Pepper is a vegetable crop widely grown in the world and has important economic value. The annual planting area accounts for about 8% of the vegetable planting area in China[26]. During the planting, growth, production and planting of pepper, it is very easy to be affected by various biological and abiotic stresses, such as cold damage, drought, light, drought, disease and insect pests, etc[27]. CRT gene family members play a crucial role in plant growth and development and in response to biotic and abiotic stresses[28]. Therefore, comprehensively identify and analyze the CRT gene family of capsicum to reveal its expression characteristics under low temperature stress. It laid a foundation for studying its response function under low temperature stress[29].
In this study, we downloaded the Capsicum annuum genome file (Zunla-1 version, https://solgenomics.net/) from the Solanaceae Genomics Network website and extracted the protein sequence file of Capsicum annuum using TBtools. A total of 4 CRT genes were identified by bioinformatics by searching the CRT gene family characteristic domains (PF00262) of pepper from Pfam database (http://pfam.xfam.org/), and the amino acid sequence of pepper CRT was screened by Simple HMM Search. The results of 3 D homology modeling of CaCRTs show that the four CRT genes are structurally similar(Fig. 1). Through genome alignment, Tbtools was used to generate chromosome distribution maps(Fig. 2). The four CaCRTs were evenly distributed on the four chromosomes. CaCRT1 is on Chr03, CaCRT2 is on Chr05, CaCRT3 is on Chr06, and CaCRT4 is on Chr08. Observation of the conserved domains of CaCRT showed that all CaCRTs contained Calreticulin domains, which proved that they were all CRT genes, and CaCRT1 contained Amidase superfamily domains in addition to Calreticulin domains, suggesting that CaCRT1 may have other functions(Fig. 3). At the same time, phylogenetic trees(Fig. 4), intra-species(Fig. 5) and inter-species(Fig. 6) collinearity were analyzed. Based on the GFF3 gene structure annotation information, TBtools was used to generate the exon/intron structure diagram of CaCRTs(Fig. 7). It was found that all CaCRTs contained introns, among which CaCRT1 had the largest number of introns, and CaCRT2 had the lowest number of introns. Conserved motif analysis showed that 15 motifs were identified from CaCRTs(Fig. 8). Among them, CaCRT2 has the fewest motifs, only 8; CaCRT1 contains the largest number of motifs with 15. Phylogenetic analysis revealed that the more recent CaCRTs contained more similar motifs.In addition, domain analysis found that Motif1, Motif3, Motif5, Motif7, Motif9 and Motif13 constituted the key CRT functional domains (Calreticulin), while the other motifs did not match the known key functional domains. The results of cis-acting element analysis showed that CRT gene may respond to multiple stresses.
As a conserved calcium-binding protein, CRT plays an extremely important role in cell signal transduction, protein sorting and biological metabolism by regulating intracellular calcium homeostasis and protein folding[30]. The main research results of CRTs gene in plants were all based on the cloning, protein expression or function analysis of CRT gene in model plants such as Arabidopsis thaliana, rice and wheat[31]. In this study, calretin gene CaCRTs was cloned from pepper. Gene families were identified and their expression patterns explored[32]. Existing evidence shows that CRTs plays an important role in plant growth and development, hormone response, physiological and immune response to stress[33]. The molecular expression characteristics of CRTs gene are closely related to the physiological and metabolic processes regulated by the plant. TaCRT1 was mainly expressed in stems, leaves, pistil, and aeng of wheat, and the expression levels were similar, indicating that the function of this gene was different during plant development[34]. The CRT gene of castor bean was expressed in the whole plant, and the expression level was higher in young and tender tissues[35]. CRTs gene treatment has tissue-specific expression and can respond to various biological and abiotic stresses, such as TaCRT1[36], AtCRT2[37], inducing or inhibiting the expression of target genes and participating in various physiological processes in plants. In this study, the expression pattern of CRT gene in pepper was explored and it was found that CRT gene was expressed in root, stem, leaf, flower, top bud and side bud, but the expression levels were different, which indicated that CRTs gene had functional differences during plant growth and development(Fig. 9a). Through protein interaction analysis, it was found that capsicum CRT gene may interact with a variety of molecular chaperones, which is consistent with previous studies, indicating that capsicum CRT gene can affect the normal folding and expression of proteins by interacting with other molecular chaperones in vivo.
In this study, the expression of CRT gene in pepper under low temperature stress was analyzed by fluorescence quantitative method after applying different stresses to pepper seedlings, and the results showed that the expression of CRT1 and CRT3 was stronger than that of CRT4 under low temperature biological stress, indicating that CRT1 and CRT3 may specifically respond to low temperature stress(Fig. 9b). At the same time, the VIGS silencing technology was used to obtain CaCRT1 silencing plants. After 12 h of low temperature stress, the silent plants began to wilt, and after 24 h, the silent plants were seriously wilted(Fig. 9c). The control of the positive vaccine was observed(Fig. 9d), and the silencing efficiency was analyzed(Fig. 9e). REL (Fig. 9f) and MDA (Fig. 9g) were measured, and it was found that the TRV2:00 control plants were lower than those of silent plants after low temperature treatment, indicating that the degree of cell membrane damage was smaller. The activities of SOD, POD and CAT and the corresponding genes in the silent plants and the control plants were measured(Fig. 11def), indicating that the ROS content was too much, which represented low degradation and affected the plant's resistance to stress, and the expression of several cold-responsive genes was measured(Fig. 11ghi), and the results showed that the content was reduced compared with the control plants, indicating that the silencing genes may be involved in the process of plants resisting low temperature stress, and the basic information of CRT genes in pepper can be understood through this study. The information obtained is of great significance for the use of CRT gene to improve plant stress resistance.