CN113912698B - 咖啡短体线虫Pc-CD蛋白、其编码基因及应用 - Google Patents
咖啡短体线虫Pc-CD蛋白、其编码基因及应用 Download PDFInfo
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Abstract
本发明涉及一种咖啡短体线虫Pc‑CD蛋白、其编码基因及应用，旨在解决咖啡短体线虫难以防治的技术问题。本发明筛选得到一种Pc‑CD蛋白，获取自咖啡短体线虫，其氨基酸序列如SEQ ID NO:1所示，编码该蛋白的Pc‑cd基因核苷酸序列如SEQ ID NO:2所示。本发明利用dsRNA处理沉默Pc‑cd基因后，咖啡短体线虫的繁殖力及其对寄主玉米的致病力与对照GFP dsRNA处理组相比均显著降低，表明此基因在咖啡短体线虫的生殖和侵染致病过程中具有重要作用，可作为植物抗线虫工程的靶标基因；本发明对于短体线虫致病机理研究以及抗线虫植物制备具有重大应用价值。
Description
技术领域
本发明涉及生物基因工程技术领域，具体涉及一种咖啡短体线虫Pc-CD蛋白、其编码基因及应用。
背景技术
咖啡短体线虫(Pratylenchus coffeae)是一种重要的迁移性内寄生植物病原线虫，主要通过口针穿刺寄主根部皮层进入根内寄生取食，造成植物根系出现坏死、腐烂等症状，进而导致寄生植物对水分和营养的吸收与运输受阻，该线虫侵染所造成的伤口还为土壤中其它病原物的入侵创造条件，造成复合侵染现象，对农业生产造成巨大的经济损失。咖啡短体线虫是危害最为严重的的短体线虫之一，在世界各地普遍发生，寄主范围非常广泛，在我国严重危害小麦、玉米、大豆、芝麻、烟草、番茄、山药等多种作物。咖啡短体线虫的广泛分布和对农业的巨大危害使其受到越来越多的关注和研究，但因其定殖和适生能力强，寄主范围广，至今对该线虫的防治仍是一个世界性难题，缺少有效的靶标和方法。
效应蛋白是一类由病原物产生的，有助于病原物从寄主获得营养物质，能够改变寄主结构细胞和功能的蛋白质或小分子物质。植物寄生线虫的效应蛋白主要由线虫食道腺细胞分泌，在线虫侵染致病等过程中具有重要作用。近年来，植物寄生线虫效应蛋白的筛选和功能逐渐成为研究的热点，但多数研究主要集中在定居型寄生的根结和孢囊线虫上，而在短体线虫这一类迁移性寄生线虫上的研究较少见。
因此，挖掘和筛选咖啡短体线虫新的效应蛋白，研究其在咖啡短体线虫侵染致病等过程中的生物学功能及其与寄主互作的分子机制，将其作为靶标用于抗线虫植物的制备，具有重大的价值和应用前景。
公开于该背景技术部分的信息仅仅旨在加深对本发明总体背景技术的理解，而不应当被视为承认或以任何形式暗示该信息构成本领域技术人员所公知的现有技术。
发明内容
本发明的目的在于提供一种来源于咖啡短体线虫Pc-CD蛋白及其编码基因，以解决咖啡短体线虫难以防治的技术问题。
为解决上述技术问题，本发明采用如下技术方案：
本发明筛选得到一种咖啡短体线虫的Pc-CD蛋白，长度为481个氨基酸，其氨基酸序列如SEQ ID NO：1所示。
克隆得到编码所述咖啡短体线虫Pc-CD蛋白的pc-cd基因，长度为1446个核苷酸，其核苷酸序列如SEQ ID NO：2所示。
基于所述咖啡短体线虫Pc-cd基因核苷酸序列设计合成原位杂交探针，其核苷酸序列如SEQ ID NO: 3所示。
基于所述咖啡短体线虫Pc-cd基因核苷酸序列设计合成了一个dsRNA片段， 其核苷酸序列如SEQ ID NO：4所示。
所述Pc-cd基因或所述Pc-CD蛋白能够有效应用于咖啡短体线虫的防控中。
基于所述Pc-cd序列设计dsRNA或其片段，可使咖啡短体线虫摄入dsRNA片段影响咖啡短体线虫的生殖及致病力，从而起到防治咖啡短体线虫的作用。
上述编码基因的杂交探针、dsRNA，含有Pc-cd基因及其同源基因的重组表达载体、超表达载体、干扰载体、重组病毒、转基因细胞系、转基因植物或组织、重组菌、重组基因表达盒及其应用，均属于本发明构思之范畴。
含Pc-cd基因的重组表达载体包括双元农杆菌载体，病毒载体，细菌表达载体及酵母表达载体等。含Pc-cd基因的载体在构建过程中，可以单独或者多个组合使用诱导型、组成型、增强型、组织特异型等；载体可以包括抗生素或抗化学试剂的抗性筛选标记，也可以含有产生颜色变化的酶，比如GUS、或者红色或绿色荧光蛋白等；构建的载体可以转化单双子叶植物、真菌、细菌等，具体可以为大肠杆菌、酵母、烟草、拟南芥、番茄、小麦、玉米等。
Pc-CD蛋白在咖啡短体线虫病害防控中的应用。
蛋白质的功能应用研究常用RNA干扰、体内异位表达等基于编码基因操作的方法，本发明使用RNA干扰有效影响了Pc-cd基因的表达水平，并且在线虫的生殖和致病力方面表现出差异。此差异建立在对咖啡短体线虫敏感的寄主上，例如玉米。
Pc-cd基因的应用主要包括：
(1)在咖啡短体线虫不同发育阶段表达水平变化；
(2)在咖啡短体线虫不同组织部位表达水平。
短体线虫不同阶段其表达的蛋白质有差异，并且反映在编码基因的转录水平上。在涉及到对寄主的寄生致病和抗逆方面，往往在侵染和寄生的虫态表达，而与自生发育相关的蛋白质，其编码基因很可能在后期表达。此外编码基因在线虫的转录表达部位也与其应用有关，例如食道腺细胞中表达的蛋白很可能与寄生植物有关，而在头感器或者表皮中表达则可能与寻找寄主或者抑制寄主免疫反应有关。
本发明所述Pc-cd基因，从表达水平上来看，在侵染阶段的雌虫和幼虫上的表达量最高；而从表达部位来看，其主要集中在线虫的食道腺细胞中。
抑制Pc-cd基因的表达为本发明的构思范围，本发明构思还涉及用于抑制Pc-cd基因的表达的物质在制备产品中的应用。所述产品功能为抑制短体线虫对植物的侵染和/或抑制短体线虫对植物的致病和/或抑制短体线虫的生殖；用于抑制Pc-cd基因表达的物质具体可以为抑制Pc-cd基因表达的dsRNA，干扰载体，以及病毒载体等；所述植物可以是单子叶植物或双子叶植物，具体可以为玉米，烟草，番茄等。
本发明Pc-CD蛋白在抑制短体线虫对植物的寄生与危害，和/或抑制短体线虫对植物的致病性，和/或抑制短体线虫生殖中的应用。
本发明还涉及用于抑制Pc-cD蛋白活性的物质在制备产品中的应用；所述产品功能为抑制短体线虫对植物的寄生和/或抑制短体线虫对植物的致病，和/或抑制短体线虫的生殖；所述植物可以为单子叶植物或双子叶植物，具体可以为烟草，番茄等。
本发明还涉及Pc-CD蛋白在植物中的表达及应用，所述寄主植物具体可以为烟草、番茄和玉米。
食道腺是植物寄生线虫非常重要的腺体，植物寄生线虫食道腺分泌物被公认是与线虫的侵染和致病等过程密切相关的，食道腺分泌物中含有大量的效应蛋白并可以通过线虫的口针的释放到寄主植物的根组织细胞，在线虫侵染、致病等过程中发挥重要作用。本发明Pc-cd基因主要在咖啡短体线虫的食道腺表达，在雌虫和幼虫中的表达量显著高于在雄虫和卵中的表达量；亚细胞定位试验表明Pc-CD蛋白定位于烟草叶片细胞的细胞膜和细胞核上。本发明涉及到的寄主品种包括但不限于玉米，也可为其它感病作物。
本发明通过基因沉默，将Pc-cd dsRNA导入咖啡短体线虫体内，测定了Pc-cd基因沉默对咖啡短体线虫生殖以及对寄主玉米致病力的影响，实验结果表明Pc-cd基因经dsRNA处理沉默后，咖啡短体线虫的繁殖力与对照GFP dsRNA相比下降了64.8 %，线虫的致病力与对照GFP dsRNA相比也显著下降（t检验，置信区间95%），表明该基因在咖啡短体线虫生殖和致病等过程中发挥重要作用，可作为植物抗线虫工程的靶标基因。本发明对于短体线虫致病机理研究以及抗性线虫植物的制备具有重大价值。
与现有技术相比，本发明的主要有益技术效果在于：
1. 本发明筛选得到一种咖啡短体线虫Pc-CD蛋白及其编码基因，该蛋白基因与咖啡短体线虫的繁殖和致病有关，可以利用该蛋白或其编码基因进行生物防控。
2. 本发明对于短体线虫致病机理研究以及抗线虫植物制备具有重大价值。
附图说明
图1为Pc-cd基因在咖啡短体线虫中的组织定位图；其中，A: 正义链探针无杂交信号（负对照）；B-C: 反义链探针杂交结果显示pc-cd基因主要表达在咖啡短体线虫的食道腺细胞中，各图中，s:口针；m：中食道球；eg：食道腺。
图2为Pc-cd基因在咖啡短体线虫4种不同虫态中的表达定量分析图；通过qRT-PCR方法进行检测，相对表达分析的方法为2-△△Ct法，内参基因为egfp；卵的RQ= l；egg：卵；juvenile：幼虫；male：雄虫；female：雌虫。
图3为Pc-CD蛋白的亚细胞定位显微观察照片；其中，A：叠加结果；B：DAPI蓝色荧光下加细胞核marker观察结果；C：GFP绿色荧光下观察结果； D：明场下观察结果。
图4为Pc-cd基因的沉默效率检测结果；其中，CK为未处理的线虫；G12、G24、G36和G48分别为线虫经过egfp dsRNA 处理12、24、36和48 h；R12、R24、R36 和R48分别为线虫经过Pc-cd dsRNA 处理12、24、36和48 h。
图5为不同处理的线虫接种胡萝卜愈伤组织60天后的虫量统计结果；其中，CK为未处理的线虫；G12为经过egfp dsRNA 处理12 h的线虫；R12为经过Pc-cd dsRNA 处理12 h的线虫。
图6为Pc-cd dsRNA浸泡处理12h后，咖啡短体线虫对玉米的致病性测定结果；其中，A:株高；B：地上部鲜重；C：根鲜重；D：根际虫量。
具体实施方式
下面结合附图和实施例来说明本发明的具体实施方式，但以下实施例只是用来详细说明本发明，并不以任何方式限制本发明的范围。
下述实施例中所用的试验材料及试剂，如无特别说明，均购买于常规生化试剂公司；所涉及的仪器设备如无特别说明，均为常规的实验室仪器设备；以下实施例中的定量试验，如无特别说明均设置三次重复实验，结果取平均值。
实施例一、Pc-CD蛋白以及pc-cd基因的获取
1. 收集咖啡短体线虫约50 mg于离心管中，使用depc处理水洗2～3次，加入1 mlTrizol (Invitrogen)，参照Trizol Reagent操作步骤提取总RNA，去除总RNA中残余的DNA后，采用Takara公司cDNA合成试剂盒反转录获得cDNA。
2. 以cDNA为模板，采用引物Pc-cd-F 和Pc-cd-R进行PCR扩增，具体序列如下：
上游引物：Pc-cd-F：5′-GACTCTCTTTAGAAAATGGCC-3′；
下游引物： Pc-cd-R：5′-CCTTCTTTTTCTTCACATCGTG -3′。
扩增体系为2× Taq PCR Mix，12.5 µL；cDNA第一链模板，1 µL；正向引物，1 µL；反向引物，1 µL；ddH2O补至25 μL。
PCR扩增程序：94 ℃ 预变性2 min，（94℃ 变性30s，59 ℃退火30 s，72 ℃ 延伸90 s）35个循环；72 ℃ 终延伸8 min。
3. 将pMD18-T载体与步骤2中的PCR扩增产物连接，然后转化JM109大肠杆菌感受态细胞中，测序。
测序结果表明，扩增产物中具有如SEQ ID NO:2所示的开放阅读框，编码如SEQ IDNO:1所示的蛋白质。将序列表的SEQ ID NO: 1命名为Pc-CD蛋白，将其编码基因命名为Pc- cd基因。
实施例二、Pc-cd基因的组织定位分析
1. 以Pc-cd基因克隆载体为模板通过常规PCR扩增靶标序列，采用的引物如下：
ISH-T7F: 5′-GGATCCTAATACGACTCACTATAGGGTTTCGGTTCTCGGCGTCA-3′；
ISH-R: 5′-GCAATCAACGATGTTCCCG-3′；
ISH-F1: 5′-TTTCGGTTCTCGGCGTCA-3′ ；
ISH-T7R1: 5′-GGATCCTAATACGACTCACTATAGGGGCAATCAACGATGTTCCCG-3′。
以ISH-T7F/ISH-R和ISH-F1/ISH-T7R1为引物，分别合成RNA探针体外转录需要的正义链和反义链DNA 模板，具体步骤参照KOD FX DNA 聚合酶（TOYOBO）说明书配制反应体系进行PCR反应。
PCR反应条件如下：94 ℃ 预变性2 min，（98℃ 变性10s，58.2 ℃退火30 s，68℃延伸90 s）35个循环；72℃ 终延伸10 min。PCR产物用DNA凝胶回收试剂盒（Bioflux）进行纯化回收。
2. 参照DIG RNA labeling Mix（Roche）说明书合成地高辛标记的正链探针和反链探针。
以步骤1回收的正义链和反义链片段为模板，在RNA Polymerase 的作用下37℃孵育2 h，得到正义链探针/反义链探针。
体系如下：正/反义链DNA 模板(上步骤获得)，1 μL；10×DIG RNA labeling Mix，2 μL；10×Transcription Buffer, 2 μL; Protector RNase Inhibitor，0.25 μL；RNAPolymerase，2 μL，RNase-free water，补至10 μL，轻轻混匀后，置于PCR仪中37℃温育2 h；加入2 μL DNase I（RNase-free），37℃温育15 min；最后加入2 μL 0.2M的EDTA（pH=8.0）终止反应。按照说明书进行原位杂交。
结果如图1所示，反链探针有杂交信号，杂交信号主要位于咖啡短体线虫的食道腺，表明pc-cd基因为食道腺细胞表达基因。
实施例三、咖啡短体线虫pc-cd基因在不同虫态中的表达量分析
参考Li等(Li Y, Wang K, Xie H, Wang D W, Xu C L, Huang X, Wu W J, Li DL. Cathepsin B cysteine proteinase is essential for the development andpathogenesis of the plant parasitic nematode Radopholus similis.International Journal of Biological Sciences, 2015, 11(9):1073-1087.)所述方法，从胡萝卜愈伤组织上分离获得咖啡短体线虫混合虫态，分别提取雄虫、雌虫、幼虫和卵四种虫态的RNA，反转录合成cDNA作为模板，采用primer 5.0软件设计pc-cd基因特异性引物如下：
上游引物qPCR-F1: 5′-AATTGCCTGTAGCGACGGATGC-3′,
下游引物qPCR-R1: 5′-GGAATTGCGCCGATGTATTGTTGG-3′。
采用Real time PCR相对定量技术检测pc-cd基因在不同虫态的表达量，以18s基因作为内参基因，采用TB Green Premix Ex Tap Kit（Takara），在荧光定量PCR仪上进行Real time PCR检测，用2-△△Ct 法（△Ct=目的基因的平均Ct值－内参基因的平均Ct值）对样本基因进行表达差异相对定量分析。
反应体系（12 μL）：SYBR Green PCR Mix，6 μL；PCR Forward Primer (10 μM)，0.5 μL；PCR Reverse Primer (10 μM)，0.5 μL；cDNA模板，1 μL；RNase-free water，4 μL。
反应程序：95 °C，2 min； (95 °C，15 s；Tm，30 s；72 °C，30 s) 40个循环；95 °C，15 s；60 °C，60 s；95 °C, 30 s；60°C，15 s。
结果如图2所示，相对于卵时期的表达量，pc-cd基因在雌虫和幼虫时期的表达量显著增高（t检验，置信区间95%），其中雌虫时期的表达量最高，即初步推测表明pc-cd基因主要在咖啡短体线虫的侵染时期表达。
实施例四、pc-cd基因的亚细胞定位
1. 以pc-cd基因cDNA为模板通过常规PCR扩增靶标序列，引物具体序列如下：
PGWC-Pc-CD-F: 5′- AGCAGGCTTTGACTTTAGGTCGGACTCTCTTTAGAAAATGGCC-3′
PGWC-Pc-CD-R: 5′- TGGGTCTAGAGACTTTAGGTCCCTTCTTTTTCTTCACATCGTGG-3′
PCR反应体系为：2×Phanta Max Buffer，12.5 μL；dNTP Mix (10 Mm each)，0.5μL；上游引物，1 μL；下游引物，1 μL；Phanta Max Super-Fidelity DNA Polymerase，0.5 μL；cDNA模板,1 μL; ddH2O，补至25 μL。
PCR反应条件：预变性95℃，3 min；变性95℃，15 s；退火58℃，15 s；延伸72℃，1min，共35个循环；终延伸72℃，5 min；4℃保存。
反应结束后，PCR产物用1%琼脂糖凝胶电泳检测目的条带，并对扩增片段进行回收、连接、转化、测序。
2. 利用 Invitrogen 公司的LR 重组酶将重组入门载体 pGWC-Pc-CD与表达载体pEarleyGate104 重组，获得重组表达载体pEarleyGate104-Pc-CD。采用冻融法将重组质粒转化农杆菌GV3101感受态，取适量摇培菌液涂布于含有相应抗生素的LB平板，28℃下培养36-48 h。
3. 将上述转化成功的农杆菌重新划线培养，挑取单菌落接种于适量LB液体培养基(含相应的抗生素)，28°C振荡培养，室温下离心4000rpm/15min，弃上清后用悬浮缓冲液(含10mM MgCl2+ 10 mM MES+200 μM As的无菌水)重悬菌体，并用悬浮缓冲液调节0D600至0.8-1.0。混匀后静置3h后，用1ml注射器向烟草(本氏烟)幼苗注射混合菌液,每棵苗注射3-4片叶(苗龄4-5叶期) 。注射48-72 h后，制作切片利用激光共聚焦显微镜观察浸润部位是否有荧光产生。激发光的波长为488 nm，接收光的波长为520～550 nm。
试验结果（如图3所示）表明，Pc-CD蛋白定位于烟草叶片细胞的细胞核和细胞膜上。
实施例五、通过体外RNA干扰沉默Pc-cd基因验证其作为靶标在抗线虫中的应用
1. 采用primer 5.0软件设计靶基因的特异引物，并在特异引物前面加上T7启动子序列（下划线显示），通过PCR扩增获得合成dsRNA的模板，纯化后用于下一步实验，同时以外源基因绿色荧光蛋白egfp作为对照，具体引物如下：
Pc-cd正-T7S: 5′-GGATCCTAATACGACTCACTATAGGGTCGCCGAGGCAGTCAGTG-3′，
Pc-cd正-A: 5′-CAATGGCTTGGCATCCGTC-3′，
Pc-cd反-S: 5′-TCGCCGAGGCAGTCAGTG-3′，
Pc-cd反-T7A: 5′-GGATCCTAATACGACTCACTATAGGGCAATGGCTTGGCATCCGTC-3′；
egfp正-T7S: 5′-GGATCCTAATACGACTCACTATAGGGAAACGGCCACAAGTTCAGCG-3′，
egfp正-A: 5′-TGATGCCGTTCTTCTGCTTGTC′-3′，
egfp反-S: 5′-AAACGGCCACAAGTTCAGCG-3′，
egfp反-T7A:5′-GGATCCTAATACGACTCACTATAGGGTGATGCCGTTCTTCTGCTTGTC-3′。
2. 分别以上述获得的DNA为模板，参照Script MaxTM Thermo T7 Transcriptionkit的操作手册进行体外转录，合成Pc-cd基因特异的dsRNA，以egfp dsRNA和清水处理作为对照。将从胡萝卜愈伤组织上分离获得的混合虫态的咖啡短体线虫经DEPC水清洗后，加入到含有Pc-cd dsRNA的浸泡液中，在摇床(100 rpm, 25°C)中分别孵育12 h、24 h、36 h和48h后进行以下试验：
(I)不同处理组的线虫经DEPC水清洗3次后分别提取其RNA，反转录后按照上述方法利用qPCR检测Pc-cd基因的沉默效率，试验设置三次生物重复；
(II)挑取30条不同处理的雌虫分别接种于胡萝卜愈伤组织上，在25 ℃的黑暗培养箱中培养60 d后分离统计线虫的繁殖量，试验设置5次生物重复。
以egfp dsRNA (2.0 mg/mL)浸泡相同时间的线虫做为对照，未经任何处理的线虫作为空白对照。为了检测Pc-cd的沉默是否影响咖啡短体线虫对寄主的致病力，将沉默效率最高处理组（Pc-cd dsRNA浸泡处理12 h）的线虫接种玉米幼苗(约20 cm高)进行盆栽试验，每株的接种量为1000条。在温室中生长60 d后测量不同处理玉米的株高、地上部鲜重和根重，并按照已报到的方法分离和统计植株根际的线虫数量(Kaplan D T, Vanderspool MC, Opperman C H. Sequence tag site and host range assays demonstrate thatRadopholus similis and R. citrophilus are not reproductively isolated.Journal of Nematology, 1997, 29: 421-429.) (Zhang C, Xie H, Xu C L, Cheng X,Li K E, Li Y. Differential expression of Rs-eng-1b in two populations ofRadopholus similis (Tylenchida: Pratylecnchidae) and its relationship topathogenicity. European Journal of Plant Pathology, 2012, 133: 899-910.)。
以egfp dsRNA浸泡12 h和未经任何处理的线虫接种的玉米植株作为对照。试验设置5次生物重复。
试验结果见图4、5、6，其结果表明Pc-cd dsRNA浸泡处理后能有效抑制靶基因的表达，浸泡处理12 h靶基因的沉默效率最高；经Pc-cd dsRNA的浸泡处理12 h后，咖啡短体线虫的繁殖力及其对寄主玉米的致病力与对照处理组相比均显著降低（t检验，置信区间95%）；表明Pc-cd基因在咖啡短体线虫的生殖和致病过程中具有重要作用，可作为植物抗线虫工程的靶标基因。
上面结合附图和实施例对本发明作了详细的说明；但是，所属技术领域的技术人员能够理解，在不脱离本发明构思的前提下，所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，从而形成多个具体的实施例，均为本发明的常见变化范围，在此不再一一详述。
SEQUENCE LISTING
<110> 河南农业大学
<120> 咖啡短体线虫Pc-CD蛋白、其编码基因及应用
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<160> 4
<170> PatentIn version 3.2
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<211> 481
<212> PRT
<213> 咖啡短体线虫Pc-CD蛋白
<400> 1
Met Ala Lys Phe Leu Ile Leu Ala Leu Pro Ala Ile Phe Leu Leu Phe
1 5 10 15
Ala Phe His Ser Met Glu Cys Gly Arg Ile His Gly Asp Gly His His
20 25 30
Arg His Arg Gln His His Leu Gln Arg Val Pro Leu Tyr Arg Met Ser
35 40 45
Ser Ile Arg Glu Arg Leu Ala Asp Ser Asp Ser Leu Glu Gln Phe Ala
50 55 60
Lys His Arg Gln Glu Ala Leu Arg Gln Arg Leu Ala Arg Phe Thr Ala
65 70 75 80
Asn Asp Glu Gly Asp Ser Ser Ser Glu Glu Asp Asp Gly Leu Ile Glu
85 90 95
Gly Asn Gly Ala Ala Val Gly Thr Glu Ile Asp Glu Leu Leu Lys Asn
100 105 110
Tyr Met Asp Ala Gln Tyr Tyr Gly Pro Ile Ser Ile Gly Thr Pro Gly
115 120 125
Gln Asn Phe Thr Val Ile Phe Asp Thr Gly Ser Ser Asn Leu Trp Val
130 135 140
Pro Ser Lys Lys Cys Pro Ile Tyr Asp Ile Ala Cys Leu Leu His His
145 150 155 160
Lys Tyr Asp Ser Thr Lys Ser Ser Ser Tyr Lys Glu Asp Gly Arg Lys
165 170 175
Met Gln Ile Gln Tyr Gly Thr Gly Ser Met Lys Gly Phe Ile Ser Lys
180 185 190
Asp Thr Val Cys Val Ala Gly Ile Cys Val Gln Gln Gln Glu Phe Ala
195 200 205
Glu Ala Val Ser Glu Pro Gly Leu Thr Phe Val Ala Ala Lys Phe Asp
210 215 220
Gly Ile Leu Gly Met Ala Phe Pro Glu Ile Ser Val Leu Gly Val Thr
225 230 235 240
Pro Val Phe Gln Gln Met Val Ala Gln Gln Lys Val Gln Gln Pro Val
245 250 255
Phe Ala Phe Trp Leu Asn Arg Asp Pro Asn Ala Asp Phe Gly Gly Glu
260 265 270
Ile Thr Ile Gly Gly Thr Asp Gln Arg Arg Tyr Val Asp Pro Ile Thr
275 280 285
Tyr Thr Pro Val Thr Arg Lys Ala Tyr Trp Gln Phe Lys Met Asp Ser
290 295 300
Val Asn Gly Ala Ser Gly Lys Ile Ala Cys Ser Asp Gly Cys Gln Ala
305 310 315 320
Ile Ala Asp Thr Gly Thr Ser Leu Ile Ala Gly Pro Lys Ala Gln Val
325 330 335
Glu Lys Ile Gln Gln Tyr Ile Gly Ala Ile Pro Leu Phe His Gly Glu
340 345 350
Tyr Met Val Ser Cys Glu Arg Val Pro Ser Leu Pro Glu Ile Ser Phe
355 360 365
Val Ile Ala Gly Lys Ser Tyr Thr Leu Lys Gly His Asp Tyr Ile Leu
370 375 380
Asn Val Ser Ala Met Gly Lys Ser Ile Cys Leu Ser Gly Phe Met Gly
385 390 395 400
Ile Asp Leu Pro Pro Lys Val Gly Glu Leu Trp Ile Leu Gly Asp Val
405 410 415
Phe Ile Gly Arg Tyr Tyr Thr Val Phe Asp Val Gly Gln Gln Arg Leu
420 425 430
Gly Phe Ala Gln Ala Arg Asp Ala Glu Arg Thr Pro Ile Glu Pro Val
435 440 445
Val Lys Glu Cys Ile Ser Gly Gly Arg Leu Cys Phe Glu Pro Asp Ala
450 455 460
Ser Ile Asp Asp Thr Glu Gln Ala Arg Glu Ser Asp Phe Phe Ala Thr
465 470 475 480
Met
<210> 2
<211> 1446
<212> DNA
<213> 咖啡短体线虫Pc-cd 基因
<400> 2
atggccaaat tcttgatttt ggcactgccg gcaattttct tgctttttgc cttccattca 60
atggaatgtg gccgcatcca tggcgatggc catcatcgcc atcggcaaca tcatctgcaa 120
cgcgttcctc tctaccgaat gagcagcatt cgtgagcggc tggctgacag cgattcgctg 180
gaacaattcg ccaaacatcg gcaagaggcc cttcgtcaac gtttggcccg attcaccgca 240
aatgatgagg gggattcgtc gtcggaagag gatgatggac tgatcgaagg gaatggggca 300
gcggttggga cagaaattga cgagctgctc aaaaattaca tggatgccca atattatggc 360
ccaatttcga ttggcacgcc gggtcaaaat ttcaccgtaa ttttcgacac tggctcctcc 420
aatctttggg tgccctcaaa aaaatgtcca atctacgata ttgcatgcct cctccaccac 480
aaatatgaca gcacaaaatc gtcgagctac aaggaggatg gccgaaaaat gcaaattcaa 540
tacgggaccg ggtcgatgaa gggattcatt tcgaaggaca ccgtttgcgt ggccggaatt 600
tgcgttcaac agcaggaatt cgccgaggca gtcagtgaac cgggtctcac atttgtggcc 660
gccaaattcg acggcatttt gggcatggcc ttccccgaaa tttcggttct cggcgtcacc 720
ccagttttcc agcaaatggt cgcccaacaa aaagtccaac agcccgtttt tgccttttgg 780
ctaaatcgtg atccaaacgc ggattttggc ggcgaaatca ccattggagg cactgatcaa 840
cgccgatatg tggatccgat cacatatacc ccagtgaccc ggaaggcata ttggcaattt 900
aaaatggaca gcgtgaatgg ggcaagcggc aaaattgcct gtagcgacgg atgccaagcc 960
attgccgaca cgggaacatc gttgattgcg gggccaaagg cgcaggtcga aaaaatccaa 1020
caatacatcg gcgcaattcc cctcttccat ggcgagtaca tggtttcgtg tgaacgcgtc 1080
ccatcgctgc cggaaatttc cttcgtcatt gccggcaagt cctacaccct caaaggccat 1140
gactacattt tgaacgtgtc ggccatgggc aagagcattt gcctctccgg cttcatgggc 1200
atcgatttgc cgccaaaagt cggcgaactt tggattctgg gcgatgtgtt tattggccgt 1260
tattataccg tttttgatgt tggccaacaa cgccttggat ttgcgcaggc tcgggatgcg 1320
gaacgcaccc caattgagcc cgtggtgaag gaatgcatca gcgggggacg cctctgcttc 1380
gagcccgatg cttcaattga cgacacggaa caggcccggg agagcgactt ttttgccacg 1440
atgtga 1446
<210> 3
<211> 289
<212> DNA
<213> Pc-cd基因原位杂交探针
<400> 3
tttcggttct cggcgtcacc ccagttttcc agcaaatggt cgcccaacaa aaagtccaac 60
agcccgtttt tgccttttgg ctaaatcgtg atccaaacgc ggattttggc ggcgaaatca 120
ccattggagg cactgatcaa cgccgatatg tggatccgat cacatatacc ccagtgaccc 180
ggaaggcata ttggcaattt aaaatggaca gcgtgaatgg ggcaagcggc aaaattgcct 240
gtagcgacgg atgccaagcc attgccgaca cgggaacatc gttgattgc 289
<210> 4
<211> 345
<212> DNA
<213> Pc-cd基因的dsRNA
<400> 4
ucgccgaggc agucagugaa ccgggucuca cauuuguggc cgccaaauuc gacggcauuu 60
ugggcauggc cuuccccgaa auuucgguuc ucggcgucac cccaguuuuc cagcaaaugg 120
ucgcccaaca aaaaguccaa cagcccguuu uugccuuuug gcuaaaucgu gauccaaacg 180
cggauuuugg cggcgaaauc accauuggag gcacugauca acgccgauau guggauccga 240
ucacauauac cccagugacc cggaaggcau auuggcaauu uaaaauggac agcgugaaug 300
gggcaagcgg caaaauugcc uguagcgacg gaugccaagc cauug 345
Claims (8)
1.一种咖啡短体线虫Pc-CD蛋白，其特征在于，其氨基酸序列如SEQ ID NO: 1所示。
2.编码权利要求1所述咖啡短体线虫Pc-CD蛋白的Pc-cd基因，其特征在于，其核苷酸序列如SEQ ID NO: 2所示。
3.抑制权利要求2所述Pc-cd基因表达的dsRNA。
4.根据权利要求3所述的dsRNA，其特征在于，其核苷酸序列如SEQ ID NO: 4所示。
5.含有权利要求2所述Pc-cd基因的重组表达载体、超表达载体、干扰载体、重组病毒、转基因细胞系、重组菌或重组基因表达盒。
6.权利要求2所述Pc-cd基因的原位杂交探针，其核苷酸序列如SEQ ID NO: 3所示。
7.权利要求1所述Pc-CD蛋白或权利要求2所述Pc-cd基因在防治咖啡短体线虫中的应用。
8.一种防治咖啡短体线虫的方法，其特征在于，包括如下步骤：
（1）基于权利要求2所述基因序列设计合成Pc-cd基因特异的dsRNA；
（2）使咖啡短体线虫摄入所述dsRNA，以降低咖啡短体线虫的繁殖力及其对寄主植物的侵染致病能力。
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