張承啟，王曉妍，陳莉

肌動(dòng)蛋白結(jié)合蛋白FgAbp1參與禾谷鐮孢生長(zhǎng)、發(fā)育和毒素體形成

張承啟，王曉妍，陳莉

安徽農(nóng)業(yè)大學(xué)植物保護(hù)學(xué)院/作物有害生物綜合治理安徽省重點(diǎn)實(shí)驗(yàn)室/植物病蟲害生物學(xué)與綠色防控安徽普通高校重點(diǎn)實(shí)驗(yàn)室，合肥 230036

【】Abp1作為肌動(dòng)蛋白結(jié)合蛋白家族成員之一，在各種真核生物肌動(dòng)蛋白骨架形成過程中具有核心作用。本研究旨在分析禾谷鐮孢（）中肌動(dòng)蛋白結(jié)合蛋白FgAbp1在生長(zhǎng)發(fā)育、對(duì)新型殺菌劑氰烯菌酯敏感性以及毒素體形成等生物學(xué)過程中的功能。利用融合PCR和酵母空隙修復(fù)技術(shù)分別構(gòu)建基因敲除打靶片段和融合熒光蛋白載體，再通過聚乙二醇介導(dǎo)的原生質(zhì)體轉(zhuǎn)化的方法獲取基因缺失突變體ΔFgAbp1和熒光標(biāo)記菌株。觀察比較野生型PH-1、突變體ΔFgAbp1和回補(bǔ)體ΔFgAbp1-C的菌絲生長(zhǎng)、有性生殖以及無性繁殖，并測(cè)定基因敲除突變體ΔFgAbp1對(duì)殺菌劑氰烯菌酯的敏感性。通過融合綠色熒光蛋白，明確FgAbp1在菌絲中的分布情況；利用透射電子顯微鏡觀察分析FgAbp1對(duì)細(xì)胞中液泡/囊泡形態(tài)的影響。在非產(chǎn)毒環(huán)境和誘導(dǎo)產(chǎn)毒條件下，通過雙熒光共定位分析FgAbp1在禾谷鐮孢毒素體形成過程中的作用。禾谷鐮孢中，F(xiàn)gAbp1主要呈顆粒狀定位于近細(xì)胞膜處。在MM培養(yǎng)基中，基因敲除突變體ΔFgAbp1的生長(zhǎng)速率與野生型相比降低了15%，但在營(yíng)養(yǎng)豐富的CM中ΔFgAbp1的生長(zhǎng)速率卻減慢了38%。突變體ΔFgAbp1在無性繁殖以及有性生殖過程中相較野生型沒有明顯缺陷。然而在0.5 μg·mL-1氰烯菌酯的作用下，ΔFgAbp1的菌絲生長(zhǎng)完全受到抑制，并且分生孢子萌發(fā)率顯著下降。此外，敲除基因?qū)е录?xì)胞中液泡不能正常形成且囊泡增多。在非產(chǎn)毒條件下，F(xiàn)gAbp1與DON毒素合成關(guān)鍵酶Tri1共定位于囊泡中；在誘導(dǎo)產(chǎn)毒條件下，F(xiàn)gAbp1與Tri1則共定位于毒素體中。此外，敲除基因會(huì)導(dǎo)致毒素體不能正常形成。肌動(dòng)蛋白結(jié)合蛋白FgAbp1在禾谷鐮孢營(yíng)養(yǎng)生長(zhǎng)、發(fā)育、氰烯菌酯敏感性以及毒素體形成過程中發(fā)揮著重要作用。

禾谷鐮孢；FgAbp1；氰烯菌酯；毒素體

0 引言

【研究意義】由禾谷鐮孢（）引起的小麥赤霉病是小麥上的毀滅性病害，該病害不但會(huì)導(dǎo)致小麥嚴(yán)重減產(chǎn)，而且病原菌在小麥粒中可以產(chǎn)生真菌毒素嚴(yán)重危害人和動(dòng)物健康[1]。禾谷鐮孢通常產(chǎn)生的真菌毒素為脫氧雪腐鐮刀菌烯醇（deoxynivalenol，DON）、雪腐鐮刀菌烯醇（nivalenol，NIV）以及玉米赤霉烯酮（zearalenone，ZEN），而其中又以DON的毒性最大，也是全世界范圍內(nèi)最受關(guān)注的真菌毒素之一，很多國(guó)家都已經(jīng)制定了小麥及其制品中DON毒素殘留的最高限量標(biāo)準(zhǔn)[2-3]。DON毒素可以作用于動(dòng)物細(xì)胞的核糖體，抑制蛋白質(zhì)的合成，從而使機(jī)體的免疫系統(tǒng)出現(xiàn)障礙，導(dǎo)致血液、生殖以及呼吸系統(tǒng)發(fā)生病變甚至誘發(fā)癌癥[4]。減少食品中DON毒素污染最直接有效的辦法就是防控小麥赤霉病的發(fā)生，目前農(nóng)業(yè)生產(chǎn)中防控小麥赤霉病的關(guān)鍵措施就是在小麥抽穗揚(yáng)花期施用殺菌劑[5-6]。氰烯菌酯作為一種新型殺菌劑對(duì)鐮孢菌具有特異的抑菌活性，其通過作用于肌球蛋白myosin-Ⅰ，從而實(shí)現(xiàn)抑菌和控制產(chǎn)毒的機(jī)理在一定程度上已經(jīng)得到了詳細(xì)的解析[7-8]。肌動(dòng)蛋白結(jié)合蛋白（actin binding protein，Abp1）作為肌球蛋白myosin-Ⅰ重要的互作元件之一[9]，研究其在禾谷鐮孢生長(zhǎng)發(fā)育、殺菌劑敏感性以及DON毒素生物合成過程中的作用，對(duì)于揭示該病原菌的產(chǎn)毒調(diào)控機(jī)制具有重要的理論意義。【前人研究進(jìn)展】Abp1廣泛存在于真核生物細(xì)胞中，包括真菌、果蠅、寄生蟲以及哺乳動(dòng)物，并且作為橋梁參與肌動(dòng)蛋白骨架連接的內(nèi)吞過程[10]。Abp1p首次被研究鑒定是在釀酒酵母（）中，通過親和層析試驗(yàn)發(fā)現(xiàn)Abp1p與肌動(dòng)蛋白功能的正常發(fā)揮密切相關(guān)[11]。Abp1p蛋白結(jié)構(gòu)特征主要包括N-端肌動(dòng)蛋白解聚因子同源結(jié)構(gòu)域（N-terminal actin depolymerizing factor homology，ADF-H）[12]、螺旋區(qū)域（helical region）、富含脯氨酸（proline-rich region，PRR）的中心區(qū)域以及C-端的SH3（Src-homology 3）結(jié)構(gòu)域[13]。Abp1p不與肌動(dòng)蛋白單體（actin monomer）結(jié)合，而是主要通過ADF-H結(jié)構(gòu)域結(jié)合肌動(dòng)蛋白絲（actin filament）[12,14]。Abp1p具有的兩個(gè)酸性氨基酸殘基序列是結(jié)合并激活A(yù)rp2/3復(fù)合體所必需的[14]；通過SH3結(jié)構(gòu)域與Ark1p、Scp1p以及Sjl2p等蛋白互作共同參與內(nèi)吞過程[13,15-17]。與釀酒酵母的Abp1p類似，哺乳動(dòng)物的同源蛋白mAbp1也是通過ADF-H結(jié)構(gòu)域與肌動(dòng)蛋白絲結(jié)合并參與受體介導(dǎo)的內(nèi)吞作用[18-19]。不同的是，酵母的Abp1p定位于近細(xì)胞膜側(cè)的肌動(dòng)蛋白塊（actin patch）中，而mAbp1通常定位在近細(xì)胞核區(qū)域，但是當(dāng)被Rac GTPase激活后，mAbp1被招募到動(dòng)態(tài)的肌動(dòng)蛋白束（actin cable）上，并精確地定位到Arp2/3復(fù)合體中[11,20]。此外，mAbp1還通過與其他多種蛋白互作參與背紋的形成和細(xì)胞運(yùn)動(dòng)[21-23]。在酵母中，僅僅敲除并不會(huì)影響菌體的生長(zhǎng)，只是輕微地影響了細(xì)胞的內(nèi)吞過程，但是當(dāng)參與內(nèi)吞過程的、或其中的任何一個(gè)與同時(shí)被敲除則會(huì)致死[24-25]；過表達(dá)會(huì)導(dǎo)致酵母細(xì)胞中的肌動(dòng)蛋白骨架出現(xiàn)嚴(yán)重的缺陷[11]。還有研究表明，同時(shí)敲除和肌動(dòng)蛋白形成相關(guān)的蛋白激酶時(shí)，會(huì)導(dǎo)致酵母對(duì)溫度異常敏感[26]。Abp1作為細(xì)胞內(nèi)吞過程的標(biāo)志性蛋白，在酵母中參與內(nèi)吞作用時(shí)與Sla2的功能類似[27-28]。在哺乳動(dòng)物細(xì)胞中，mAbp1介導(dǎo)肌動(dòng)蛋白骨架與內(nèi)吞作用之間的聯(lián)系，并充當(dāng)匯聚多個(gè)信號(hào)途徑的樞紐[18]。在稻瘟病菌（）中，MoAbp1與蛋白激酶MoArk1互作，調(diào)控病原菌的生長(zhǎng)、致病以及內(nèi)吞過程[29]?！颈狙芯壳腥朦c(diǎn)】Abp1普遍存在于真核生物中，雖然在蛋白結(jié)構(gòu)上具有一定的保守性，但其功能在不同的物種中又具有差異性。禾谷鐮孢中Abp1同源蛋白由基因（FGSG_01316）編碼，尚未有研究報(bào)道其在病原真菌次生代謝過程中的作用?！緮M解決的關(guān)鍵問題】基于同源重組原理，通過PEG介導(dǎo)的原生質(zhì)體轉(zhuǎn)化的方法，獲得基因敲除突變體ΔFgAbp1以及相關(guān)的熒光標(biāo)記菌株，明確FgAbp1在禾谷鐮孢生長(zhǎng)發(fā)育、對(duì)氰烯菌酯敏感性以及毒素體形成等方面的作用，為進(jìn)一步揭示DON毒素生物合成調(diào)控提供一定的理論依據(jù)。

1 材料與方法

試驗(yàn)于2019—2020年在安徽農(nóng)業(yè)大學(xué)國(guó)家農(nóng)作物品種審定特性鑒定站完成。

1.1 菌株及培養(yǎng)條件

禾谷鐮孢野生型菌株P(guān)H-1（NRRL 31084）、基因敲除突變體ΔFgAbp1和回補(bǔ)突變體ΔFgAbp1-C、質(zhì)粒neo-PYF11、pBluescript SK+以及大腸桿菌DH5等均保存在安徽農(nóng)業(yè)大學(xué)國(guó)家農(nóng)作物品種審定特性鑒定站。誘導(dǎo)產(chǎn)毒培養(yǎng)基TBI（trichothecene biosynthesis induction medium）：1 L，pH 4.5，含有蔗糖30 g、腐胺0.8 g、KH2PO41 g、MgSO4·7H2O 0.5 g、KCl 0.5 g、FeSO4·7H2O 10 mg和微量元素溶液200 μL（100 mL：檸檬酸5 g、ZnSO4·7H2O 5 g、CuSO4·5H2O 0.25 g、MnSO4·H2O 50 mg、H3BO350 mg和NaMoO4·2H2O 50 mg）。試驗(yàn)中涉及到的完全培養(yǎng)基（complete medium，CM）、基本培養(yǎng)基（minimal medium，MM）、馬鈴薯瓊脂培養(yǎng)基（potato dextrose agar，PDA）、分生孢子誘導(dǎo)培養(yǎng)基（carboxymethyl cellulose，CMC）等的配制方法來源于鐮孢菌試驗(yàn)手冊(cè)（laboratory manual）[30]。

1.2 化學(xué)試劑和儀器

高保真和普通DNA聚合酶、RNA反轉(zhuǎn)錄試劑盒、SYBR Green Ⅰ熒光染料購(gòu)自南京諾唯贊生物科技有限公司；酵母轉(zhuǎn)化試劑盒購(gòu)自美國(guó)MP Biomedicals公司；潮霉素、酵母質(zhì)粒提取試劑盒購(gòu)自北京索萊寶科技有限公司；質(zhì)粒小量提取試劑盒、膠回收試劑盒、引物以及常規(guī)生化試劑均采購(gòu)于上海生工生物工程有限公司。

移液器、小型臺(tái)式離心機(jī)、低溫冷凍離心機(jī)產(chǎn)自Eppendorf公司；PCR儀型號(hào)：C1000 Touch，Bio-Rad公司；垂直電泳儀（Powerpac HV）和多功能凝膠成像儀（Chemidoc）均為Bio-Rad公司生產(chǎn)；激光共聚焦顯微鏡Zeiss LSM780，德國(guó)；透射電子顯微鏡JEOL JEM-1230，日本。

1.3 基因敲除及回補(bǔ)

在真菌基因組數(shù)據(jù)庫(kù)（https://fungidb.org/fungidb/）中檢索出基因（FGSG_01316），并設(shè)計(jì)引物（表1）。首先，以野生型菌株P(guān)H-1的基因組DNA為模板，分別擴(kuò)增出基因編碼區(qū)上游、下游序列各約1 000 bp，以質(zhì)粒pBluescript SK+為模板擴(kuò)增出潮霉素磷酸轉(zhuǎn)移酶基因（）。其次，利用融合PCR（double-joint PCR）技術(shù)原理，按照上游--下游的順序融合成一個(gè)完整的重組DNA片段，從而構(gòu)建出基因敲除打靶片段[31]。最后，借助原生質(zhì)體轉(zhuǎn)化的方法將重組打靶DNA片段轉(zhuǎn)入野生型菌株P(guān)H-1中[32]，在含有100 μg·mL-1潮霉素B的PDA平板中篩選轉(zhuǎn)化子，并用表1中引物Abp1-ID-F/Abp1-ID-R鑒定敲除轉(zhuǎn)化子。

利用高保真酶和表1中的引物Abp1-GFP-F/Abp1- GFP-R，連同基因的啟動(dòng)子區(qū)至開放閱讀框一起擴(kuò)增，將純化后的PCR產(chǎn)物與經(jīng)Ⅰ線性化的質(zhì)粒neo-pYF11，共轉(zhuǎn)化進(jìn)入酵母菌株XK1-25細(xì)胞中[33-34]。經(jīng)PCR鑒定并提取陽(yáng)性克隆酵母中的重組質(zhì)粒，將其轉(zhuǎn)入大腸桿菌DH5中進(jìn)行質(zhì)粒擴(kuò)繁；提取大腸桿菌質(zhì)粒，通過PEG介導(dǎo)的原生質(zhì)體轉(zhuǎn)化，回補(bǔ)基因缺失突變體ΔFgAbp1，通過抗生素G418（100 μg·mL-1）篩選，同時(shí)PCR鑒定出回補(bǔ)突變體。

1.4 菌落生長(zhǎng)、無性繁殖和有性生殖測(cè)定

生長(zhǎng)速率測(cè)定：用內(nèi)徑為5 mm的打孔器切取已活化于PDA中生長(zhǎng)3 d的菌落邊緣，菌碟分別接種于含有PDA、MM和CM的9 cm培養(yǎng)皿中，25℃培養(yǎng)3 d后測(cè)量菌落直徑并拍照，每次重復(fù)3塊培養(yǎng)皿，試驗(yàn)重復(fù)3次。

產(chǎn)孢量及分生孢子形態(tài)觀測(cè)：從新鮮的菌落邊緣取5個(gè)直徑為5 mm的菌碟接種到30 mL CMC培養(yǎng)液中，每個(gè)菌株3瓶，25℃，光暗交替12 h，180 r/min搖培3 d。三層擦鏡紙過濾收集分生孢子，血球計(jì)數(shù)板統(tǒng)計(jì)產(chǎn)孢量，試驗(yàn)重復(fù)3次。分別吸取2.5 μL濾液中的分生孢子和2.5 μL熒光增白劑（calcofluor white，CFW）于潔凈的載玻片上吸打混勻，于熒光顯微鏡下觀察分生孢子的形態(tài)特征。

表1 本研究所用到的引物

有性生殖測(cè)定：將各菌株接種于胡蘿卜培養(yǎng)基中進(jìn)行有性生殖誘導(dǎo)，每個(gè)菌株5個(gè)重復(fù)。25℃普通光照培養(yǎng)箱中待各菌株完全長(zhǎng)滿整個(gè)培養(yǎng)皿，刮凈胡蘿卜培養(yǎng)基表面的氣生菌絲，并在表面均勻涂抹800 μL 0.1% Tween-20，晾干后封口并正置于黑光燈下，25℃培養(yǎng)20 d后拍照。接種針挑取胡蘿卜培養(yǎng)基表面的子囊殼于載玻片上，滴加無菌水，加上蓋玻片輕輕按壓，顯微鏡下觀察子囊及子囊孢子的形態(tài)。

1.5 氰烯菌酯敏感性測(cè)定

從新鮮的菌落邊緣打孔取直徑5 mm的菌碟分別接種于含有0.5 μg·mL-1氰烯菌酯和等量二甲基亞砜（DMSO）的PDA培養(yǎng)基中，25℃培養(yǎng)3 d后測(cè)量菌落直徑并拍照，每次重復(fù)3個(gè)皿，試驗(yàn)重復(fù)3次。觀察氰烯菌酯對(duì)分生孢子萌發(fā)的影響時(shí)，用2%的蔗糖水溶液調(diào)節(jié)分生孢子濃度至5 000個(gè)/mL，加入氰烯菌酯使之終濃度為0.5 μg·mL-1并以等量的DMSO作為對(duì)照組，25℃黑暗條件下處理5 h，各菌株分別統(tǒng)計(jì)300個(gè)孢子計(jì)算萌發(fā)率。

1.6 熒光定位以及透射電鏡觀察

將新鮮的菌碟分別接種于PDB和TBI培養(yǎng)基中25℃避光，搖培48 h，吸取菌絲于激光共聚焦顯微鏡下觀察紅、綠熒光信號(hào)，物鏡60×1.30 Oil DIC M27，激發(fā)波長(zhǎng)488、561 nm，采集8位模式的圖像。用透射電子顯微鏡觀察細(xì)胞中囊泡的方法參考Tang等[35]。

2 結(jié)果

2.1 FgAbp1參與禾谷鐮孢營(yíng)養(yǎng)生長(zhǎng)

基因敲除突變體ΔFgAbp1在培養(yǎng)基PDA和MM上的生長(zhǎng)速率與野生型PH-1、回補(bǔ)突變體ΔFgAbp1-C相比降低了約15%，但在CM培養(yǎng)基中生長(zhǎng)時(shí)，ΔFgAbp1的生長(zhǎng)速率降低了38%。雖然敲除基因在一定程度上減慢了病原菌的生長(zhǎng)速率，但是突變體ΔFgAbp1合成色素的能力并沒有發(fā)生明顯變化（圖1-a、1-b）。

2.2 ΔFgAbp1無性繁殖和有性生殖能力分析

觀察統(tǒng)計(jì)誘導(dǎo)產(chǎn)孢培養(yǎng)基CMC中分生孢子的產(chǎn)量以及形態(tài)特征，發(fā)現(xiàn)突變體ΔFgAbp1與野生型PH-1相比并沒有明顯差異（圖2-a、2-b）。此外，子囊殼和子囊孢子作為重要的初侵染源，在禾谷鐮孢侵染小麥的過程中發(fā)揮著關(guān)鍵作用[36]。通過有性生殖誘導(dǎo)發(fā)現(xiàn)，野生型PH-1、敲除突變體ΔFgAbp1和回補(bǔ)體ΔFgAbp1-C在子囊殼數(shù)量、子囊以及子囊孢子的形態(tài)特征上沒有明顯差異（圖2-c），說明基因的缺失并不影響禾谷鐮孢的無性繁殖和有性生殖過程。

a：野生型PH-1、敲除突變體ΔFgAbp1和回補(bǔ)體ΔFgAbp1-C在PDA、MM和CM中25℃培養(yǎng)3 d的菌落形態(tài)Colony morphology of the wild-type PH-1, deletion mutant ΔFgAbp1 and the complemented strain ΔFgAbp1-C on PDA, MM and CM after 3 days of incubation at 25℃；b：統(tǒng)計(jì)分析各菌株在PDA、MM和CM中25℃生長(zhǎng)3 d后的菌落直徑。誤差線表示標(biāo)準(zhǔn)差，*：P＜0.05 Statistical analysis of each strain colony diameters following growth on PDA, MM and CM at 25℃ for 3 days. Error bars represent standard deviation

a：PH-1、ΔFgAbp1和ΔFgAbp1-C的分生孢子經(jīng)熒光增白劑染色，標(biāo)尺20 μm Conidia of PH-1, ΔFgAbp1 and ΔFgAbp1-C were stained with calcofluor white. Bar = 20 μm；b：各菌株在CMC培養(yǎng)液中3 d的產(chǎn)孢量Conidiation of each strain was assayed with 3-day-old CMC cultures；c：PH-1、ΔFgAbp1和ΔFgAbp1-C在胡蘿卜培養(yǎng)基中的有性生殖。有性生殖誘導(dǎo)20 d后拍照；插入框中為子囊和子囊孢子Sexual development of PH-1, ΔFgAbp1 and ΔFgAbp1-C grown on carrot agar media. Photos were taken 20 days after sexual induction. Dissecting the perithecia exhibited the asci and ascospores of each strain (inset boxes)

2.3 敲除FgAbp1影響禾谷鐮孢對(duì)氰烯菌酯的敏感性

將野生型PH-1、突變體ΔFgAbp1和回補(bǔ)體ΔFgAbp1-C分別接種在含有0.5 μg·mL-1氰烯菌酯的PDA平板中時(shí)發(fā)現(xiàn)，突變體ΔFgAbp1對(duì)氰烯菌酯的敏感性顯著增加，氰烯菌酯對(duì)野生型PH-1的菌絲生長(zhǎng)抑制率為75.2%，而相同條件下ΔFgAbp1則幾乎不能生長(zhǎng)（圖3-a、3-b）。通過進(jìn)一步分析氰烯菌酯對(duì)突變體ΔFgAbp1分生孢子萌發(fā)的影響，發(fā)現(xiàn)在0.5 μg·mL-1氰烯菌酯作用下，ΔFgAbp1分生孢子萌發(fā)率顯著降低（圖3-c、3-d）。但是ΔFgAbp1對(duì)苯并咪唑類殺菌劑多菌靈、三唑類殺菌劑戊唑醇的敏感性與野生型相比沒有明顯差異（數(shù)據(jù)未顯示）。

a：PH-1、ΔFgAbp1和ΔFgAbp1-C在含有0.5 μg·mL-1氰烯菌酯的PDA中25℃培養(yǎng)3 d的生長(zhǎng)比較。溶劑DMSO作為對(duì)照組Comparison of PH-1, ΔFgAbp1 and ΔFgAbp1-C following incubation at 25℃ for 3 days on PDA plates supplemented 0.5 μg·mL-1phenamacril. DMSO was used as a control treatment；b：各菌株在0.5 μg·mL-1氰烯菌酯處理下的生長(zhǎng)抑制率。誤差線表示標(biāo)準(zhǔn)差，**：P＜0.01 The growth inhibition rate of each strain under 0.5 μg·mL-1phenamacril treatment. Error bars represent standard deviation；c：氰烯菌酯對(duì)PH-1、ΔFgAbp1和ΔFgAbp1-C分生孢子萌發(fā)的抑制作用。激光共聚焦顯微鏡拍攝微分干涉（DIC）和熒光增白劑（CFW）染色照片。標(biāo)尺50 μm Inhibitory effect of 0.5 μg·mL-1 phenamacril on conidial germination of PH-1, ΔFgAbp1 and ΔFgAbp1-C. Differential interference contrast (DIC) images of conidia stained with calcofluor white (CFW) were taken with a confocal fluorescence microscope. Bar = 50 μm；d：0.5 μg·mL-1氰烯菌酯處理下各菌株分生孢子的萌發(fā)率。誤差線表示標(biāo)準(zhǔn)差**：P＜0.01 The conidial germination rate of each strain under 0.5 μg·mL-1 phenamacril treatment. Error bars represent standard deviation

2.4 FgAbp1參與囊泡/液泡的形成

通過融合綠色熒光蛋白（green fluorescent protein，GFP）回補(bǔ)基因缺失突變體ΔFgAbp1，激光共聚焦顯微鏡觀察發(fā)現(xiàn)回補(bǔ)菌株ΔFgAbp1-C中GFP熒光信號(hào)主要呈顆粒狀定位于細(xì)胞膜附近（圖4-a）。為了進(jìn)一步明確突變體ΔFgAbp1細(xì)胞內(nèi)部結(jié)構(gòu)的變化，利用透射電子顯微鏡觀察發(fā)現(xiàn)，野生型菌株P(guān)H-1的細(xì)胞中液泡形態(tài)正常，而基因敲除突變體ΔFgAbp1細(xì)胞中則沒有大液泡，而是出現(xiàn)了很多囊泡（圖4-b）。

a：FgAbp1的亞細(xì)胞定位。DIC，微分干涉；GFP，綠色熒光蛋白。標(biāo)尺10 μm Subcellular localization of FgAbp1. DIC, differential interference contrast. GFP, green fluorescent protein. Bar = 10 μm；b：突變體ΔFgAbp1細(xì)胞中形成大量的囊泡和異常的液泡。透射電子顯微鏡拍攝PH-1和ΔFgAbp1細(xì)胞超微結(jié)構(gòu)中液泡/囊泡的形態(tài)The mutant ΔFgAbp1 caused a high vesicle number and abnormal vacuole. The ultrastructural morphology of vacuole/vesicle in PH-1 and ΔFgAbp1 was visualized by transmission electron microscopy

2.5 FgAbp1參與毒素體的形成

近年來研究發(fā)現(xiàn)，DON毒素生物合成途徑中關(guān)鍵氧化酶Tri1定位于一種被稱為毒素體（toxisome）的囊泡膜上[37]。在禾谷鐮孢中FgAbp1與囊泡/液泡的形成密切相關(guān)，為了明確FgAbp1是否參與了毒素體的形成，進(jìn)行了共定位試驗(yàn)。將FgAbp1融合紅色熒光蛋白（red fluorescent protein，RFP），參與DON毒素合成的關(guān)鍵酶Tri1融合GFP。在非誘導(dǎo)產(chǎn)毒培養(yǎng)基PDB中，Tri1-GFP與FgAbp1-RFP共定位于囊泡中（圖5-a），但在誘導(dǎo)產(chǎn)毒培養(yǎng)基TBI中，發(fā)現(xiàn)Tri1-GFP與FgAbp1-RFP則完全共定位于毒素體膜上（圖5-b）。為了進(jìn)一步證實(shí)該結(jié)果，將Tri1-GFP分別轉(zhuǎn)入野生型PH-1和突變體ΔFgAbp1中，在誘導(dǎo)產(chǎn)毒條件下，發(fā)現(xiàn)PH-1細(xì)胞中能產(chǎn)生形態(tài)正常的毒素體，而突變體ΔFgAbp1則不能正常形成毒素體（圖5-c）。

3 討論

肌動(dòng)蛋白作為細(xì)胞骨架的重要組成部分，在真核生物中起著至關(guān)重要的作用。肌動(dòng)蛋白在細(xì)胞形態(tài)、極性的產(chǎn)生和維持，內(nèi)吞作用和物質(zhì)運(yùn)輸，細(xì)胞的收縮性、運(yùn)動(dòng)性以及細(xì)胞分裂等生物學(xué)過程中發(fā)揮功能[38]。但是，肌動(dòng)蛋白的組裝和拆卸，以及它們編織為功能性高級(jí)網(wǎng)絡(luò)的過程，又都受到肌動(dòng)蛋白結(jié)合蛋白的調(diào)節(jié)[39]。本研究通過基因敲除和回補(bǔ)以及亞細(xì)胞定位等方法研究了肌動(dòng)蛋白結(jié)合蛋白FgAbp1在禾谷鐮孢中的生物學(xué)功能。

基因敲除突變體ΔFgAbp1與野生型相比生長(zhǎng)速率有一定程度降低，這與稻瘟病菌中Δ突變體的生長(zhǎng)缺陷相類似[29]。然而，釀酒酵母突變體和構(gòu)巢曲霉（）同源基因缺失突變體Δ則并沒有明顯的生長(zhǎng)缺陷[24,40]，這說明Abp1 在絲狀真菌和酵母中的功能存在一定的分化。肌動(dòng)蛋白通常在細(xì)胞中主要的存在形式有絲狀肌動(dòng)蛋白和斑點(diǎn)狀肌動(dòng)蛋白[9]，通過C-端融合GFP發(fā)現(xiàn)，禾谷鐮孢中FgAbp1主要呈顆粒狀定位于近細(xì)胞膜側(cè)或彌散在細(xì)胞質(zhì)中，這種現(xiàn)象的出現(xiàn)與細(xì)胞中肌動(dòng)蛋白的形態(tài)相吻合。研究已發(fā)現(xiàn)，無論是在酵母、哺乳動(dòng)物還是絲狀真菌中，Abp1都直接參與了細(xì)胞內(nèi)吞過程[24,29,40-41]。突變體ΔFgAbp1細(xì)胞中液泡/囊泡的形態(tài)異常，很可能是因?yàn)榍贸蚝髮?dǎo)致菌體內(nèi)吞過程受阻所致，因?yàn)閮?nèi)吞過程需要囊泡/液泡的參與。在真核生物中，肌球蛋白myosin-Ⅰ與Abp1互作共同參與肌動(dòng)蛋白的組裝以及內(nèi)吞過程[9,42]，對(duì)鐮孢菌特異的新型殺菌劑氰烯菌酯的作用靶標(biāo)即為肌球蛋白myosin-Ⅰ（FgMyo1）[8,43]。最新研究表明，F(xiàn)gMyo1與肌動(dòng)蛋白互作調(diào)控禾谷鐮孢毒素體的形成進(jìn)而影響DON毒素的生物合成[7,44]。筆者發(fā)現(xiàn)突變體ΔFgAbp1專一性地對(duì)氰烯菌酯表現(xiàn)敏感，可能是由于ΔFgAbp1中肌動(dòng)蛋白骨架受損，影響了肌球蛋白FgMyo1正常的功能，間接導(dǎo)致ΔFgAbp1表現(xiàn)出對(duì)氰烯菌酯的敏感性。有趣的是，在非產(chǎn)毒環(huán)境中FgAbp1與DON毒素生物合成過程中關(guān)鍵酶Tri1共定位于囊泡中；而在誘導(dǎo)產(chǎn)毒條件下，F(xiàn)gAbp1與Tri1共定位于毒素體膜上，并且的缺失會(huì)導(dǎo)致毒素體不能正常形成，該結(jié)果表明FgAbp1在禾谷鐮孢毒素體的形成過程中發(fā)揮著重要作用。關(guān)于FgAbp1調(diào)控毒素體形成的機(jī)制有待于進(jìn)一步研究。

a：PDB培養(yǎng)液中生長(zhǎng)48 h后Tri1-GFP和FgAbp1-RFP共定位情況。標(biāo)尺5 μm Co-localization of Tri1-GFP with FgAbp1-RFP in liquid PDB medium for 48 h. Bar = 5 μm；b：TBI培養(yǎng)基中誘導(dǎo)48 h后，Tri1-GFP和FgAbp1-RFP共定位于毒素體。標(biāo)尺5 μm Tri1-GFP and FgAbp1-RFP co-localized in toxisomes after 48 h incubation in TBI medium. Bar = 5 μm；c：TBI培養(yǎng)基中誘導(dǎo)48 h后，PH-1和ΔFgAbp1菌絲中毒素體的形成狀態(tài)。Tri1-GFP指示毒素體的形成。標(biāo)尺5 μm The toxisome formation patterns in the mycelia of PH-1 and ΔFgAbp1 after 48 h incubation in TBI medium. The toxisome formation was visualized using Tri1-GFP as the indicator. Bar = 5 μm

4 結(jié)論

FgAbp1參與禾谷鐮孢的營(yíng)養(yǎng)生長(zhǎng)，但不影響其無性繁殖和有性生殖。更為重要的是，F(xiàn)gAbp1特異性地參與了禾谷鐮孢對(duì)殺菌劑氰烯菌酯的敏感性，并且與毒素體的形成密切相關(guān)。

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The actin binding protein FgAbp1 is involved in growth, development and toxisome formation in

ZHANG Chengqi, WANG Xiaoyan, CHEN Li

School of Plant Protection, Anhui Agricultural University/Anhui Province Key Laboratory of Integrated Pest Management on Crops/Key Laboratory of Biology and Sustainable Management of Plant Diseases and Pests of Anhui Higher Education Institutes, Hefei 230036

【】Abp1 is one of the actin binding proteins that plays a central role in actin cytoskeleton of diverse eukaryotic organisms. The objective of this study is to analyze functions of the actin binding protein FgAbp1 in growth and development, sensitivity to the novel fungicide phenamacril and toxisome formation in.【】Targeted gene deletion construct and fluorescent protein fusion vectors were generated by double-joint PCR and budding yeast gap repair system, respectively. Then, the mutant ΔFgAbp1 and fluorescently labeled strains were obtained using polyethylene glycol (PEG) mediated protoplast transformation. Mycelia growth, sexual/asexual reproduction and sensitivity to phenamacril of wild type PH-1, the mutant ΔFgAbp1 and complemented strain ΔFgAbp1-C were investigated. Localization of FgAbp1 in hyphae was examined through fusion green fluorescent protein. Transmission electron microscopy was carried out to assay the role of FgAbp1 in vacuole/vesicle morphology. Under noninducing medium and DON biosynthesis induction conditions, the role of FgAbp1 in the toxisome formation ofwas performed by dual fluorescence colocalization assay.【】FgAbp1 is primarily localized near the cell membrane in patches of. In MM medium, the growth rate of gene knockout mutant ΔFgAbp1 was reduced by 15% compared with the wild type. But in the nutrient-rich CM, the growth rate of ΔFgAbp1 was decreased by 38%. The mutant ΔFgAbp1 had no obvious defects in sexual and asexual reproduction in comparison with the wild type, while the mycelial growth of ΔFgAbp1 was completely inhibited and the conidia showed significant reduction of germination rate with 0.5 μg·mL-1phenamacril treatment. Moreover,deletion resulted in a high vesicle number and a block of normal vacuole formation. During growth in a toxinnoninducing condition, FgAbp1 and the DON biosynthetic key enzyme Tri1 co-fluoresced in vesicles. Unexpectedly, FgAbp1 and Tri1 cellular co-localized in toxisomes under DON biosynthesis inducing conditions. Furthermore, disruption ofresulted in abnormal toxisomes.【】The actin binding protein FgAbp1 plays an important role in vegetative growth, development, phenamacril tolerance and toxisome formation in

;FgAbp1; phenamacril; toxisome

10.3864/j.issn.0578-1752.2021.13.006

2020-10-11；

2020-10-28

國(guó)家自然科學(xué)基金（31701744）

張承啟，E-mail：zhcq@ahau.edu.cn。通信作者陳莉，E-mail：chenlii@ahau.edu.cn

（責(zé)任編輯 岳梅）