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作物学报 ›› 2023, Vol. 49 ›› Issue (4): 1016-1027.doi: 10.3724/SP.J.1006.2023.24037

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蓝型油菜长链烷烃合成相关基因的克隆及其与BnCER1-2的互作

柏成成**(), 姚小尧**(), 王雨璐, 王赛玉, 李金莹, 蒋有为, 靳舒荣, 陈春杰, 刘渔, 魏星玥, 徐新福, 李加纳, 倪郁*()   

  1. 西南大学农学与生物科技学院/西南大学农业科学研究院, 重庆 400716
  • 收稿日期:2022-02-13 接受日期:2022-06-07 出版日期:2023-04-12 网络出版日期:2022-06-21
  • 通讯作者: *倪郁, E-mail: nmniyu@126.com
  • 作者简介:柏成成, E-mail: 1316502363@qq.com;
    姚小尧, E-mail: 823706108@qq.com
    **同等贡献
  • 基金资助:
    国家自然科学基金项目(32171938);国家自然科学基金项目(31771694);重庆市自然科学基金项目(cstc2021jcyj-msxmX0332);财政部和农村农业部国家现代农业产业技术体系建设专项(CARS-12)

Cloning of genes involved in cuticular very-long-chain alkane synthesis and its interaction with BnCER1-2 in Brassica napus

BAI Cheng-Cheng**(), YAO Xiao-Yao**(), WANG Yu-Lu, WANG Sai-Yu, LI Jin-Ying, JIANG You-Wei, JIN Shu-Rong, CHEN Chun-Jie, LIU Yu, WEI Xing-Yue, XU Xin-Fu, LI Jia-Na, NI Yu*()   

  1. College of Agronomy and Biotechnology/Academy of Agricultural Sciences, Southwest University, Chongqing 400716, China
  • Received:2022-02-13 Accepted:2022-06-07 Published:2023-04-12 Published online:2022-06-21
  • Contact: *E-mail: nmniyu@126.com
  • About author:**Contributed equally to this work
  • Supported by:
    National Natural Science Foundation of China(32171938);National Natural Science Foundation of China(31771694);Natural Science Foundation of Chongqing, China(cstc2021jcyj-msxmX0332);China Agriculture Research System of MOF and MARA(CARS-12)

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摘要：

长链烷烃是甘蓝型油菜角质层蜡质的优势组分, 在阻止植株的非气孔性水分散失中起主要作用。BnCER1-2催化甘蓝型油菜长链烷烃的生物合成, 但BnCER1-2是否通过与其他蛋白互作调控长链烷烃合成还不清楚。前期通过甘蓝型油菜蜡质差异材料转录组筛选获得4个长链烷烃合成相关基因BnCER3.a10、BnCER3.c02、BnCYTB5B.c09、BnCER1-L2.a05。本研究克隆了这4个基因的编码序列, 序列分析表明BnCER3.a10/c02和BnCER1-L2.a05前体蛋白具有典型的脂肪酸羟化酶与WAX2 C末端结构域, 而BnCYTB5B.c09具有Cyt_B5蛋白家族保守结构域。亚细胞定位结果表明, BnCER3.a10/c02、BnCYTB5B.c09和BnCER1-L2.a05均定位于细胞内质网, 与BnCER1-2共定位。双分子荧光互补(bimolecular fluorescent complementation, BiFC)与萤火素酶互补试验(luciferase complementation assay, LCA)检测结果表明, BnCER3.a10、BnCYTB5B.c09、BnCER1-L2.a05与BnCER1-2蛋白存在相互作用, 而BnCER3.c02与BnCER1-2蛋白不互作。实时荧光定量PCR结果显示, 与BnCER1-2的表达模式一致, BnCER3.a10和BnCYTB5B.c09主要在甘蓝型油菜茎/叶中表达, 并受干旱胁迫诱导显著上调。BnCER3.a10在NaCl与低温胁迫下表达量显著减少, 其中BnCER3.a10受MeJA、ACC诱导显著下调, BnCYTB5B.c09表达受ABA诱导上调。BnCER1-L2.a05在花中的表达量最高, 在茎和叶片中的表达量最低, 在干旱、低温及NaCl胁迫下转录水平均显著下降, 其中SA诱导BnCER1-L2.a05表达上调, 而MeJA诱导其表达下调。蜡质差异材料荧光定量PCR结果证实, BnCER3.a10与BnCYTB5B.c09在高蜡(烷)油菜中的表达量显著高于低蜡(烷)油菜, 而BnCER1-L2.a05则呈相反变化。综合分析认为, BnCER3.a10和BnCYTB5B.c09可能通过与BnCER1-2互作而促进甘蓝型油菜长链烷烃的生物合成, BnCER1-L2.a05可能通过与BnCER1-2互作负调控长链烷烃的合成。

关键词: 甘蓝型油菜, 角质层蜡质, 蛋白互作, 长链烷烃

Abstract:

Very-long-chain (VLC) alkanes are the main components of cuticular wax in Brassica napus, which play a key role in preventing non-stomatal water loss. BnCER1-2 is the core enzyme that catalyzes the synthesis of VLC alkanes in B. napus. However, it is unclear whether BnCER1-2 protein regulates VLC alkane synthesis by interacting with other proteins. Four genes potentially involved in VLC alkane synthesis, BnCER3.a10, BnCER3.c02, BnCYTB5B.c09, and BnCER1-L2.a05, were screened previously by the transcriptome in B. napus with differential wax load. In this study, their coding sequences were cloned from B. napus. A typical fatty acid hydroxylase domain and a wax2 C-terminal domain were detected in the predicted BnCER3.a10/c02 and BnCER1-L2.a05 proteins, while cyt_b5 protein family conserved domain was in the predicted BnCYTB5B.c09. Subcellular localization showed that BnCER3.a10/c02, BnCYTB5B.c09, and BnCER1-L2.a05 were all located in the endoplasmic reticulum, which were co-located with BnCER1-2. Bimolecular fluorescence complementary (BiFC) and luciferase complementation assay (LCA) revealed that BnCER3.a10, BnCYTB5B.c09, and BnCER1-L2.a05 interacted with BnCER1-2 protein, while BnCER3.c02 did not interact with BnCER1-2. The RT-qPCR indicated that BnCER3.a10 and BnCYTB5B.c09 were mainly expressed in the stems or leaves of B. napus, and the relative expression was significantly up-regulated under drought stress, which were consistent with the expression pattern of BnCER1-2. The relative expression levels of BnCER3.a10 decreased significantly under NaCl and low temperature stresses. Further, the relative expression level of BnCER3.a10 was significantly down-regulated by MeJA and ACC, while the relative expression level of BnCYTB5B.c09 was up-regulated by ABA. The highest relative expression of BnCER1-L2.a05 was in flowers and the lowest in stems and leaves, and its expression was significantly down-regulated under drought, cold, and NaCl stresses. Further, the relative expression level of BnCER1-L2.a05 was up-regulated by SA, while down-regulated by MeJA. The relative expression levels of BnCER3.a10 and BnCYTB5B.c09 in B. napus with high wax/alkane load were significantly higher than that with low wax/alkane load, while BnCER1-L2.a05 had the opposite change. Comprehensive analysis suggested that BnCER3.a10 and BnCYTB5B.c09 may promote the biosynthesis of VLC alkanes in B. napus by interacting with BnCER1-2, while BnCER1-L2.a05 may negatively regulate the synthesis of VLC alkanes by interacting with BnCER1-2.

Key words: Brassica napus, cuticular wax, protein interaction, very-long-chain alkanes

表1

BiFC重组载体构建引物"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)		 酶切位点
Restriction site		 重组载体
Vector
FBnCER1-2 TCTGAGGAGGATCTTCCCGGGATGGCTACGAAACCAGGCATCC Sma I nYFP
RBnCER1-2 AGGGCATGCCTGCAGGTCGACTCAGGGGTATTGGAAGTGATGTGG Sal I
FBnCER3.a10 TCTAGGAGCTCGGTACCCGGGATGGTAGCTTTTTCAGCTTGGCCT Sma I cYFP
RBnCER3.a10 ATCGTATGGGTACATACTAGTTCTTGTGAGTGAAGAAACAGAGCGAA Spe I
FBnCER3.c02 TCTAGGAGCTCGGTACCCGGGATGGTTACTTTATCAGTCTGGCCTTGG Sma I cYFP
RBnCER3.c02 ATCGTATGGGTACATACTAGTATTAGTGAGTGAAGACACAGAGCTAAGGC Spe I
FBnCYTB5B.c09 TCTAGGAGCTCGGTACCCGGGATGGGCGGAGACGGCAAAGT Sma I cYFP
RBnCYTB5B.c09 ATCGTATGGGTACATACTAGTAGAAGAAGAAGGAGTCTTGGTGTAAGAACG Spe I
FBnCER1-L2.a05 TCTAGGAGCTCGGTACCCGGGATGGCGTCGAGACCAGGTTTTC Sma I cYFP
RBnCER1-L2.a05 ATCGTATGGGTACATACTAGTGTAACCATTGCTCAATCCTTTGGTCTC Spe I

表2

LCA重组载体构建引物"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)		 酶切位点
Restriction site		 重组载体
Vector
FBnCER1Bi AACACGGGGGACGAGCTCGGTACCATGGCTACGAAACCAGGCAT Kpn I nLUC
RBnCER1Bi GGACGCGTACGAGATCTGGTCGACGGGGTATTGGAAGTGATGTGG Sal I
FBnCER3.a10Bi TACGCGTCCCGGGGCGGTACCATGGTAGCTTTTTCAGCTTGGCCT Kpn I cLUC
RBnCER3.a10Bi ACGAAAGCTCTGCAGGTCGACTCATCTTGTGAGTGAAGAAACAGAGCG Sal I
FBnCER3.c02Bi TACGCGTCCCGGGGCGGTACCATGGTTACTTTATCAGTCTGGCCTTGG Kpn I cLUC
RBnCER3.c02Bi ACGAAAGCTCTGCAGGTCGACATTAGTGAGTGAAGACACAGAGCTAAGGC Sal I
FBnCYTB5.c09Bi TACGCGTCCCGGGGCGGTACCATGGGCGGAGACGGCAAAGT Kpn I cLUC
RBnCYTB5.c09Bi ACGAAAGCTCTGCAGGTCGACTCAAGAAGAAGAAGGAGTCTTGGTGTA Sal I
FBnCER1-L2.a05Bi TACGCGTCCCGGGGCGGTACCATGGCGTCGAGACCAGGTTTTC Kpn I cLUC
RBnCER1-L2.a05Bi ACGAAAGCTCTGCAGGTCGACCTAGTAACCATTGCTCAATCCTTTGGTCT Sal I

表3

亚细胞定位引物"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)		 酶切位点
Restriction site
FBnCER3.a10S GCTGCGGCAGCGGCCGAATTCATGGTAGCTTTTTCAGCTTGGCCT EcoR I
RBnCER3.a10S TTATCTAGATCCGGTGGATCCTCATCTTGTGAGTGAAGAAACAGAGCG BamH I
FBnCER3.c02S GCTGCGGCAGCGGCCGAATTCATGGTTACTTTATCAGTCTGGCCTTGG EcoR I
RBnCER3.c02S TTATCTAGATCCGGTGGATCCTCAATTAGTGAGTGAAGACACAGAGCTA BamH I
FBnCYTB5.c09S GCTGCGGCAGCGGCCGAATTCATGGGCGGAGACGGCAAAGT EcoR I
RBnCYTB5.c09S TTATCTAGATCCGGTGGATCCTCAAGAAGAAGAAGGAGTCTTGGTGTAAGAA BamH I
FBnCER1-L2.a05S GCTGCGGCAGCGGCCGAATTCATGGCGTCGAGACCAGGTTTTC EcoR I
RBnCER1-L2.a05S TTATCTAGATCCGGTGGATCCCTAGTAACCATTGCTCAATCCTTTGGTC BamH I

表4

荧光定量PCR引物"

引物名称
Primer name		 上游引物序列
Forward sequence (5°-3°)		 引物名称
Primer name		 下游引物序列
Reverse sequence (5°-3°)		 扩增片段大小
Size (bp)
qFBnCER3.a10 AGATTCGGGTTCCAATACTT qRBnCER3.a10 TAACACCAATCTTATCAGCCCTA 94
qFBnCYTB5.c09 GGATTTCCTCATCAAGATCCTTCA qRBnCYTB5.c09 GGAGTCTTGGTGTAAGAACG 87
qFBnCER1-L2.a05 TGTCTTTGAGAGAAACCGC qRBnCER1-L2.a05 GTTGATAGATTCTTTACACTGCTG 135

图1

BnCER3.a10/c02、BnCYTB5B.c09和BnCER1-L2.a05蛋白的结构域分析 A: 脂肪酸羟化酶结构域; B: WAX2 C末端结构域; C: Cyt_B5蛋白家族保守结构域。参与氨基酸序列比对的有拟南芥(Arabidopsis thaliana) CER1 (AT1G02205)、CER1-L1 (AT1G02190)、CER1-L2 (AT2G37700)、CER3 (AT5G57800), 参与比对的CYTB5分别来自拟南芥(Arabidopsis thaliana) (AT5G48810)、甘蓝(Brassica oleracea) (XP_013609519.1)、埃塞俄比亚芥(Brassica carinata) (KAG2316461.1)、荠菜(Capsella rubella) (XP_006279708.1)、萝卜(Raphanus sativus) (XP_018457867.1)。"

图2

BnCER3.a10/c02、BnCYTB5B.c09、BnCER1-L2.a05与其他十字花科物种相关蛋白的系统进化树分析 参与系统进化树分析的氨基酸序列包括拟南芥AtCER1 (AT1G02205)、AtCER1-L1 (AT1G02190)、AtCER1-L2 (AT2G37700)、AtCER3 (AT5G57800)、AtCYTB5 (AT5G48810), 白菜BrCER1 (XP_009118866.2)、BrCER1-L2 (XP_009143457.1)、BrCER3 (XP_033137694.1)、 BrCYTB5B (XP_009151679.1), 甘蓝BoCER1 (QCO76034.1)、BoCER1-L2 (XP_013632332.1)、BoCER3 (XP_013621098.1)、BoCYTB5B"

图3

BnCER3.a10/c02、BnCYTB5B.c09和BnCER1-L2.a05在烟草叶片中的亚细胞定位 标尺为20 μm。"

图4

BiFC检测BnCER1-2与BnCER3.a10、BnCYTB5B.c09、BnCER1-L2.a05蛋白互作 pC1300-YN-OsHAL3 + pC2300-OsHAL3-YC为阳性对照, pC1300-YN-BnCER1-2 + pC2300-OsHAL3-YC、pC1300-YN-OsHAL3 + pC2300-BnCER3.a10-YC、pC1300-YN-OsHAL3 + pC2300-BnCYTB5B.c09-YC、pC1300-YN-OsHAL3 + pC2300-BnCER1-L2.a05-YC为阴性对照。标尺为20 μm。"

图5

LCA检测BnCER1-2与BnCER3.a10、BnCYTB5B.c09、BnCER1-L2.a05蛋白互作 A: BnCER3.a10与BnCER1-2互作检测; B: BnCYTB5B.c09与BnCER1-2互作检测; C: BnCER1-L2.a05与BnCER1-2互作检测; NLuc空载体与含目的基因的CLuc载体组合、CLuc空载体与含目的基因的NLuc载体组合以及均为空载体的CLuc/NLuc载体组合分别作为阴性对照。"

图6

甘蓝型油菜BnCER3.a10、BnCYTB5B.c09、BnCER1-L2.a05的表达模式 A: 组织器官特异性表达; B: 蜡质差异材料表达; C: 非生物胁迫和激素诱导表达。SA: 水杨酸; MeJA: 茉莉酸甲酯; ACC: 1-氨基环丙烷-1-羧酸; ABA: 脱落酸。*代表P < 0.05。"

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