桃PpCLH1基因过表达载体构建与功能鉴定  

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

作　　者：刘鑫 张晓煜 孟君仁 李昂 段文宜 孙世航 潘磊[1] 曾文芳[1] 王志强[1] 牛良[1] LIU Xin;ZHANG Xiaoyu;MENG Junren;LI Ang;DUAN Wenyi;SUN Shihang;PAN Lei;ZENGWenfang;WANG Zhiqiang;NIU Liang(Zhengzhou Fruit Research Institute,Chinese Academy of Agricultural Sciences/National Peach&Grape Improvement Center/Zhongyuan Research Center,Zhengzhou 450009,Henan,China)

机构地区：[1]中国农业科学院郑州果树研究所·国家桃、葡萄改良中心·中原研究中心,郑州450009

出　　处：《果树学报》2025年第3期476-485,共10页Journal of Fruit Science

基　　金：国家桃产业技术体系(CARS-30);中国农业科学院科技创新工程专项经费项目(CAAS-ASTIP-2023-ZFRI);河南省重大公益科技专项(No.201300110500)。

摘　　要：【目的】探究PpCLH1基因在叶绿素降解中的功能。果皮叶绿素降解是桃果实成熟过程中的标志性信号之一。叶绿素酶(Chlorophyllase,CLH)一直被人们认为是叶绿素降解过程中的第一种酶,CLH是参与植物叶片和果实褪绿的重要基因。然而,桃CLH基因在果皮叶绿素降解中的功能尚不清晰。【方法】以中蟠102为试验材料,克隆PpCLH1基因,分析PpCLH1的氨基酸序列,检测桃果实成熟过程中果皮PpCLH1的转录水平,研究PpCLH1基因在桃叶绿素降解过程中的功能。【结果】PpCLH1编码区全长为972 bp,编码323个氨基酸,在蔷薇科植物中表现出高度的保守性。PpCLH1基因的表达水平随着果实成熟褪绿呈现逐渐上升的趋势,在成熟前12 d达到最高,之后表达量不断降低。瞬时过表达PpCLH1基因后,烟草叶片颜色出现明显褪绿。PpCLH1是桃果皮转色期叶绿素降解的关键基因。【结论】通过对PpCLH1基因进行鉴定和功能研究,发现果实成熟前12 d PpCLH1明显高表达,在烟草叶片中瞬时过表达PpCLH1会导致叶片明显褪绿,这为进一步解析桃果实发育过程中叶绿素降解的分子机制提供了参考。【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-time quantitative 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

关 键 词：桃 叶绿素降解 叶绿素酶 PpCLH1基因 

分 类 号：S662.1[农业科学—果树学]