烟草苯丙氨酸解氨酶基因NtPAL的克隆及功能分析  

Cloning and functional analysis of the phenylalanine ammonia-lyase gene NtPAL in tobacco

作　　者：许力 许永 向海英 黄海涛 米其利 陈钰栋 蒋佳芮 李雪梅 高茜 XU Li;XU Yong;XIANG Haiying;HUANG Haitao;MI Qili;CHEN Yudong;JIANG Jiarui;LI Xuemei;GAO Qian(Technology Centre of China Tobacco Yunnan Industrial Co.,Ltd.,Kunming 650231,China)

机构地区：[1]云南中烟工业有限责任公司技术中心,昆明市五华区科医路41号650231

出　　处：《中国烟草学报》2025年第2期93-103,共11页Acta Tabacaria Sinica

基　　金：中国烟草总公司科技项目“多酚类物质代谢相关新品系配方适用性评价及应用”(110202201015(JY-15));云南大观实验室课题、云南中烟工业有限责任公司科技项目“低芸香苷新品系的品质保障技术优化及烟叶配伍性研究”(YNDG202301YZ01、2023YJ02)。

摘　　要：【背景和目的】烟草苯丙氨酸解氨酶参与苯丙氨酸代谢途径,不仅跟烟叶香气品质相关,而且对烟草抗病、抗逆等防御功能也有影响。为鉴定苯丙氨酸解氨酶参与烟草抗花叶病途径的基因功能。【方法】采用同源克隆的方法,在栽培烟草红花大金元中克隆了苯丙氨酸解氨酶基因NtPAL,利用生物信息学方法分析NtPAL蛋白结构和进化关系。采用CRISPR/cas9基因编辑技术获得NtPAL基因编辑植株PAL-KO,在植株成熟期测定多酚含量和苯丙氨酸解氨酶活性。利用本氏烟草瞬时表达系统,进行NtPAL亚细胞定位分析。【结果】烟草NtPAL基因的全长CDS序列为2016bp,编码一个由671个氨基酸组成的PAL蛋白。NtPAL含有典型的苯丙氨酸解氨酶结构域PLN02457,是一个新的烟草NtPAL基因。亚细胞定位实验表明NtPAL定位在细胞核区域。与对照植株相比,编辑植株PAL-KO对烟草花叶病毒(TMV)的抗性降低,成熟期叶片中新绿原酸、莨菪亭、隐绿原酸、绿原酸及芸香苷5种多酚含量均显著升高,苯丙氨酸解氨酶活性降低。【结论】烟草NtPAL通过调控多酚含量与苯丙氨酸解氨酶活性响应烟草花叶病抗性。该研究结果为深入阐明NtPAL在烟草抗病性增强和遗传改良方面的潜力奠定了理论基础。Tobacco phenylalanine ammonia-lyase(PAL)participates in the phenylalanine metabolism pathway,which is related to tobacco aroma quality and plays a role in defense functions,including disease and stress resistance.The aim is to identify the gene function of phenylalanine ammonia-lyase in the pathway of resistance to tobacco mosaic virus(TMV).To identify the gene function of phenylalanine ammoniase involved in tobacco anti-mosaic pathway,the NtPAL gene was identified and cloned from cultivated tobacco(cv.:Hong Hua Da Jin Yuan)by homologous cloning.The protein structures and evolutionary relationships of NtPAL in tobacco were analyzed using bioinformatics method.CRISPR/cas9 gene editing technology was employed to obtain NtPAL gene-edited plants(PAL-KO),and polyphenol content and PAL activity were measured at the plant’s mature stage.Subcellular localization of NtPAL was analyzed using a transient expression system in Nicotiana benthamiana.The protein coding sequence of the tobacco NtPAL gene is 2016bp,encoding a PAL protein consisting of 671 amino acids.NtPAL contains a typical phenylalanine ammonia-lyase domain(PLN02457),identifying it as a novel NtPAL gene in tobacco.Subcellular localization experiments showed that NtPAL is localized in the nucleus.Compared with CK,,the PAL-KO edited plants showed reduced resistance to TMV.In mature leaves,the contents of five polyphenols—neochlorogenic acid,scopoletin,cryptochlorogenic acid,chlorogenic acid,and rutin—were significantly increased,while PAL activity was decreased.Tobacco NtPAL responds to TMV resistance by regulating polyphenols content and phenylalanine ammoniase activity.The results of this study provide a theoretical basis for further elucidating the potential of NtPAL in enhancing disease resistance and genetic improvement of tobacco.

关 键 词：烟草 NtPAL 亚细胞定位 基因功能 抗病性 

分 类 号：S572[农业科学—烟草工业]