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作物学报 ›› 2022, Vol. 48 ›› Issue (12): 3080-3090.doi: 10.3724/SP.J.1006.2022.14244

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蔗易化子家族蛋白ScZIFL1与6K2互作应答SCMV侵染

刘淑娴(), 杨宗桃, 程光远, 张海, 周营栓, 商贺阳, 黄国强, 徐景升()   

  1. 农业农村部福建甘蔗生物学与遗传育种重点实验室 / 福建农林大学国家甘蔗工程技术研究中心 / 教育部作物遗传育种与综合利用重点实验室, 福建福州 350002
  • 收稿日期:2021-12-22 接受日期:2022-03-25 出版日期:2022-12-12 网络出版日期:2022-04-19
  • 通讯作者: 徐景升
  • 作者简介:E-mail: Liushuxian1010@163.com
  • 基金资助:
    国家自然科学基金项目(31971991);福建农林大学科技创新基金(CXZX2018026)

Interaction of sugarcane main facilitator superfamily member ScZIFL1 with 6K2 in response to Sugarcane mosaic virus infection

LIU Shu-Xian(), YANG Zong-Tao, CHENG Guang-Yuan, ZHANG Hai, ZHOU Ying-Shuan, SHANG He-Yang, HUANG Guo-Qiang, XU Jing-Sheng()   

  1. Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs / National Engineering Research Center for Sugarcane, Fujian Agriculture and Forestry University / Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Fuzhou 350002, Fujian, China
  • Received:2021-12-22 Accepted:2022-03-25 Published:2022-12-12 Published online:2022-04-19
  • Contact: XU Jing-Sheng
  • Supported by:
    National Natural Science Foundation of China(31971991);Science and Technology Innovation Project of Fujian Agriculture and Forestry University(CXZX2018026)

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摘要：

易化子超家族转运蛋白(major facilitator superfamily, MFS)在生物中普遍存在, 锌诱导类辅助因子(zinc induced facilitator like, ZIFL)是MFS成员, 参与小分子有机物运输。本课题组前期利用酵母双杂交(yeast two-hybrid, Y2H)技术从甘蔗(Saccharum spp. hybrid)中分离鉴定了1个与甘蔗花叶病毒(Sugarcane mosaic virus, SCMV)编码蛋白6K2互作的ZIFL, 命名为ScZIFL1。本研究利用双分子荧光互补技术(bimolecular fluorescence complementation, BiFC)进一步验证了ScZIFL1与SCMV-6K2的互作。生物信息学分析表明, ScZIFL1长度为484个氨基酸, 无信号肽, 具有12个跨膜结构域, 为不稳定的疏水性蛋白。序列比对分析表明, ScZIFL1具有MFS保守的半胱氨酸模体、特征基序及反向运输基序。系统进化树分析表明, 该蛋白在单子叶和双子叶植物之间, 以及单子叶C3植物和C4植物之间存在明显分化。亚细胞定位试验表明, ScZIFL1定位于液泡膜, 部分与SCMV-6K2共定位。实时荧光定量PCR分析发现, ScZIFL1基因的表达具有明显的组织特异性, 在茎中表达最高, 叶中次之, 根中最低; 在完成形态建成且处于旺盛工作状态的+1叶和第8节间中的相对表达量显著高于未成熟的心叶、第3节间和渐衰叶片+7叶; 接种SCMV后, ScZIFL1表达量在侵染早期显著上调, 随后持续高表达。

关键词: 甘蔗花叶病毒, 6K2, ZIFL1, 蛋白互作

Abstract:

The major facilitator superfamily (MFS) members are extensively distributed in the organisms. The zinc induced facilitator like proteins (ZIFL), members of the MFS, are involved in the transport of small organic molecules. In our previous study, the ZIFL homologue was screened from sugarcane (Saccharum spp. hybrid) and identified to interact with Sugarcane mosaic virus (SCMV) coding protein 6K2 by yeast two-hybrid (Y2H), then named as ScZIFL1. In the present study, the interaction of ScZIFL1 with SCMV-6K2 was further confirmed by bimolecular fluorescence complementation (BiFC) assays. Bioinformatics analysis showed that ScZIFL1 coded 484 aa, and was an unstable hydrophobic protein without signal peptide but 12 transmembrane domains. Sequences alignment revealed the conserved cysteine-containing motif, canonical MFS signature, and anti-porter signatures in ScZIFLl. Phylogenetic tree indicated that ZIFLl was divergent between monocotyledons and dicotyledons, as well as C3 and C4 plants. Subcellular localization referred that ScZIFL1 was localized in tonoplast but partially co-localized with SCMV-6K2. ScZIFL1 gene had obvious tissue specificity in sugarcane by RT-qPCR. The relative expression levels of ScZIFL1 genes were the highest in stems, followed by leaves, and the lowest in roots. Specifically, the relative expression levels of ScZIFL1 genes in the established morphogenesis and fully functional tissues such as the leaf +1 and the 8th internode were significantly higher than those in the immature tissues, i.e., leaf roll and the 3rd internode, or the fading tissues, i.e., leaf +7. Upon the infection of SCMV, ScZIFL1 was significantly up-regulated at the early stage and remained higher expression with time going on.

Key words: Sugarcane mosaic virus, 6K2, ZIFL1, protein interaction

表1

本研究使用的引物"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 策略
Strategy
221-ScZIFL1-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGGCGGTGGCGGCGA 双分子荧光互补载体构建
221-ScZIFL1-R GGGGACCACTTTGTACAAGAAAGCTGGGTCTGAATGCCTCATTGAATTTG Vector generation for BiFC
ScZIFL1-qF TGGACGAGGCGAACGAGACC 定量PCR
ScZIFL1-qR AGCGGCGGTGAGGCAGAC Real-time qPCR
GAPDH-F CACGGCCACTGGAAGCA 内参基因
GAPDH-R TCCTCAG GGTTCCTGATGCC Reference gene
Actin-F CCTGAAGATCACCCTGTGCT 内参基因
Actin-R GCAGTCTCCAGCTCCTGTTC Reference gene
SCMV-CP-F TACAGAGAGACACACAGCTG SCMV检测
SCMV-CP-R ACGCTACACCAGAAGACACT Detection of SCMV

图1

ScZIFL1跨膜结构域预测"

图2

甘蔗ScZIFL1与其他植物ZIFL1蛋白的氨基酸序列比对 MFS蛋白的半胱氨酸motif CPGC, 特征基序WG(V/M/I)(F/V/A/I)AD(K/R)(Y/I//H/L)GRKP和逆向转运基序S(x8)G(x3)GP(A/T/G) (L/I)GG (x代表任意氨基酸)用红色字体表示, 并以黄色高亮标记。序号I包含的序列来自单子叶植物; 序号II包含的序列来自双子叶植物。ZmZIFL1 (NP_001141824.1): 玉米; SbZIFL1 (XP_002457667.1): 高粱; PhZIFL1 (XP_025817905.1): 黍; SvZIFL1 (XP_034593088.1): 狗尾草; BdZIFL1 (XP_003567304.1): 二穗短柄草; HvZIFL1 (KAE8779043.1): 大麦; TaZIFL1 (KAF7029361.1): 小麦; OsZIFL1 (XP_025880517.1): 水稻; PtZIFL1 (XP_024443823.1): 毛果杨; PpZIFL1 (XP_007217932.1): 桃树; VvZIFL1 (XP_002277598.2): 葡萄; NaZIFL1 (XP_019247413.1): 烟草; AtZIFL1 (OAP02507.1): 拟南芥; StZIFL1 (XP_006347685.1): 马铃薯; GmZIFL1 (XP_006594342.1): 大豆; CaZIFL1 (XP_016582203.1): 辣椒。"

图3

ScZIFL1与其他物种ZIFL1蛋白的系统进化树分析 红色框、绿色框和橘色框分别代表亚群I-1、亚群I-2和群II。"

图4

ScZIFL1-YFP在本氏烟表皮细胞中的定位 A: ScZIFL1-YFP亚细胞定位; B: ScZIFL1-YFP和6K2-CFP亚细胞共定位。比例尺为 25 μm。"

图5

BiFC检测ScZIFL1与SCMV-6K2的互作 A: YN融合于ScZIFL1的N末端, YC融合于SCMV-6K2的C末端; B: YN融合于SCMV-6K2的N末端, YC融合于ScZIFL1的C末端。将6K2-YC和ScZIFL1-YN (A), ScZIFL1-YC和6K2-YN (B)分别共注射到本氏烟叶片中进行瞬时表达, 48 h后激光共聚焦观察。标尺为25 μm。"

图6

ScZIFL1基因的组织特异性表达分析 LR: 心叶; +1 L: +1叶; +7 L: +7叶; +3 I: 第3节间; +8 I: 第8节间; R: 根。误差线为每组处理的标准误差(n = 3)。柱上不同的小写字母表示在P < 0.05时显著性差异。"

图7

ScZIFL1基因应答SCMV侵染的表达模式 误差线为每组处理的标准误差(n = 3)。柱上不同的小写字母表示在P < 0.05时显著性的差异。"

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