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Pumpkin (Cucurbita moschata) HSP20 Gene Family Identification and Expression Under Heat Stress

ABSTRACT: Pumpkin (Cucurbita moschata) is an important cucurbit vegetable crop that has strong resistance to abiotic stress. While heat shock protein 20 (HSP20) has been implicated in vegetable response to heat stress, little is known regarding activity of HSP20 family proteins in C. moschata. Here, we performed a comprehensive genome-wide analysis to identify and characterize the functional dynamics of the Cucurbita moschata HSP20 (CmoHSP20) gene family. A total of 33 HSP20 genes distributed across 13 chromosomes were identified from the pumpkin genome. Our phylogenetic analysis determined that the CmoHSP20 proteins fell into nine distinct subfamilies, a division supported by the conserved motif composition and gene structure analyses. Segmental duplication events were shown to play a key role in expansion of the CmoHSP20 gene family. Synteny analysis revealed that 19 and 18 CmoHSP20 genes were collinear with those in the cucumber and melon genomes, respectively. Furthermore, the expression levels of pumpkin HSP20 genes were differentially induced by heat stress. The transcript level of CmoHSP20-16, 24 and 25 were down-regulated by heat stress, while CmoHSP20-7, 13, 18, 22, 26 and 32 were up-regulated by heat stress, which could be used as heat tolerance candidate genes. Overall, these findings contribute to our understanding of vegetable HSP20 family genes and provide valuable information that can be used to breed heat stress resistance in cucurbit vegetable crops.

SUBMITTER: Hu Y

PROVIDER: S-EPMC8553033 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Heat shock protein 70s (HSP70s) are highly conserved proteins that are involved in stress responses. These chaperones play pivotal roles in protein folding, removing the extra amounts of oxidized proteins, preventing protein denaturation, and improving the antioxidant system activities. This conserved family has been characterized in several crops under drought stress conditions. However, there is no study on HSP70s in pumpkin (Cucurbita moschata). Therefore, we performed a comprehensive analysis of this gene family, including phylogenetic relationship, motif and gene structure analysis, gene duplication, collinearity, and promoter analysis. In this research, we found 21 HSP70s that were classified into five groups (from A to E). These genes were mostly localized in the cytoplasm, chloroplast, mitochondria, nucleus, and endoplasmic reticulum (ER). We could observe more similarity in closely linked subfamilies in terms of motifs, the number of introns/exons, and the corresponding cellular compartments. According to the collinearity analysis, gene duplication had occurred as a result of purifying selection. The results showed that the occurrence of gene duplication for all nine gene pairs was due to segmental duplication (SD). Synteny analysis revealed a closer relationship between pumpkin and cucumber than pumpkin and Arabidopsis. Promoter analysis showed the presence of various cis-regulatory elements in the up-stream region of the HSP70 genes, such as hormones and stress-responsive elements, indicating a potential role of this gene family in stress tolerance. We furtherly performed the gene expression analysis of the HSP70s in pumpkin under progressive drought stress. Pumpkin is widely used as a rootstock to improve stress tolerance, as well as fruit quality of cucumber scion. Since stress-responsive mobile molecules translocate through vascular tissue from roots to the whole plant body, we used the xylem of grafted materials to study the expression patterns of the HSP70 (potentially mobile) gene family. The results indicated that all CmoHSP70s had very low expression levels at 4 days after stress (DAS). However, the genes showed different expression patterns by progressing he drought period. For example, the expression of CmoHSP70-4 (in subgroup E) and CmoHSP70-14 (in subgroup C) sharply increased at 6 and 11 DAS, respectively. However, the expression of all genes belonging to subgroup A did not change significantly in response to drought stress. These findings indicated the diverse roles of this gene family under drought stress and provided valuable information for further investigation on the function of this gene family, especially under stressful conditions.

Project description:Background:Quantitative real-time PCR (qRT-PCR) is a commonly used high-throughput technique to measure mRNA transcript levels. The accuracy of this evaluation of gene expression depends on the use of optimal reference genes. Cucumber-pumpkin grafted plants, made by grafting a cucumber scion onto pumpkin rootstock, are superior to either parent plant, as grafting conveys many advantages. However, although many reliable reference genes have been identified in both cucumber and pumpkin, none have been obtained for cucumber-pumpkin grafted plants. Methods:In this work, 12 candidate reference genes, including eight traditional genes and four novel genes identified from our transcriptome data, were selected to assess their expression stability. Their expression levels in 25 samples, including three cucumber and three pumpkin samples from different organs, and 19 cucumber-pumpkin grafted samples from different organs, conditions, and varieties, were analyzed by qRT-PCR, and the stability of their expression was assessed by the comparative ?Ct method, geNorm, NormFinder, BestKeeper, and RefFinder. Results:The results showed that the most suitable reference gene varied dependent on the organs, conditions, and varieties. CACS and 40SRPS8 were the most stable reference genes for all samples in our research. TIP41 and CACS showed the most stable expression in different cucumber organs, TIP41 and PP2A were the optimal reference genes in pumpkin organs, and CACS and 40SRPS8 were the most stable genes in all grafted cucumber samples. However, the optimal reference gene varied under different conditions. CACS and 40SRPS8 were the best combination of genes in different organs of cucumber-pumpkin grafted plants, TUA and RPL36Aa were the most stable in the graft union under cold stress, LEA26 and ARF showed the most stable expression in the graft union during the healing process, and TIP41 and PP2A were the most stable across different varieties of cucumber-pumpkin grafted plants. The use of LEA26, ARF and LEA26+ARF as reference genes were further verified by analyzing the expression levels of csaCYCD3;1, csaRUL, cmoRUL, and cmoPIN in the graft union at different time points after grafting. Discussion:This work is the first report of appropriate reference genes in grafted cucumber plants and provides useful information for the study of gene expression and molecular mechanisms in cucumber-pumpkin grafted plants.

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Pumpkin (Cucurbita moschata) HSP20 Gene Family Identification and Expression Under Heat Stress

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