Construction and functional identification of overexpression vector of the peach PpCLH1 gene

【Objective】Pericarp chlorophyll degradation is a prominent signal during the ripening of peach fruit. Chlorophyllase (CLH) is considered the primary enzyme in the process of chlorophyll degradation and is a crucial gene involved in the degreening of plant leaves and fruits. The degradation of chlorophyll is an important signal for the onset of fruit ripening and the chlorophyll content plays a significant role in fruit appearance, impacting consumer choice. However, the role of the CLH gene in peach fruit pericarp chlorophyll degradation during development remains unclear. Therefore, the objective of this study was to investigate the transcript level of the PpCLH1 gene prior to fruit ripening and its role in pericarp chlorophyll degradation during fruit development.【Methods】Zhongpan 102 （Prunus persica L.）was selected as the experimental material. It has been observed that the expression level of the PpCLH1 increases during the fruit color transition period. However, there have been no reports on the functional study of the chlorophyll degradation gene PpCLH1 in peach. To address this issue, the CDS of the related gene was obtained from the genomic database, and the amino acid sequence of the PpCLH1 was analyzed. The function of the PpCLH1 gene was demonstrated using tobacco transient expression, and the overexpression of the PpCLH1 gene in tobacco was determined by real-timequantitative polymerase chain reaction (qRT- PCR).【Results】The coding sequence of the PpCLH1 gene spaned 972 base pairs and encoded a protein comprising 323 amino acids. An initial examination of the physicochemical properties of the PpCLH1 protein, was conducted utilizing the Expasy tool, disclosed an isoelectric point of 6.12, with a complement of 26 positively charged and 31 negatively charged residues. The mean hydrophilicity index stood at 0.065, surpassing the global average, while the instability index was computed to be 44.56, hinting at potential protein instability. Additionally, the aliphatic index registered at 88.76, further supporting the notion of protein instability. The advanced secondary structure analysis of the peach- derived PpCLH1 protein, executed on the Sopma platform, revealed a structural composition dominated by α-helices (29.72%) and β-sheets (4.64%), supplemented by extended strands constituting 16.10% and irregular coils accounting for 49.54% of the structure. The tertiary structure predictions, facilitated by the SWISS-MODEL server, predominantly depicted irregular coil conformations, corroborating the findings from secondary structure analysis and additionally noting the presence of extended chains and β- sheet elements.To investigate homology, a BLASTp search against the NCBI database was performed, identifying a closely related protein sequence. A comparative amino acid sequence alignment generated via DNAMAN software highlighted that the peach PpCLH1 shared the highest degree of homology, 99.2%, with the apricot PdCLH1, underscoring their genetic affinity. Similarly, cherry PaCLH1 exhibited a notable similarity of 96.6% to PpCLH1, indicative of a conserved CLH1 function across the Rosaceae family, specifically within the genus Prunus. The phylogenetic analysis clustered peach PpCLH1, apricot PdCLH1, and cherry PaCLH1 into a single clade, evidencing their close evolutionary proximity. Notably, these proteins also demonstrated elevated homology with the apple MdCLH1 protein, reinforcing the conservation hypothesis of CLH1 proteins throughout the Rosaceae family. Conversely, CLH1 proteins from tomato and tobacco showed more remote phylogenetic relationships. To ascertain the transcriptional dynamics of PpCLH1 during the preripening phase of peach fruit, RNA was isolated and reverse-transcribed into cDNA for subsequent qRTPCR quantification. This analysis unveiled a low expression level of PpCLH1 30 days prior to fruit maturation, characterized by a distinct green pericarp. As the ripening process unfolded, the pericarp transitioned to a paler green hue, paralleled by a surge in PpCLH1 transcript abundance. Specifically, at 16 days preceding fruit ripening, a marked elevation in PpCLH1 expression was observed, peaking at 12 days before ripening—a twelvefold increase compared to its baseline at 30 days prior. An overexpression construct for PpCLH1 was engineered through homologous recombination techniques and transiently introduced into tobacco leaves to assess functional implications. The phenotypic evaluation of transfected tobacco leaves displayed a lighter green phenotype relative to the dark green control leaves, accompanied by a substantial reduction in chlorophyll content. Collectively, these observations imply that PpCLH1 would be instrumental in facilitating chlorophyll breakdown in tobacco, thereby providing indirect evidence for its regulatory role in chlorophyll degradation and concomitant color transformations in peach fruit during ripening. Consequently, it could be postulated that the PpCLH1 gene would act as a pivotal regulator of chlorophyll metabolism and be associated with visual changes in peach fruits approaching maturity.【Conclusion】This study conducted identification and functional studies on the PpCLH1 gene, discovering that PpCLH1 is significantly highly expressed during the fruit color transition period. The transient overexpression of the PpCLH1 in tobacco leaves led to a noticeable chlorosis of the leaves. These findings would provide new insights into the degradation of chlorophyll in peach skin throughout the entire fruit development process. Furthermore, they would offer valuable references for exploring the molecular mechanisms of chlorophyll degradation during peach fruit development.