Gene Silencing (VIGS)
The successfully constructed pTRV2-ShCDPK6 and pTRV2-ShCDPK26 were transformed into Agrobacterium tumefaciens strain GV3101 together with pTRV1 and pTRV2-PDS. Cell culture containing the appropriate plasmid was injected into the tomato seedlings through the leaves with a syringe. Agrobacterium tumefaciens containing PTRV1 was mixed with agrobacterium tumefaciens containing pTRV2-ShCDPK6, pTRV2-ShCDPK26 and pTRV2-PDS respectively at 1:1 and placed at room temperature for 3 h. Plant leaves were inoculated with a 1 mL syringe to fill the injected leaves with bacterial solution to the greatest extent (the leaves are not damaged). Hence, 50 seedlings were infected at one time, among which 5 seedlings were used as the pTRV2-PDS plants,15 seedlings used for the drought treatment, 15 seedlings used for cold treatment, and 15 seedlings used for the Botrytis cinerea treatment. Each plant was inoculated 3 times, with 1 mL each time. At the same time, the pTRV2-PDS empty vector was inoculated into plant leaves as the positive control group. The soaked tomatoes were incubated overnight in dark conditions at 22 • C, and then incubated in a constant temperature incubator with a light cycle of 16 h/8 h. Other conditions were not changed. To ensure successful infiltration, tomato plant materials were inoculated with different combinations of fluid in each 4 min vacuum process. After the positive control plants showed the phenomenon of melatonin, the leaves of the experimental group and the control group were taken to detect efficiency of silencing gene using qRT-PCR [51]. The experiment was repeated three times. After the target fragment was successfully silenced, five physiological indexes of superoxide dismutase (SOD), catalase (CAT), ascorbate oxidase (AAO), peroxidase (POD), and polyphenol oxidase (PPO) were determined.

Conclusions
In this study, 33 CDPK gene family members of Solanum habrochaites were identified, which can be divided into four subgroups, mainly distributed in the cytoplasm with a few in the outer membrane. Phylogenetic analysis showed that the CDPK gene family had similar genes between Solanum habrochaites and cultivated tomato. Structural analysis showed that there was intron enrichment in the CDPK family of Solanum habrochaites. Motif analysis showed that the gene motifs with high homology of the CDPK gene family in Solanum habrochaites had strong similarity. According to the analysis of cis acting elements, the CDPK gene family of Solanum habrochaites is related to defense and stress response, anaerobic induction, drought stress, and other functions. The results of qRT-PCR showed that the CDPK gene family could respond to low temperature, drought, and Botrytis cinerea stress, and ShCDPK6 and ShCDPK26 were significantly expressed under the three stresses. The stress resistance of ShCDPK6 and ShCDPK26 silenced plants decreased significantly, indicating that ShCDPK6 and ShCDPK26 play an important role in response to abiotic and biotic stress.