桃PpSGR基因功能鉴定及其对乙烯合成的调控

Function identification of PpSGR gene and its regulation of ethylene syn-thesis in peach

【目的】乙烯合成及果肉褪绿是桃果实成熟过程中相伴出现的两个生理事件。STAY-GREEN(SGR)是参与植物叶片和果实褪绿的重要基因。然而,桃SGR基因在果实成熟及褪绿过程中的功能尚不清晰,旨在初步探究PpSGR基因在桃果实成熟及褪绿过程中的功能。【方法】以秋蜜红为试验材料,对PpSGR基因进行克隆,对PpSGR的核苷酸及氨基酸序列进行分析,对不同发育时期桃果肉中PpSGR的转录水平进行检测,并对PpSGR基因调控叶绿素降解及乙烯合成的功能进行研究。【结果】PpSGR编码区全长为831 bp;该基因编码的蛋白序列含有1个高度保守的SGR域。PpSGR基因的表达水平随着果肉逐渐褪绿呈现上升的趋势。瞬时过表达PpSGR基因后,桃叶片颜色明显褪绿,并且在300 mmol·L-1NaCl的盐胁迫下,过表达PpSGR的叶片褪绿更加明显。此外,过表达PpSGR后,桃苗乙烯合成的限速基因PpACS1、PpACS4及PpACS6均表达上调,且内源乙烯释放量显著增多。【结论】对PpSGR的基因功能进行鉴定和研究,并分析了其对乙烯的调控作用,为进一步解析桃果实成熟及果肉褪绿提供了新的思路,也为不同成熟期桃品种的选育提供了理论基础。

【Objective】Ethylene synthesis and disappearance of peel green color are two physiological events associated with fruit ripening of peach.STAY-GREEN(SGR)is a crucial gene that promotes dis-appearance of leaf and fruit green color in the plant kingdom.In various plant species,SGR genes exhib-it similar functions in regulating chlorophyll.Peach(Prunus persica)is a significant fruit globally.How-ever,the function of the PpSGR gene in the peach genome has not been identified,and its potential im-pact on green color disappearance and ripening of peach fruit remains unclear.This study aims to identi-fy the SGR gene in the peach genome,analyze the expression level of the PpSGR gene in different fruit maturity stages,and preliminarily investigate the function of the PpSGR gene.【Methods】The nucleo-tide sequences of the PpSGR gene were cloned from the Qiumihong cultivar.Bioinformatics analysis was conducted to analyze the gene structure of PpSGR in peach,the secondary and tertiary structure of the PpSGR protein was predict,the amino acid sequences of SGR in different species were compared,and a phylogenetic tree was constructed.Subsequently,the transcriptional level of the PpSGR gene in peach fruits at different maturity stages was analyzed using Quantitative Real-time PCR(qRT-PCR).The function of the PpSGR gene in chlorophyll degradation was preliminarily verified by transient over-expression in peach seedlings.Numerous studies have reported that the SGR gene can promote chloro-phyll degradation under salt stress,leading to leaf yellowing.To verify the function of SGR under salt stress,the Fv/Fm values were measured and analyzed using a chlorophyll fluorescence analyzer after dark treatment of the whole peach seedlings transiently infected with the PpSGR gene.Additionally,there is evidence suggesting that ethylene can promote the expression of the SGR gene,but whether the SGR gene has a direct effect on ethylene synthesis remains to be studied.Therefore,we measured ethyl-ene production and the transcriptional level of PpACSs genes involved in ethylene synthesis in the peach seedlings transiently overexpressing the PpSGR gene.【Results】The results of the bioinformatics analysis revealed that the length of the PpSGR gene sequencewas 1115 bp,consisting of 4 exons and 3 introns.The coding region of PpSGR spanned a total length of 831 bp,encoding 277 amino acids.Com-parative analysis of SGR proteins from different species demonstrated that the PpSGR protein shared high homology with SGR proteins from other species.The middle region of the SGR protein contained a highly conserved SGR domain,while the N-terminal region contained a conserved chloroplast transit peptide.The C-terminal region of the SGR protein exhibited variability across different species.The amino acid sequence similarities between PpSGR and MdSGR,FvSGR or CsSGR were found to be 77.63%,69.83%and 63.05%,respectively.A phylogenetic tree was constructed using amino acid se-quences of SGR proteins from 13 different plant species,revealing that the PpSGR protein from peach was closely related to apples and strawberries,which belonged to the same Rosaceae family.The pre-dicted tertiary structure of the PpSGR protein consisted mainly ofα-helices and random graph curls,which was consistent with the predicted secondary structure.qRT-PCR results indicated that the tran-scription level of the PpSGR gene was lower during the early stage of peach fruit development when the flesh was still green.As the fruit matured gradually,the flesh turned green,and the expression level of PpSGR increased continuously.At 120 days after the full-bloom stage,the expression of PpSGR in-creased rapidly,reaching a level of 16 times higher than that at 80 days after the full-bloom stage,with the highest expression observed at 180 days after the full-bloom stage.This suggested that the expres-sion level of the PpSGR gene gradually increased with the decolorization of peach fruit.Phenotypic analysis of PpSGR transgenic plants revealed that the leaves of transient PpSGR plants were light green,whereas the control plants had dark green leaves.This suggested that transient overexpression of the PpSGR gene led to chlorosis in peach seedlings.Under salt stress conditions(300 mmol·L-1 NaCl),the leaves of peach plants transiently overexpressing PpSGR exhibited a slower rate of yellowing,com-pared to control plants.The Fv/Fm value of the leaves in PpSGR-overexpressing plants was significantly lower than that of the control plants,indicating that the PpSGR gene may play an important role in leaf yellowing induced by the abiotic stress.Furthermore,the expression levels of genes involved in ethyl-ene production were analyzed in peach seedlings transiently overexpressing PpSGR.The results demon-strated that the ethylene synthesis genes PpACS1,PpACS4 and PpACS6 were significantly induced and up-regulated in these seedlings.The expression of PpACS1 in peach seedlings with PpSGR overexpres-sion was more than 32 times higher than that in control seedlings.Additionally,the ethylene production of peach seedlings with PpSGR overexpression showed a gradual increase.【Conclusion】Based on the aforementioned findings,it can be concluded that the PpSGR gene plays a role in promoting chlorosis in peach leaves.Moreover,our preliminary analysis suggests that PpSGR may enhance the function of ethylene synthesis in peach seedlings.This finding holds great significance for understanding the molec-ular mechanism of SGR in regulating ethylene synthesis.Additionally,it also provides some theoretical references for SGR gene function research,peach fruit ripening and post-harvest preservation.