木薯MeNPR4互作蛋白筛选及其抗病功能分析

Screening of cassava interacting proteins of MeNPR4 and analysis of the disease-resistant functions

  • 摘要: 【目的】 筛选木薯水杨酸(SA)受体MeNPR4的互作蛋白,并分析互作蛋白的抗病功能,为探究MeNPR4与其他激素互作介导的防卫机制及木薯遗传改良和育种提供科学依据。【方法】 以木薯品种华南124的叶片cDNA为模板克隆MeNPR4基因,利用酵母双杂交(Y2H)系统筛选MeNPR4的候选互作蛋白,再结合双分子荧光互补试验(BiFC)进行验证,并通过构建pCAMBIA2300-MeERS2-GFP融合表达载体对筛选出的互作蛋白乙烯(ET)受体(MeERS2)进行亚细胞定位。在MeERS2基因的沉默植株中接种木薯普通花叶病毒(CsCMV)侵染性克隆,通过实时荧光定量PCR检测侵染过程中CsCMV积累量,使用叶绿素荧光成像系统测定光合参数,分析抗病功能。【结果】 从木薯中克隆获得MeNPR4基因,长度为1698 bp,与Phytozome数据库的参考序列(登录号:Manes.14G155900)一致。构建诱饵表达载体pGBKT7-MeNPR4,利用Y2H试验筛选到63个候选互作蛋白,通过Y2H试验证实MeERS2与MeNPR4在体外直接互作。将MeNPR4和MeERS2分别构建到双分子荧光互补载体pFGC-cYFP和pFGC-nYFP中进行BiFC试验,结果发现MeNPR4与MeERS2在体内直接互作。亚细胞定位结果显示,MeERS2蛋白定位于细胞核和细胞质。将MeERS2基因特异性片段与pTRV2载体连接,成功构建沉默植株。对CsCMV侵染性克隆注射木薯叶片后进行实时荧光定量PCR检测,发现2个沉默植株中的CsCMV积累量显著高于对照(P<0.05,下同),且随着时间的延长,CsCMV积累量也逐渐增高。沉默植株的最大光合效率(Fv/Fm)、实际光合效率Y(II)、光化学淬灭系数(qP)显著低于对照组,而非调节性能量耗散的量子产额Y(NO)显著高于对照组,且沉默MeERS2植株与对照相比叶片黄化更明显。【结论】 SA受体MeNPR4及ET受体MeERS2互作,且MeERS2基因正向调控木薯对普通花叶病的抗性。

     

    Abstract: 【Objective】 This study aimed to screen cassava interacting proteins of the salicylic acid (SA) receptor MeNPR4,and the disease-resistant functions were analyzed,thereby providing scientific basis for exploring the defense mechanism mediated by the interaction between MeNPR4 and other hormones as well as genetic improvement and bree-ding of cassava. 【Method】 Taking the MeNPR4 gene cloned using cDNA from leaves of cassava variety Hunan 124 as a template, the yeast two-hybrid (Y2H) system was used to screen the candidate interacting proteins of MeNPR4,and bimolecular fluorescence complementation (BiFC) assays were used for validation. The fusion expression vector pCAMBIA2300-MeERS2-GFP was constructed for subcellular localization of the ethylene (ET) receptor MeERS2. The silenced plants of MeERS2 gene were inoculated with the infectious clone of cassava common mosaic virus (CsCMV),and the CsCMV accumulation was determined via real-time fluorescence quantitative PCR. The chlorophyll fluorescence imaging system was employed to analyze the disease-resistant functions of silenced plants. 【Result】 The MeNPR4 gene was cloned from cassava, and its length was 1698 bp, which was consistent with the reference sequence (Manes.14G155900) in the Phytozome database. A bait expression vector pGBKT7-MeNPR4 was constructed, and 63 candidate interacting proteins were screened by Y2H test, confirming a direct in vitro interaction between MeERS2 and MeNPR4. For BiFC assay,the MeNPR4 and MeERS2 were individually cloned into bimolecular fluorescence complementation vectors pFGC-cYFP and pFGC-nYFP,and the results indicated a direct in vivo interaction between MeERS4 and MeNPR2. Subcellular localization analysis revealed that the MeERS2 protein localized at the nucleus and cytoplasm. A MeERS2-specific fragment was ligated into the pTRV2 vector to generate silencing plants. Real-time quantitative PCR analysis of cassava leaves injected with the CsCMV infectious clone revealed that the CsCMV accumulation in the two silenced plants was significantly higher than that in the control (P<0.05, the same below), and it gradually increased over time. The maximum photochemical efficiency (Fv/Fm), actual photochemical efficiency Y(II),and photochemical quenching coefficient (qP) of silenced plants were significantly lower than those in control, but non-regulated energy dissipation Y(NO) of silenced plants was higher than this in control. Silenced plants exhibited more pronounced leaf etiolation than the control. 【Conclusion】 The SA receptor MeNPR4 interacts with the ET receptor MeERS2,and the MeERS2 gene positively regulates the immune response of cassava to common mosaic disease.

     

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