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作物学报 ›› 2025, Vol. 51 ›› Issue (9): 2527-2537.doi: 10.3724/SP.J.1006.2025.44216

• 研究简报 • 上一篇    下一篇

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 接受日期:2025-06-04 出版日期:2025-09-12 网络出版日期:2025-06-16
  • 通讯作者: *文柳璎, E-mail: wenliuying@caas.cn; 张强, E-mail: 283240782@qq.com; 高军平, E-mail: gaojp0104@hngy.tobacco.com
  • 作者简介:吉白璐, E-mail: ji15935932310@163.com;孙艺文, E-mail: 312888167@qq.com
    **同等贡献
  • 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);中国烟草总公司陕西省公司科技项目(KJ-2023-02)

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. 1Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao 266101, Shandong, China
    2China Tobacco Hunan Industrial Co., Ltd., Changsha 410007, Hunan, China
    3School of Agriculture, Yunnan University, Kunming 650500, Yunnan, China
    4Tobacco Institute of Shaanxi Province, Xi’an 710061, Shaanxi, China
  • Received:2024-12-24 Accepted:2025-06-04 Published:2025-09-12 Published online:2025-06-16
  • Contact: *E-mail: wenliuying@caas.cn; E-mail: 283240782@qq.com; E-mail: gaojp0104@hngy.tobacco.com
  • About author:**Contributed equally to this work
  • Supported by:
    Agricultural Science and Technology Innovation Program of CAAS(ASTIP-TRIC01);Natural Science Foundation of Shandong Province, China(ZR2023MC139);Natural Science Foundation of Tobacco Genome Project of State Tobacco Monopoly Administration (110202201010(JY-17);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试验依据。

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

图1

NtLPAT基因序列对比以及表达模式分析 图A中, M为DL 2000 marker, 1为阴性对照, 2为目的条带。图B为烟草、马铃薯、辣椒与番茄LPAT序列的比对。图C为NtLPAT在烟草不同组织中的表达量, Root: 地下部; Shoot: 地上部; Shoot apex: 苗端。图D为NtLPAT响应黑胫病的表达模式, 0、6、12、24 hpi表示接菌后0、6、12、24 h, R表示烟草抗黑胫品种BH1000-1, S表示感黑胫烟草品种小黄金1025。图E为NtLPAT响应青枯病表达模式, 3、9、24 hpi表示接菌后3、9、24 h, R表示烟草抗青枯品种反帝三号-丙, S表示烟草感青枯品种红花大金元, CK表示未接菌对照, RS表示接种青枯菌。*、**和***分别表示在0.05、0.01和0.001水平差异显著。"

图2

ntlpat对青枯病抗性鉴定 图A为转基因材料鉴定峰图; 图B为K326与ntlpat接种青枯病菌后14、18、22、25、28、32、35 d时的病情指数; 图C为K326与ntlpat接种青枯0 d与35 d时的烟苗状态。*表示P < 0.05。"

表1

ntlpat株系病毒病抗性分析"

样品Sample TMV PVY CMV
K326 38.26±8.03 b 60.93±3.51 a 39.23±8.85 b
ntlpat 63.75±11.36 a 66.35±4.31 a 20.43±4.66 a

表2

ntlpat突变株系主要农艺性状"

样品
Sample		 株高
Plant height
(cm)		 茎围
Stem circumference (cm)		 节距
Stem pitch
(cm)		 叶数
Leaf number		 腰叶长
Middle leaf length (cm)		 腰叶宽
Middle leaf width (cm)
K326 103.60±2.07 a 8.28±4.25 a 5.05±0.45 a 17.40±0.89 a 67.10±2.08 a 24.00±1.96 a
ntlpat 82.50±8.89 b 8.48±0.45 a 3.40±0.65 b 16.75±1.26 a 63.60±4.59 a 23.38±2.43 a

表3

ntlpat突变株系烟叶外观评价"

样品
Sample		 颜色
Color		 成熟度
Maturity		 油分
Oil		 身份
Body		 结构
Structure		 色度
Color intensity		 总分
Score		 量化总分
Quantify the total score
K326 7 8 8 9 8 6 46 7.71
ntlpat 7 8 6 9 8 5 43 7.47

图3

ntlpat突变株系脂质组分析图 图A为差异代谢物KEGG分类图; 图B为脂类代谢途径中的差异脂类示意图。TG: 甘油三脂; PG: 磷脂酰甘油; MGDG: 单半乳糖二酰基甘油; DGDG: 二半乳糖二酰基甘油; Cer: 神经酰胺; SPH: 鞘磷脂; PC: 磷脂酰甘油; LPA: 溶血磷脂酸; LPC: 溶血磷脂酰胆碱。"

图4

ntlpat突变株系代谢组分析图 图A为差异表达基因GO富集柱状图; 图B为差异表达基因KEGG富集气泡图; 图C为磷脂合成途径和光合系统复合体差异表达基因示意图。"

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