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烟草脂类合成关键基因NtLPAT的功能验证

吉白璐1,3,**，孙艺文1,**，刘万峰2，钱亚新1,3，蒋彩虹1，耿锐梅1，刘旦1，程立锐1，杨爱国1，黄立钰3，李晓旭2，蒲文宣2，高军平2,*，张强4,*，文柳璎1,*   

  1. 1中国农业科学院烟草研究所, 山东青岛266101; 2湖南中烟工业有限责任公司, 湖南长沙410007; 3云南大学农学院, 云南昆明650500; 4陕西省烟草科学研究所, 陕西西安710061
  • 收稿日期:2024-12-24 修回日期:2025-06-04 接受日期:2025-06-04 网络出版日期:2025-06-16
  • 基金资助:
    本研究由中国农业科学院科技创新工程项目(ASTIP-TRIC01), 山东省自然科学基金项目(ZR2023MC139), 烟草基因组计划和生物育种重大科技项目(110202201010(JY-17))和中国烟草总公司陕西省公司科技项目(KJ-2023-02)资助。

Functional verification of the key gene NtLPAT involved in lipid biosynthesis in tobacco

JI Bai-Lu1,3,**,SUN Yi-Wen1,**,LIU Wan-Feng2,QIAN Ya-Xin1,3,JIANG Cai-Hong1,GENG Rui-Mei1,LIU Dan1,CHENG Li-Rui1,YANG Ai-Guo1,HUANG Li-Yu3,LI Xiao-Xu2,PU Wen-Xuan2,GAO Jun-Ping2,*,ZHANG Qiang4,*,WEN Liu-Ying1,*   

  1. 1 Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao 266101, Shandong, China; 2 China Tobacco Hunan Industrial Co., Ltd., Changsha 410007, Hunan, China; 3 School of Agriculture, Yunnan University, Kunming 650500, Yunnan, China; 4 Tobacco Institute of Shaanxi Province, Xi’an 710061, Shaanxi, China
  • Received:2024-12-24 Revised:2025-06-04 Accepted:2025-06-04 Published online:2025-06-16
  • Supported by:
    This study was supported by the Agricultural Science and Technology Innovation Program of CAAS (ASTIP-TRIC01), the Natural Science Foundation of Shandong Province, China (ZR2023MC139), the Natural Science Foundation of Tobacco Genome Project of State Tobacco Monopoly Administration (110202201010(JY-17)), and the Technology Project of the Shaanxi Provincial Company of China National Tobacco Corporation (KJ-2023-02). 

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摘要：

溶血磷脂酰基转移酶(LPAT)是负责催化溶血磷脂酸(LPA)和脂肪酰基-辅酶A (Acyl-CoA)酯化生成磷脂酸(PA)合成途径的关键酶，尚不清楚烟草中LPAT的基因功能。本研究从K326中克隆得到NtLPAT基因，利用CRISPR/Cas9技术获得NtLPAT敲除植株ntlpat，对其农艺性状和抗病性以及外观质量进行鉴定，结合脂质组、转录组分析NtLPAT的功能。结果表明，NtLPAT受青枯病菌和黑胫病病菌的诱导。ntlpat株高降低，对黄瓜花叶病毒病(CMV)与青枯病的抗性提高。脂质组分析表明，ntlpat中甘油酯代谢发生了重排，三酰甘油含量显著降低，组成类囊体膜的甘油糖脂含量(MGDG，DGDG)随之上升，并且鞘脂和磷脂酰肌醇含量也发生变化。转录组分析表明，ntlpat的光合作用、碳固定代谢、鞘脂合成和磷脂酰肌醇信号转导途径的相关基因发生重编程，因此推测NtLPAT具有脂酰基转移酶活性，调控三酰甘油的从头合成途径，并参与细胞信号传导，影响烟草的生长和对CMV、青枯病菌的抗性，研究结果为挖掘烟草株型及抗性改良提供基因资源和实验依据。

关键词: 溶血磷脂酰基转移酶, 烟草, 基因敲除, 脂质组学, 转录组学

Abstract:

Lysophosphatidyl transferase (LPAT) is a key enzyme in the lipid biosynthesis pathway, catalyzing the transfer of a fatty acyl group from fatty acyl-CoA (Acyl-CoA) to lysophosphatidic acid (LPA) to produce phosphatidic acid (PA). However, the functional role of LPAT in tobacco remains largely unexplored. In this study, we cloned the NtLPAT gene from the tobacco cultivar K326 and generated an NtLPAT knockout mutant (ntlpat) using CRISPR/Cas9 technology. The ntlpat mutant was evaluated for agronomic traits, disease resistance, and phenotypic appearance. In addition, lipidomic and transcriptomic analyses were conducted to assess the impact of NtLPAT loss of function. Our results showed that NtLPAT expression was induced by infection with Ralstonia solanacearum and Phytophthora parasitica. The ntlpat mutant exhibited reduced plant height but enhanced resistance to cucumber mosaic virus (CMV) and bacterial wilt. Lipidomic analysis revealed altered glyceride metabolism in the mutant: triacylglycerol (TAG) levels were significantly decreased, while the contents of two major glycerol glycolipids—monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), both critical components of the thylakoid membrane—were increased. Additionally, changes were observed in sphingolipid and phosphatidylinositol compositions. Transcriptomic analysis indicated that genes involved in photosynthesis, carbon fixation, sphingolipid biosynthesis, and phosphatidylinositol signaling pathways were reprogrammed in ntlpat. These findings suggest that NtLPAT possesses acyltransferase activity, regulates de novo TAG biosynthesis, and plays a role in cellular signaling pathways, thereby affecting tobacco growth and resistance to CMV and R. solanacearum. This study provides valuable genetic resources and an experimental basis for tobacco breeding improvement.

Key words: lysophosphatidyl transferase, tobacco, gene knock-out, lipidomics, transcriptome

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