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作物学报 ›› 2026, Vol. 52 ›› Issue (6): 1618-1630.doi: 10.3724/SP.J.1006.2026.54151

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蔗类四跨膜蛋白ScTSPAN18与6K2互作应答SCMV侵染研究

崔致远(), 秦晨展, 刘星雨, 张海, 曾康, 黄国强, 徐景升*()   

  1. 福建农林大学国家甘蔗工程技术研究中心 / 农业农村部福建甘蔗生物学与遗传育种重点实验室, 福建福州 350002
  • 收稿日期:2025-12-23 接受日期:2026-02-27 出版日期:2026-06-12 网络出版日期:2026-03-04
  • 通讯作者: * 徐景升, E-mail: xujingsheng@126.com
  • 作者简介:崔致远, E-mail: 1946397717@qq.com
  • 基金资助:
    国家重点研发计划项目(2023YFD1200703);福建农林大学科技创新基金项目(KFB24033)

Interaction between the sugarcane tetraspanin-like protein ScTSPAN18 and 6K2 in response to SCMV infection

Cui Zhi-Yuan(), Qin Chen-Zhan, Liu Xing-Yu, Zhang Hai, Zeng Kang, Huang Guo-Qiang, Xu Jing-Sheng*()   

  1. National Engineering Research Center for Sugarcane, Fujian Agriculture and Forestry University / Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs, Fuzhou 350002, Fujian, China
  • Received:2025-12-23 Accepted:2026-02-27 Published:2026-06-12 Published online:2026-03-04
  • Contact: * Xu Jing-Sheng, E-mail: xujingsheng@126.com
  • Supported by:
    National Key Research and Development Program of China(2023YFD1200703);Science and Technology Innovation Project of Fujian Agriculture and Forestry University(KFB24033)

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摘要：

四跨膜蛋白(tetraspanin, TET)是一类保守的跨膜蛋白, 在细胞生物中普遍存在并具有重要功能。TET可以通过形成TET富集微域(tetraspanin enriched microdomain, TEM)的方式来执行功能。本课题组前期以甘蔗花叶病毒(sugarcane mosaic virus, SCMV)的6K2蛋白为诱饵, 从甘蔗栽培种(Saccharum spp. hybrid) cDNA酵母文库中分离并鉴定了1个与其互作的类TET蛋白(ScTSPAN18), 并鉴定了甘蔗品种新台糖22的TET家族基因。本研究通过双分子荧光互补(bimolecular fluorescence complementation, BiFC)和荧光素酶互补(luciferase complementation assay, LCA)试验进一步验证SCMV-6K2与ScTSPAN18的互作关系。生物信息学分析表明, ScTSPAN18长度为251 aa, 有4个跨膜结构域, 但在结构上与典型的TET存在差异, 且酵母双杂交(yeast two-hybrid, Y2H)试验表明, ScTSPAN18蛋白不能自互作, 也不与典型的TET蛋白互作。系统进化树分析表明, 类TET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, ScTSPAN18定位于内质网中。实时荧光定量PCR分析发现, ScTSPAN18基因的表达具有明显的组织特异性, 在叶片和根中相对表达量较高; 在完成形态建成且处于旺盛工作状态的正1叶的相对表达量显著高于第3节间和第8节间; 接种SCMV后, ScTSPAN18表达量在侵染早期显著上调, 随后持续高表达, 在第14天下调至初始水平。

关键词: 甘蔗花叶病毒, 6K2, 四跨膜蛋白, 类四跨膜蛋白, 蛋白互作

Abstract:

Tetraspanins (TETs) are a conserved class of transmembrane proteins that are widely distributed across cellular organisms and play important roles. TETs function in part by assembling tetraspanin-enriched microdomains (TEMs). In our previous study, we screened and identified a TET-like protein, ScTSPAN18, from a sugarcane cDNA yeast library using the 6K2 protein of sugarcane mosaic virus (SCMV) as bait, and we also characterized the TET gene family in the sugarcane (Saccharum spp. hybrid) cultivar Xintaitang 22 (XTT22). Here, the interaction between SCMV-6K2 and ScTSPAN18 was further validated using bimolecular fluorescence complementation (BiFC) and luciferase complementation assay (LCA). Bioinformatic analyses showed that ScTSPAN18 is 251 amino acids long and comprises four transmembrane domains. Structural modeling indicated that ScTSPAN18 differs from classical TETs, and yeast two-hybrid (Y2H) assays showed that ScTSPAN18 neither self-interacts nor interacts with classical TETs. Phylogenetic analysis suggested that TET like proteins are divergent between monocotyledonous and dicotyledonous species, as well as between C3 and C4 plants. Subcellular localization assays indicated that ScTSPAN18 is localized to the endoplasmic reticulum. RT-qPCR analysis showed that ScTSPAN18 exhibits tissue-specific expression, with relatively high transcript levels in leaves and roots; expression in the +1 leaf was significantly higher than that in the 3rd and 8th internodes. Following SCMV challenge, ScTSPAN18 expression increased significantly at early infection stages and remained high until day 14, after which it decreased to the basal level.

Key words: sugarcane mosaic virus, 6K2, tetraspanin, tetraspanin-like protein, 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
ScTSPAN18-221-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGCGTCGCCGGAGC 双分子荧光互补载体构建
ScTSPAN18-221-R GGGGACCACTTTGTACAAGAAAGCTGGGTCCTGAGACGGTGCTGCATTTGC Vector generation for BiFC
ScTSPAN18-qF CACCTCAGCGACCTCCTAAGC 定量PCR
ScTSPAN18-qR TGTAGAAGCAGAGGCAGCATCC Real-time qPCR
eEF-1α-F TTTCACACTTGGAGTGAAGCAGAT 内参基因[21]
eEF-1α-R GACTTCCTTCACAATCTCATCATAA Reference gene [21]
Actin-F CCTGAAGATCACCCTGTGCT 内参基因[42]
Actin-R GCAGTCTCCAGCTCCTGTTC Reference gene [42]
SCMV-CP-F TACAGAGAGACACACAGCTG SCMV检测
SCMV-CP-R ACGCTACACCAGAAGACACT Detection of SCMV
ScTSPAN18-nLuc-F GGGGACGAGCTCGGTACCATGGCGCGTCGCCGGA 荧光素酶互补载体构建
ScTSPAN18-nLuc-R CGCGTACGAGATCTGGTCGACCTGAGACGGTGCTGCATTTGCATC Vector generation for LCA
SCMV-6K2-cLuc-F GTCCCGGGGCGGTACCATGGGAATGGACGCTACGGCTGC 荧光素酶互补载体构建
SCMV-6K2-cLuc-R CTAGTGGACTGCAGGTCGACTTGGTGTGACACTTCTGTCT Vector generation for LCA
ScTSPAN18-pPR3-F TGGCCATTACGGCCCGGGATGGCGCGTCGCCGGAGC 酵母双杂交载体构建
ScTSPAN18-pPR3-R GCTTGATATCGAATTCTCTCACTGAGACGGTGCTGCATTTGC Vector generation for Y2H
ScTSPAN18-pBT-F ATGTAATGGCCATTACGATGGCGCGTCGCCGGAGC 酵母双杂交载体构建
ScTSPAN18-pBT-R TCCTGCAGATGGCCGATCACTGAGACGGTGCTGCATTTGC Vector generation for Y2H

图1

TET与类TET蛋白结构模拟 AtTET1: 拟南芥TET蛋白(NP_199482.1); OsTET1: 水稻TET蛋白(XP_015630772.1); AtTET-like 1: 拟南芥类TET蛋白(NP_565468.1); ScTSPAN18: 甘蔗类TET蛋白(QHD26891); ScTET2: 甘蔗TET蛋白(PX136847)。LEL: 大胞外环状结构; SEL: 小胞外环状结构。"

图2

使用TMHMM预测TET与类TET蛋白的跨膜结构域 供试蛋白及GenBank登录号同图1。"

图3

甘蔗ScTSPAN18与其他植物类TET蛋白的氨基酸序列比对 类TET的保守基序用黑色框标记, 典型TET的保守基序用红色框标记。LEL: 大胞外环状结构; SEL: 小胞外环状结构。"

图4

ScTSPAN18与其他物种类TET蛋白的系统进化树分析 I-1: 群I的亚群1; I-2: 群I的亚群2; II: 群II。系统进化树结点处标记的数字为最大似然自展值。距离标尺为0.10。"

图5

ScTSPAN18-YFP 在本氏烟草表皮细胞中的定位 比例尺为20 μm。"

图6

ScTSPAN18与SCMV-6K2的互作验证 A: YN融合于SCMV-6K2的N末端, YC融合于ScTSPAN18的C末端; YN融合于ScTSPAN18的N末端, YC融合于SCMV-6K2的C末端。将YN-SCMV-6K2和ScTSPAN18-YC, YN-ScTSPAN18和SCMV-6K2-YC分别共注射到本氏烟草叶片中进行瞬时表达, 48 h后激光共聚焦观察。比例尺为25 μm。B: ScTSPAN18构建至nLuc, SCMV-6K2构建至cLuc, 并转入农杆菌GV3101。设置cLuc和ScTSPAN18-nLuc、SCMV-6K2-cLuc和cLuc和nLuc为阴性对照。共注射至本氏烟草叶片, 48 h后进行观察。C: 使用AlphaFold3预测ScTET8和ScTSPAN18与SCMV-6K2及拟南芥AtTET8和AtTET-like 1与TuMV-6K2的互作。图中蓝色蛋白为TET或TET-like蛋白; 紫色部分为6K2蛋白; 黄线表示氢键并标注互作氨基酸。"

图7

Y2H试验检测ScTSPAN18的自互作及其与9个甘蔗ScTET的互作 将pBT-ScTSPAN18分别与pPR3-ScTET2、pPR3-ScTET8、pPR3-ScTET13、pPR3-ScTET23、pPR3-ScTET34、pPR3-ScTET55、pPR3-ScTET67、pPR3-ScTET78、pPR3-ScTET96、pPR3-ScTSPAN18共转酿酒酵母菌株NYM51, 在二缺和四缺培养基上培养。以共转pTUS2-APP和pNubG-Fe65的酵母菌株为阳性对照, 以共转pTUS2-APP和pPR3-N的酵母菌株为阴性对照。TET序列登录号: ScTET2 (PX136847)、ScTET8 (PX136848)、ScTET13 (PX136849)、ScTET23 (PX136850)、ScTET34 (PX136851)、ScTET55 (PX136852)、ScTET67 (PX136853)、ScTET78 (PX136854)、ScTET96 (PX136855)。DDO: 缺少亮氨酸和色氨酸的酵母合成限定基本培养基; QDO: 缺少亮氨酸、色氨酸、组氨酸和腺嘌呤的酵母合成限定基本培养基。"

图8

ScTSPAN18基因的组织特异性和应答SCMV侵染的表达模式分析 A: LR: 心叶; +1 L: 正一叶; +7 L: 正七叶; +3 I: 第3节间; +8 I: 第8节间; R: 根。B: 在SCMV侵染后0 h、12 h、1 d、3 d、7 d、14 d取样, 分析SCMV的侵染下ScTSPAN18的表达水平。误差线为每组处理的标准误差(n = 3)。柱上不同小写字母表示在P < 0.05时差异显著。"

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