王從麗，李春杰，胡巖峰

(中國科學(xué)院 東北地理與農(nóng)業(yè)生態(tài)研究所 黑土區(qū)農(nóng)業(yè)生態(tài)院重點(diǎn)實(shí)驗(yàn)室，黑龍江 哈爾濱 150081)

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利用Pluronic三維膠系統(tǒng)研究植物寄生線蟲的趨化性

王從麗，李春杰，胡巖峰

(中國科學(xué)院 東北地理與農(nóng)業(yè)生態(tài)研究所 黑土區(qū)農(nóng)業(yè)生態(tài)院重點(diǎn)實(shí)驗(yàn)室，黑龍江 哈爾濱 150081)

摘要：化學(xué)趨向性是植物寄生線蟲尋找寄主的重要機(jī)制。文章在綜述植物寄生線蟲對(duì)寄主及其土壤根圍中潛在的一些化學(xué)物質(zhì)趨性研究進(jìn)展的基礎(chǔ)上，詳細(xì)介紹了利用可模擬土壤三維空間的、透明的、熱可逆的Pluronic膠系統(tǒng)在線蟲趨化性研究中的應(yīng)用及相關(guān)研究進(jìn)展。通過該凝膠系統(tǒng)明確了根結(jié)線蟲趨于偏酸性(pH4.5~5.4)；CO2對(duì)根結(jié)線蟲的吸引是由于CO2改變了介質(zhì)的pH值而導(dǎo)致的；低濃度的HCN(15 μM~22 μM)對(duì)根結(jié)線蟲具有集聚作用，并且這個(gè)聚團(tuán)基因已被標(biāo)記到根結(jié)線蟲的基因組上；植物激素中乙烯的信號(hào)轉(zhuǎn)導(dǎo)途徑介導(dǎo)了線蟲的趨化性；由線蟲分泌參與調(diào)控植物防御和病原抗性的保守小分子化合物蛔甙可能也參與了線蟲聚團(tuán)的調(diào)控。鑒定來自植物或線蟲中參與介導(dǎo)線蟲的趨性物質(zhì)是目前研究的熱點(diǎn)。圖1，參150。

關(guān)鍵詞：植物寄生線蟲；趨化性；Pluronic膠；吸引

0引言

線蟲是一種假體腔、不分節(jié)的蠕蟲性動(dòng)物，歸屬于線蟲動(dòng)物門(Nematoda)，是動(dòng)物界中數(shù)量最豐富者之一，被記錄的物種已超過28 000個(gè)，尚有大量種未被命名。絕大多數(shù)線蟲體小呈圓柱形，又稱圓蟲(Roundworms)。它們?cè)诘?、海水和陸地上隨處可見，不論是個(gè)體數(shù)量或物種數(shù)量都超過其他動(dòng)物，其中大多數(shù)是自由生活線蟲(Free-living nematodes)，主要取食細(xì)菌和真菌，并在南極或熱噴泉等極端環(huán)境中均有存在[1-2]。土壤中自由生活線蟲是地下生態(tài)系統(tǒng)的重要組成部分，在土壤食物網(wǎng)中占重要地位，直接或間接參與生態(tài)系統(tǒng)的物質(zhì)循環(huán)和能量流動(dòng)等生物過程，因此，它們也被作為土壤污染調(diào)控的指示物種[3]。另外一種取食細(xì)菌的有益線蟲能夠侵染靶標(biāo)昆蟲，然后在昆蟲體內(nèi)釋放共生細(xì)菌，細(xì)菌能在昆蟲體內(nèi)繁殖，通常在48 h內(nèi)殺死昆蟲，因此在農(nóng)業(yè)上常作為一種生防制劑防治隱蔽性害蟲，常見的有斯氏線蟲屬(Steinernema)和異小桿線蟲屬(Heterorhabditis)[4-5]。除此之外，對(duì)人類影響最大的線蟲是寄生于植物以及寄生于動(dòng)物(包括人類)的線蟲。針對(duì)植物寄生線蟲的研究表明，每年由植物寄生線蟲引起的農(nóng)業(yè)損失達(dá)到1 500億美元[6]，其中約80%的經(jīng)濟(jì)損失由根結(jié)線蟲(Meloidogynespp.)和孢囊線蟲(Heteroderaspp.和Globoderaspp.)所致，且發(fā)展中國家由線蟲病害引起的產(chǎn)量損失遠(yuǎn)大于發(fā)達(dá)國家[7-8]。

植物寄生線蟲在土壤中能夠識(shí)別由植物根部或者根際微生物所釋放的化學(xué)物質(zhì)而定位寄主，然后侵染寄主，對(duì)于移居性的寄生線蟲(Migratory nematodes)取食植物細(xì)胞后會(huì)轉(zhuǎn)移到另外一個(gè)細(xì)胞，而對(duì)于定居性的內(nèi)寄生線蟲(Sedentary nematodes)，線蟲侵入后會(huì)在寄主植物根內(nèi)形成復(fù)雜的取食結(jié)構(gòu)，如根結(jié)線蟲形成的巨細(xì)胞和孢囊線蟲形成的合胞體[8-10]，利用這些永久的取食位點(diǎn)取食植物，使植物根系正常的生理功能發(fā)生改變，阻礙植物對(duì)水分和營養(yǎng)的吸收，使植物生長(zhǎng)受限，產(chǎn)量降低甚至絕產(chǎn)[11-12]。在線蟲與植物復(fù)雜的互作過程中，線蟲卵的孵化及線蟲對(duì)寄主的尋找和識(shí)別是線蟲對(duì)寄主預(yù)寄生階段的早期反應(yīng)。大多數(shù)植物寄生線蟲生活在土壤中，由于缺乏有效的檢測(cè)手段使得多數(shù)研究主要集中在線蟲侵染植物后期，有些綜述已經(jīng)很詳細(xì)的把近些年對(duì)線蟲侵染后與植物互作的研究做了總結(jié)[9-10,13-16]。

目前，植物寄生線蟲的防治仍以化學(xué)防治為主，雖然效果很好，但化學(xué)藥品殘留期長(zhǎng)且毒性極高，禁用或限制使用溴甲烷薰蒸劑及其它高劇毒的殺線劑，使得對(duì)植物寄生線蟲防治工作提出了新的挑戰(zhàn)。因?yàn)榫€蟲對(duì)寄主的尋找和識(shí)別是線蟲成功寄生植物的第一步，所以很多學(xué)者認(rèn)為如果能干擾或切斷線蟲尋找和識(shí)別寄主的信號(hào)，那么將對(duì)線蟲的成功防治起著重要作用。文章詳細(xì)介紹了利用可模擬土壤三維空間的、透明的、熱可逆的Pluronic膠系統(tǒng)在線蟲趨化性研究中的應(yīng)用及相關(guān)研究進(jìn)展，并對(duì)線蟲趨化性研究現(xiàn)狀進(jìn)行總結(jié)和展望，旨在為進(jìn)一步完善線蟲趨化性基礎(chǔ)理論研究及探索新的線蟲防治策略提供理論參考和應(yīng)用基礎(chǔ)。

1早期的線蟲趨化性研究進(jìn)展

長(zhǎng)期以來趨化性(Chemotaxis)被認(rèn)為是線蟲定位寄主植物的主要原因。線蟲趨化性是指線蟲隨著植物或根際微生物所釋放的化學(xué)信號(hào)物質(zhì)(Semiochemicals)的濃度梯度而移動(dòng)。線蟲是利用其頭部感應(yīng)器官—化感器(Amphids)感應(yīng)由根本身或根圍微生物產(chǎn)生的水溶性或氣體的引誘劑被吸引和定位到植物的根部[17-22]。

1925年，Steiner提出了植物寄生線蟲依靠化學(xué)感應(yīng)來定位它們的寄主，隨后更多的研究證明了線蟲的趨化性使植物寄生線蟲聚集于植物的根部[23-26]。有研究發(fā)現(xiàn)，一系列的植物寄生線蟲都能被植物根或釋放的物質(zhì)吸引或排斥，如甜菜孢囊線蟲(Heteroderaschachtii)、北方根結(jié)線蟲(Meloidogynehapla)[27]、爪哇根結(jié)線蟲(M.javanica)[28]、水稻干尖線蟲(Aphelenchoidesbesseyi)[29]、水稻潛根線蟲(Hirschmanniellaoryzae)[30]及其它線蟲[31]。從銀葉茄(Solanumelaeagnifolium)的葉子提取物中發(fā)現(xiàn)其對(duì)莖線蟲(Orrinaphyllobia)有吸引作用[32]，根腐線蟲(Pratylenchusspp.)被吸引到根部的引誘劑最可能是根滲出物[33-34]。

此后，吸引線蟲的一些引誘劑被報(bào)道，如研究發(fā)現(xiàn)月桂烯(β-myrcene)可能是吸引松材線蟲(Bursaphelenchussylophillus)的引誘劑[35]，簡(jiǎn)單的無機(jī)鹽如鈉離子、鎂離子、氯離子和醋酸離子及環(huán)形腺苷一磷酸(cAMP)均能夠吸引腎線蟲(Rotylenchulusreniformis)[28]，柑橘線蟲(Tylenchulus semipenetrans)能被萜烯類化學(xué)物質(zhì)(geijerene，limonene和pregeijerene)[36]及離子[37]吸引。馬鈴薯金線蟲(Globoderarostochiensis)和馬鈴薯白線蟲(G.pallida)被氨基丁酸和谷氨基酸所吸引[38]。一個(gè)來自洋蔥根分泌物其分子量大于700 kDa的水溶性引誘劑被鑒定出對(duì)莖線蟲(Ditylenchusdipsaci)有吸引[39]。有研究表明最普遍的趨性物質(zhì)是二氧化碳[40-45]和氧氣[46]，大豆孢囊線蟲的雄蟲引誘劑[46]及馬鈴薯金線蟲對(duì)同種線蟲的性激素的吸引[47]也被檢測(cè)到。有學(xué)者針對(duì)線蟲的這些識(shí)別機(jī)制進(jìn)行了分析[48-49]。除了植物根以外，一些食線蟲的真菌能夠吸引線蟲[49-52]。

根滲出物既包括吸引線蟲的引誘劑，也可能包括排斥線蟲的驅(qū)蟲劑，其作用取決于兩者誰占主導(dǎo)力。Zhao等[53]發(fā)現(xiàn)南方根結(jié)線蟲被植物根的吸引或排斥存在著寄主特異性，當(dāng)去掉根邊緣細(xì)胞時(shí)，沒有根結(jié)線蟲被吸引到根部，然而線蟲能被吸引到包含邊緣細(xì)胞的根尖，同時(shí)發(fā)現(xiàn)分離的邊緣細(xì)胞能使二齡幼蟲失去移動(dòng)性，身體變得僵直，但這種現(xiàn)象也是取決于線蟲寄主的特異性。對(duì)線蟲卵孵化的抑制也有一些報(bào)道[2,54-60]。Zhang等[61]從甘薯乳液中分離出的十八烷基香豆酸[Octadecyl-(Z)-p-coumarate] 對(duì)甘薯莖線蟲(Ditylenchusdestructor)有很強(qiáng)的排斥作用。

生長(zhǎng)旺盛的根圍區(qū)存在著氨基酸、離子、pH、溫度和CO2等引誘劑，它們被分成三種類型，即長(zhǎng)距離、短距離和局部的引誘劑，長(zhǎng)距離引誘劑(如揮發(fā)性物質(zhì)CO2)是指把線蟲吸引到根圍，短距離的引誘劑(如根滲出物，多數(shù)是水溶性物質(zhì))是指把線蟲吸引到根表，而局部引誘劑是指能使內(nèi)寄生線蟲定位到喜歡的侵染位點(diǎn)，如根結(jié)線蟲和孢囊線蟲能夠被吸引到植物根尖部位的伸長(zhǎng)區(qū)域，但這個(gè)區(qū)域潛在的物理和化學(xué)引誘劑仍不清楚，另外根的這個(gè)伸長(zhǎng)區(qū)域溫度升高可能影響線蟲的識(shí)別[2,20,62-64]。

2Pluronic三維膠系統(tǒng)研究方法

很久以前線蟲的趨化性已被各種研究方法證實(shí)，如瓊脂膠法[28，39,65-72]、沙柱法[48]，還有利用線蟲揮發(fā)性物質(zhì)影響線蟲行為的氣體流動(dòng)室[73-74]。但是這些方法有其不足，如瓊脂膠比較硬，使線蟲的運(yùn)動(dòng)空間受阻；沙柱法的不可視性，無法實(shí)時(shí)觀察到線蟲的移動(dòng)狀態(tài)。而土壤線蟲是以三維空間識(shí)別化學(xué)信號(hào)的。Wang等[75]建立了一套可模擬土壤三維空間的Pluronic膠系統(tǒng)，該系統(tǒng)操作簡(jiǎn)便、快捷、高效，可實(shí)時(shí)觀測(cè)到線蟲的運(yùn)動(dòng)軌跡，現(xiàn)已成功觀察到了根結(jié)線蟲被吸引到植物根部(圖1A)的全過程，并證明了這個(gè)系統(tǒng)不僅能用于研究線蟲與寄主的關(guān)系，也能用于研究線蟲的其它行為學(xué)。Pluronic膠是環(huán)氧丙烷和環(huán)氧乙烷的共聚體，被廣泛地應(yīng)用于醫(yī)藥和化妝品領(lǐng)域[76-80]。Pluronic膠曾被用于研究線蟲、細(xì)菌、真菌和植物組織的介質(zhì)[81]，23%的膠在室溫時(shí)是半固態(tài)的，在低于15℃時(shí)呈液態(tài)且高度透明，這些特點(diǎn)使得它優(yōu)于瓊脂、沙子和其它介質(zhì)[75]。首先，高度透明的膠讓我們?nèi)菀子^察線蟲在植物根存在時(shí)的移動(dòng)；其次，Pluronic膠模擬土壤三維空間[73]，能夠讓線蟲以三維空間識(shí)別信號(hào)梯度并在膠中移動(dòng)；第三，這種膠能形成穩(wěn)定的信號(hào)梯度使得這個(gè)檢測(cè)系統(tǒng)具有很高的可重復(fù)性，且能實(shí)時(shí)定量的觀測(cè)線蟲對(duì)根的反應(yīng)；第四，線蟲在低于15℃能被分散到液態(tài)膠中，這個(gè)溫度對(duì)線蟲無害，相比高溫熔化瓊脂膠更有利于線蟲的分散，并且還可以把固化的膠置于低溫下融化，回收被吸引在根圍的線蟲來研究線蟲基因的表達(dá)及功能分析；第五，Pluronic膠與瓊脂膠相比不易受微生物污染，短時(shí)間試驗(yàn)可以不用滅菌，但是如果試驗(yàn)要持續(xù)3 d或者更長(zhǎng)時(shí)間則需要滅菌。這個(gè)系統(tǒng)已被證明可以應(yīng)用于研究除植物寄生線蟲外的其它線蟲的行為及其與寄主的關(guān)系，如昆蟲病原線蟲與寄主宿主植物的關(guān)系[82]及自由生活線蟲(待發(fā)表)的研究。

Wang等[75]通過實(shí)時(shí)觀察、檢測(cè)番茄根部對(duì)不同種根結(jié)線蟲(Meloidogynespp.)的吸引和侵染，發(fā)現(xiàn)南方根結(jié)線蟲(M.incognita)和爪哇根結(jié)線蟲比北方根結(jié)線蟲更快的移動(dòng)到植物根部并對(duì)其造成更嚴(yán)重的危害；檢測(cè)發(fā)現(xiàn)南方和爪哇根結(jié)線蟲被吸引現(xiàn)象的最佳時(shí)間是接種后2 h~3 h，而北方根結(jié)線蟲大約是5 h~6 h，見圖1A；不僅番茄根對(duì)根結(jié)線蟲有吸引，模式生物擬南芥(Arabidopsis)和苜蓿(M.truncatula)的根也對(duì)根結(jié)線蟲有吸引，并且苜蓿的根齡對(duì)線蟲的吸引有很大的影響，同3 d的根齡相比，5 d的根齡顯著降低了對(duì)線蟲的吸引；抗病和感病番茄品種對(duì)根結(jié)線蟲的吸引沒有差異。Dutta等[83]利用Pluronic膠發(fā)現(xiàn)南方根結(jié)線蟲對(duì)番茄根的趨性與對(duì)水稻和芥末根相比更強(qiáng)，而水稻上的根結(jié)線蟲(M.graminicola)對(duì)水稻根的趨性強(qiáng)于番茄和芥末根的趨性，說明特異性寄主引誘劑參與了線蟲的趨性。此外，利用這個(gè)系統(tǒng)研究了小麥孢囊線蟲對(duì)不同小麥品種抗感根系的吸引差異[84-85]。Danquah等[86]在大蒜的提取物中發(fā)現(xiàn)一種為水楊醛(Salicylaldehyde)的化學(xué)物質(zhì)，它能夠顯著降低對(duì)馬鈴薯孢囊白線蟲(G.palida)的吸引。

3趨化性信號(hào)物質(zhì)

3.1酸

Wang等[87]利用Pluronic膠建立了一套簡(jiǎn)單的線蟲對(duì)化學(xué)物質(zhì)的趨化性研究系統(tǒng)。所檢測(cè)的化學(xué)物質(zhì)用Pluronic膠配制成所需濃度，然后注入兩端都被剪切的稱為化學(xué)分配器(Chemical dispenser)的移液器槍頭(200 ul或10 ul)內(nèi)，再將槍頭放入含有線蟲的培養(yǎng)皿中心使其凝固，實(shí)時(shí)檢測(cè)線蟲對(duì)化學(xué)物質(zhì)的吸引或排斥。首先利用醋酸和pH指示劑證明了在第4 h和第24 h化學(xué)分配器兩端形成了穩(wěn)定的酸性梯度，見圖1 B-C。利用這個(gè)系統(tǒng)檢測(cè)到根結(jié)線蟲被吸引到含醋酸的化學(xué)分配器兩端，適宜的趨酸pH范圍在4.5~5.4之間[87]，見圖1 D-F。結(jié)果也表明根結(jié)線蟲的不同種和小種對(duì)pH都有趨性，但趨性的程度有所差異[87]。根結(jié)線蟲、孢囊線蟲和其他植物病原/共生體能夠被吸引到根的伸長(zhǎng)區(qū)域，而生長(zhǎng)的根細(xì)胞釋放出H+能使根的伸長(zhǎng)區(qū)成為最酸性的區(qū)域[88-90]。Mulkey和Evans[88]根據(jù)pH指示染色劑，發(fā)現(xiàn)在瓊脂膠里玉米幼苗根的伸長(zhǎng)區(qū)域pH<5。利用Pluronic膠和pH指示染色劑，模式植物苜宿和番茄的幼苗根部產(chǎn)生一個(gè)酸性梯度，使pH值酸化到5或更低[87]。根部更酸的區(qū)域和線蟲最喜歡被吸引到根尖部說明了pH梯度是線蟲趨化的信號(hào)，這些信號(hào)被利用來介導(dǎo)線蟲移動(dòng)到生長(zhǎng)根的伸長(zhǎng)區(qū)域。不同種線蟲聚集的最適宜的pH范圍是不同的，北方根結(jié)線蟲集聚到所喜歡的酸性區(qū)域比南方和爪哇根結(jié)線蟲快很多，而這個(gè)結(jié)果卻與南方根結(jié)線蟲、爪哇根結(jié)線蟲比北方根結(jié)線蟲移動(dòng)到番茄根速率快的結(jié)果不一致，說明pH不是獨(dú)一的趨化信號(hào)，它的相對(duì)重要性可能取決于線蟲的株系和寄主[87]。北方根結(jié)線蟲對(duì)不同Bronsted酸的反應(yīng)也不同，強(qiáng)酸(鹽酸、硫酸、次氯酸和甲磺酸)和一元羧酸(醋酸、蟻酸和丙酸)能夠吸引線蟲到化學(xué)分配器的大頭端形成暈圈，并在小頭端的開口處聚集，見圖1D-E。然而在檸檬酸、乳酸和琥珀酸處理中線蟲卻移動(dòng)到化學(xué)分配器內(nèi)。不同的檸檬酸鹽在pH4.5或在5.5的檸檬酸緩沖液中，線蟲表現(xiàn)出和檸檬酸同樣的趨化性，進(jìn)入到化學(xué)分配器內(nèi)，而線蟲對(duì)醋酸鹽沒有反應(yīng)。這些結(jié)果說明檸檬酸、乳酸和琥珀酸的離子可能是特殊的引誘劑。同時(shí)發(fā)現(xiàn)同醋酸pKa(4.8)很相似的吡啶離子(吡啶鹽酸鹽和吡啶甲磺酸鹽)和檸檬酸一樣也引起線蟲聚集到化學(xué)分配器內(nèi)，說明吡啶(Pyridine)也可能是一個(gè)引誘劑[87]。這個(gè)結(jié)果和早期報(bào)道的吡啶是模式生物秀麗隱桿線蟲(Caenorhabditiselegans)的引誘劑[91]一樣。此結(jié)果也表明了線蟲在自然環(huán)境中對(duì)這些錯(cuò)綜復(fù)雜的信號(hào)產(chǎn)生協(xié)同或拮抗的反應(yīng)，因此有必要對(duì)這些潛在的引誘劑進(jìn)行鑒定并研究其互作關(guān)系。

3.2二氧化碳

長(zhǎng)期以來二氧化碳一直被認(rèn)為是自由生活、昆蟲寄生和植物寄生線蟲的引誘劑[42,74,92-93]，然而在這些實(shí)驗(yàn)中沒有用到緩沖液，而CO2在水溶液里能夠改變其pH，所以線蟲是被CO2還是pH梯度或是兩者都吸引呢？Wang等[87]設(shè)計(jì)了一個(gè)實(shí)驗(yàn)將CO2通入到含pH值為7和4.9的緩沖液的Plruonic 膠中，在pH值為7的緩沖液膠中，通CO2后能形成CO2和pH兩個(gè)梯度；因?yàn)樗柡偷腃O2的pH值只有4.4，所以當(dāng)向pH 4.9的膠中通入CO2時(shí)，pH基本不會(huì)改變，因此在pH為4.9的緩沖液中只能形成CO2濃度。結(jié)果表明在緩沖液為pH4.9的膠中線蟲均勻分散而沒有聚集到通CO2區(qū)域的外圍處，然而在pH為7的緩沖液中，聚集發(fā)生在通CO2的外圍處，而此處的pH值在線蟲吸引范圍內(nèi)，說明線蟲的吸引是CO2改變了膠中的pH梯度形成的，并非CO2梯度本身。

Wang等[87]在實(shí)驗(yàn)中也發(fā)現(xiàn)當(dāng)通入CO2時(shí)，通CO2區(qū)域的線蟲數(shù)量在pH為4.9和pH為7的緩沖液膠中沒有變化，然而當(dāng)停止通入CO2后，線蟲進(jìn)入CO2通入?yún)^(qū)域。發(fā)生這種現(xiàn)象可能有幾個(gè)原因：(1)CO2是麻醉劑，超過10%的濃度能夠減緩線蟲的移動(dòng)[42]，當(dāng)停止輸入CO2后，CO2濃度降低，線蟲能夠進(jìn)入這個(gè)區(qū)域；(2)中央的pH低于外圍的pH值而有助于吸引，pH計(jì)檢測(cè)的結(jié)果也證明了這一點(diǎn)；(3)通CO2區(qū)域O2的濃度比較低，線蟲被吸引到低氧區(qū)域，這與秀麗隱桿線蟲趨于低氧區(qū)報(bào)道相一致[94]。

圖1　北方根結(jié)線蟲(Meloidogyne hapla VW9)在Pluronic膠系統(tǒng)里對(duì)植物和化學(xué)物質(zhì)的趨性反應(yīng)。A：線蟲暴露于根6 h后對(duì)番茄根的趨性反應(yīng)。培養(yǎng)皿(100×15 mm)內(nèi)混有5 000條二齡幼蟲的15毫升Pluronic膠[75]。B-C:包含0.85 M的醋酸的化學(xué)分配器放進(jìn)含有pH指示劑(pH5.2~6.8，黃色到紫色)的膠里4 h(B)和24 h(C)后在Pluronic膠里形成的pH梯度[87]。D-F: 線蟲對(duì)醋酸的反應(yīng)。化學(xué)分配器里包含0.85 M的醋酸。解剖顯微鏡下顯示的化學(xué)分配器(化學(xué)分配器)的小頭端醋酸在5 h(D)和24 h(E)時(shí)對(duì)線蟲的吸引，F(xiàn)顯示24 h后化學(xué)分配器大頭端醋酸在低倍光學(xué)下對(duì)線蟲吸引情況[87]。G-I：線蟲對(duì)氰化鉀的濃度梯度反應(yīng)?；瘜W(xué)分配器里含有5 mM氰化鉀，在實(shí)驗(yàn)5 h后小頭端線蟲的反應(yīng)顯示在G，在24 h顯示在H，大頭端顯示的是試驗(yàn)后24 h低倍光學(xué)下線蟲的反應(yīng)[100]。醋酸和氰化鉀實(shí)驗(yàn)都是用每毫升膠含600條二齡幼蟲的培養(yǎng)皿中進(jìn)行，每皿20 ml膠。圖片里A、D、E、G和H的標(biāo)尺比例是1 mm。Fig.1　Root-knot Nematode (Meloidogyne hapla VW9) attraction to plants and chemicals in Pluronic gel system. A: Attraction to root tips of tomato VFNT,6 h after initiation of assay. A standard Petri dish (100×15 mm) containing 5 000 J2 in 15 ml Pluronic gel was used for the assay[75]; B-C: pH gradient formation in PF-127 gel at 4 h(B) and 24 h(C) after the dispenser was inserted into the gel. The pH indicator bromocresol purple (pH 5.2-6.8, yellow to purple) was included in the gel. The dispensers initially contained 0.85 M acetic acid[87]; D-F: Migration of nematode in acetic acid gradients in PF-127 gel. Dispensers contain 0.85 M acetic acid. Dissecting microscope images of the dispenser small end were recorded at 5 h(D) and 24 h(E) after inserting dispensers in the gel. Lower magnification edge-lighted photographs of the dispenser large end at 24 h is shown in F[87]. G-I: Response of nematodes to potassium cyanide gradients in the gel. Region of the gel around the small opening of chemical dispenser with 5 mM KCN is shown at 5 h (G) and 24 h (H) after initiation of the assay. Panel I shows a detail of large end of dispenser with 5 mM KCN at 24 h. Panels I is taken at lower magnification with backlighting for the large end of the dispenser with 5 mM KCN at 24 h after assay initiation[100].Petri dish containing 600 J2 per ml PF-127 gel was used for the acetic acid and cyanide assays. Scale bar in A, D, E, G and H is 1 mm.

3.3聚團(tuán)行為(Clumping behaviors)和氰化物(CN-)

聚團(tuán)(Clumping)或者集聚(Aggregation)行為在自由生活、植物寄生和動(dòng)物寄生線蟲中都普遍存在[95-99]。利用Pluronic膠系統(tǒng)發(fā)現(xiàn)，大部分根結(jié)線蟲(M.hapla，M.javanica，M.incognita)在有無植物根存在時(shí)都能夠形成聚團(tuán)體(clumps，線蟲聚成緊緊的一團(tuán))，無根比有根存在時(shí)線蟲形成聚團(tuán)體需要的時(shí)間更長(zhǎng)，M.hapla比M.javanica和M.incognita需要更長(zhǎng)時(shí)間[75,100]，這可能和前面提到的M.hapla比M.javanica和M.incognita移動(dòng)緩慢一致。例外的情況是從美國北卡分離到的M.hapla株系NCS線蟲能夠集聚到一起，但不會(huì)聚團(tuán)；而來源于法國的一個(gè)M.hapla株系LM卻不能積聚到一起，更不能形成聚團(tuán)。從美國葡萄上分離到的M.incognita株系Harmony也不能形成聚團(tuán)體。這些結(jié)果說明線蟲的聚團(tuán)行為依賴于線蟲種或小種的特異性。

線蟲聚團(tuán)體形成的速度和線蟲的群體密度成正相關(guān)，并且在膠表面放置蓋玻片能夠加速聚團(tuán)體的形成，這說明高的群體密度或加蓋蓋玻片可能會(huì)導(dǎo)致低氧狀態(tài)并促進(jìn)線蟲間釋放揮發(fā)性信息素(Pheromones)而造成聚團(tuán)行為[75]。高密度的線蟲也能在根尖周圍聚集成團(tuán)，說明誘發(fā)聚團(tuán)行為信號(hào)來自根或二齡幼蟲J2。植物寄生線蟲這種聚團(tuán)行為同昆蟲病原線蟲異小桿屬(Heterorhabiditisspp.)在高濕度情況下脫水干燥形成團(tuán)聚體[98]的行為相似。另外如食真菌性線蟲(Aphelenchusavenae)和莖線蟲(Ditylenchusdipsaci)在無水狀態(tài)下能夠集聚成很大的“蟲毛球(Eelworm wool)”聚團(tuán)體，這種聚團(tuán)體在干燥狀況下能存活多年[96,101]。但是根結(jié)線蟲在完全干燥狀態(tài)下不能存活，當(dāng)把新鮮植物根放在聚團(tuán)體附近，聚團(tuán)體立即解散而移動(dòng)到根部，這說明其聚團(tuán)行為可能是應(yīng)對(duì)不利環(huán)境的一種生存策略。此外，雄蟲對(duì)雌蟲的強(qiáng)烈吸引也說明了線蟲之間存在信息素。

Wang等[100]利用化學(xué)分配器系統(tǒng)檢測(cè)到氰離子(CN-)，它能夠使線蟲幾個(gè)小時(shí)后就能集聚到一起，24 h后能聚集成團(tuán)，見圖1G-I，其最適宜集聚的CN-濃度是15 μM~22 μM。氰化物對(duì)很多生物都是有毒的，常被認(rèn)為是一些根圍細(xì)菌釋放的有毒物質(zhì)如綠膿桿菌(Pseudomonasaeriginosa)釋放的氰化物能殺死秀麗隱桿線蟲[102]，所以被用來防治土壤中的病蟲害(包括線蟲)[103-104]。此外，受傷的植物組織也能釋放出HCN，此過程被認(rèn)為是一個(gè)抵抗害蟲的防御反應(yīng)[105]，已有人把產(chǎn)生氰化物的植物開發(fā)用于防治線蟲[106]。一定濃度的氰離子能吸引線蟲，可能是HCN的存在導(dǎo)致了低氧狀態(tài)而干預(yù)了對(duì)氧氣的識(shí)別；亦或是CN-是來自根圍天然的引誘劑，如根際微生物或植物釋放的CN-等。

目前對(duì)線蟲的集聚行為研究中有關(guān)調(diào)控秀麗隱桿線蟲取食行為的集聚基因NPR-1(Neuropeptide receptor 1，NPR-1)的研究最多、最清楚，NPR-1基因是一種與哺乳動(dòng)物神經(jīng)肽Y(Neuropeptide Y，NPY)受體相似的G蛋白偶聯(lián)受體。其變異的等基因位點(diǎn)能調(diào)控秀麗隱桿線蟲的聚集(Social feeding)和邊集取食行為(Boarding feeding)[107]。NPR-1通過感應(yīng)神經(jīng)元表達(dá)的鳥苷酸環(huán)化酶(Guanylatecyclases)在不同的O2濃度下調(diào)控線蟲的集聚及平衡線蟲的吸引和排斥[94,108-111]。根結(jié)線蟲被一定濃度HCN所吸引有可能是利用這個(gè)酶來調(diào)控的，對(duì)北方根結(jié)線蟲的兩個(gè)不同株系鑒定發(fā)現(xiàn)，線蟲都能夠集聚，但只能在一個(gè)親本上形成聚團(tuán)體，說明了線蟲通過像NPR-1基因變異的等基因位點(diǎn)來調(diào)控的可能性比較大。利用北方根結(jié)線蟲F2進(jìn)一步分離群體鑒定了一個(gè)單基因控制線蟲的聚團(tuán)性，并把對(duì)HCN的聚團(tuán)基因定位到線蟲的遺傳連鎖群L8上，這是第一個(gè)被標(biāo)記的專性植物寄生線蟲的表現(xiàn)型基因[100]，根結(jié)線蟲全基因組序列[6,112-113]及其高密度的SNP遺傳圖譜[114]的發(fā)表使得克隆這個(gè)基因成為可能。

此外，F(xiàn)MRF酰胺相關(guān)多肽(FMRFamide-like neuro peptides，F(xiàn)LPs)flp-18和flp-21作為NPR-1的內(nèi)源性配體參與調(diào)節(jié)線蟲對(duì)O2的感知過程[115]。大豆孢囊線蟲和秀麗隱桿線蟲的鳥苷酸環(huán)化酶基因組成員在序列和基因組表達(dá)上都具有很高的同源性，植物寄生線蟲與秀麗隱桿線蟲flp基因也有很高的同源性[6,116-119]。Kimber等[118]對(duì)馬鈴薯金線蟲flp-18基因進(jìn)行dsRNA浸泡24h后發(fā)現(xiàn)線蟲幾乎喪失了移動(dòng)的能力。RNA干擾南方根結(jié)線蟲的flp-14和flp-18兩個(gè)基因后，線蟲的移動(dòng)、吸引、侵染和繁殖都被降低，這兩個(gè)基因單獨(dú)或混合情況下在不同的時(shí)間里都能影響線蟲的趨性反應(yīng)[120-121]。參與神經(jīng)調(diào)控的這些基因成為新型殺蟲劑最具潛力的藥物靶標(biāo)之一。但是秀麗隱桿線蟲的集聚是活躍的四齡幼蟲或成蟲在取食時(shí)松散的集聚在一起，而植物寄生線蟲是非取食的早期二齡幼蟲集聚成團(tuán)，由更多的線蟲緊緊的集聚在一起，這說明了兩者集聚的原因必有其相同和不同之處。

3.4植物激素

植物激素在植物生長(zhǎng)發(fā)育及脅迫反應(yīng)中發(fā)揮著重要作用。許多研究表明植物激素也參與調(diào)控植物線蟲取食位點(diǎn)的形成[122-128]，Siddique等[129]研究發(fā)現(xiàn)，植物寄生線蟲也能通過向寄主釋放細(xì)胞分裂素來調(diào)控植物細(xì)胞分裂及促進(jìn)取食位點(diǎn)的形成。但對(duì)激素是否介導(dǎo)線蟲早期識(shí)別寄主植物的研究極少，其中原因之一就是缺乏有效的前期檢測(cè)手段。模式植物擬南芥具有基因組小、繁殖快及遺傳操作簡(jiǎn)便等優(yōu)點(diǎn)，同時(shí)也是一種植物寄生線蟲的良好寄主，作為替代型操作對(duì)象已被廣泛用于線蟲與寄主的互作機(jī)制研究。乙烯是簡(jiǎn)單的氣體化合物，能夠調(diào)控植物各個(gè)生長(zhǎng)發(fā)育過程，如種子萌發(fā)、開花、衰老和果實(shí)成熟，也包括對(duì)生物和非生物脅迫等的生理反應(yīng)過程[130-131]。乙烯含量升高不僅能誘導(dǎo)防御反應(yīng)而合成相關(guān)的蛋白[132-133]，還是產(chǎn)生拮抗微生物肽和次級(jí)代謝物所包括的植保素(Phytoalexins)所必需的[134-135]。早期Wubben等[128]利用瓊脂膠作為介質(zhì)，發(fā)現(xiàn)擬南芥乙烯過量合成突變體(eto)，根分泌物比野生型根系滲出物吸引更多的甜菜孢囊線蟲(H.schachtii)，說明乙烯途徑可能在孢囊線蟲侵染寄主早期就已經(jīng)發(fā)揮作用，上述結(jié)論得到Kannerhofer等[126]進(jìn)一步的驗(yàn)證(瓊脂膠塊法)，他們發(fā)現(xiàn)乙烯處理的擬南芥根顯著增加了對(duì)甜菜孢囊線蟲的吸引，而AVG處理的擬南芥根對(duì)線蟲的吸引與對(duì)照相比沒有區(qū)別。然而，孢囊線蟲具有相對(duì)較狹窄的寄主范圍，而根結(jié)線蟲具有更廣泛的寄主范圍，植物對(duì)兩者的吸引可能也存在差異。Fudali等[136]利用Pluronic膠系統(tǒng)研究了乙烯途徑在根結(jié)線蟲對(duì)植物早期識(shí)別過程中的作用，發(fā)現(xiàn)添加乙烯合成抑制劑AVG(2-aminoethoxyvinylglycine)增加了擬南芥根對(duì)線蟲的吸引，而乙烯過量表達(dá)突變體(eto1，eto2，eto3)卻減少了對(duì)線蟲的吸引，說明乙烯本身或乙烯途徑介導(dǎo)擬南芥根對(duì)線蟲的吸引。ETR1處于乙烯受體基因的下游位置，對(duì)下游乙烯信號(hào)傳導(dǎo)起負(fù)調(diào)控作用，其組成型表達(dá)突變體ctr1卻能降低對(duì)線蟲的吸引。與野生型擬南芥相比，乙烯受體功能獲得性突變體(etr1-3，ein4-1和ers2-1)和乙烯信號(hào)途徑正調(diào)控因子的不敏感型突變體(ein2-7)卻增加了對(duì)線蟲的吸引，這些結(jié)果進(jìn)一步證實(shí)乙烯信號(hào)途徑參與調(diào)控植物對(duì)線蟲的吸引。線蟲對(duì)植物根系滲出物表現(xiàn)出不同響應(yīng)，包括被吸引、排斥或休眠，這可能與根系釋放部位有關(guān)[53,137]，但目前仍不清楚具體是哪些化學(xué)物質(zhì)起作用。乙烯途徑調(diào)控線蟲對(duì)植物識(shí)別可能與線蟲、根分泌物的種類有關(guān)。因此，擬南芥中乙烯信號(hào)減弱可能導(dǎo)致下游次生代謝發(fā)生改變，增加了吸引根結(jié)線蟲的揮發(fā)性誘導(dǎo)物的釋放，但是卻可能減少了孢囊線蟲引誘物的分泌。這些結(jié)果也說明乙烯對(duì)廣寄主的根結(jié)線蟲與窄寄主的甜菜孢囊線蟲調(diào)控不一樣。然而當(dāng)我們利用Pluronic膠系統(tǒng)檢測(cè)乙烯對(duì)大豆孢囊線蟲的吸引影響時(shí)卻發(fā)現(xiàn)，大豆孢囊線蟲對(duì)乙烯信號(hào)的反應(yīng)和根結(jié)線蟲一樣(待發(fā)表)。大豆孢囊線蟲和甜菜孢囊線蟲屬于同一個(gè)屬，但大豆孢囊線蟲的寄主范圍比甜菜孢囊線蟲更狹窄，從這個(gè)角度來講，甜菜孢囊線蟲、根結(jié)線蟲與大豆孢囊線蟲的吸引差異是否由于所用方法的不同而導(dǎo)致的結(jié)果差異值得探討。研究者是利用包含根系分泌物的瓊脂塊檢測(cè)甜菜孢囊線蟲的吸引，是否能代表活體根還需要進(jìn)一步研究，因此，很有必要利用Pluronic膠系統(tǒng)來證實(shí)乙烯對(duì)甜菜孢囊線蟲的吸引。

Feng等[138]利用Pluronic膠系統(tǒng)發(fā)現(xiàn)高濃度生長(zhǎng)素增加了對(duì)水稻干尖線蟲(A.besseyi)的吸引，可能是因?yàn)樯L(zhǎng)素這類化合物的pH呈酸性。內(nèi)源生長(zhǎng)素含量高的水稻品種比內(nèi)源生長(zhǎng)素低的品種更易被水稻干尖線蟲侵染，外源噴施萘乙酸后稻穗種子內(nèi)的線蟲數(shù)量明顯增加，但是生長(zhǎng)素運(yùn)輸抑制劑處理的稻穗種子內(nèi)有很少的線蟲，說明生長(zhǎng)素對(duì)根腐線蟲的吸引、侵入及繁殖都起著重要的作用。Kannerhofer等[126]利用瓊脂膠發(fā)現(xiàn)茉莉酸激活植物對(duì)線蟲的早期防御反應(yīng)，而水楊酸在后期線蟲合胞體的形成和雌蟲的發(fā)育過程中起到負(fù)調(diào)控作用。我們利用Pluronic膠系統(tǒng)檢測(cè)了擬南芥生長(zhǎng)素、茉莉酸和水楊酸等激素突變體對(duì)根結(jié)線蟲和大豆孢囊線蟲的吸引，發(fā)現(xiàn)突變體和野生型間沒有差異，說明這些激素可能在線蟲侵染后期起作用(待發(fā)表)。結(jié)果表明線蟲的不同屬、種甚至小種對(duì)植物信號(hào)物質(zhì)有不同的反應(yīng)，說明了反應(yīng)特異性。植物激素茉莉酸、水楊酸、乙烯和生長(zhǎng)素等信號(hào)途徑存在復(fù)雜的交互作用[139]，目前的研究?jī)H局限于單個(gè)激素的研究，激素之間的交互作用對(duì)線蟲早期信號(hào)的識(shí)別將是下一步研究的目標(biāo)。

3.5線蟲蛔甙(Ascarosides)信息素(Pheronmones)

蛔甙(Ascarosides)是起初從秀麗隱桿線蟲中分離的小分子信息素，能夠調(diào)控雌雄吸引、排斥、集聚、嗅覺可塑性及進(jìn)入抵抗壓力的休眠(Dauer)等行為[140-144]，近期引起了植物線蟲及病理學(xué)家的廣泛關(guān)注?；走笆蔷€蟲特有的一種攜帶親脂側(cè)鏈脂肪酸的雙脫氧蛔糖基(Dideoxysugar ascarolyse)的糖苷，廣泛存在于自由生的秀麗隱桿線蟲[145]、Pristionchuspacificus[46]、寄生昆蟲的昆蟲病原線蟲異小桿線蟲(Heterorhabditisbacteriophora)[147]、寄生植物的根結(jié)線蟲(M.incognita、M.javanica和M.hapla)、大豆孢囊線蟲(H.glycine)和最短尾短體線蟲(Pratylenchusbrachyurus)中[148]，不同種特異性蛔甙混合物能夠調(diào)控線蟲進(jìn)入抵抗壓力的分散(Dispersal)或侵染幼蟲階段。蛔甙的生物活性與其構(gòu)象有關(guān)，而且極低的蛔甙濃度能夠引起生物學(xué)活性[142]。目前已經(jīng)從20多種不同線蟲里鑒定出200多種不同的蛔甙結(jié)構(gòu)，這說明蛔甙代表著線蟲高度保守的分子特征。其中，蛔甙ascr#18廣泛存在于自由生活、昆蟲寄生和植物寄生性線蟲體內(nèi)，根結(jié)線蟲中它的含量最豐富；蛔甙ascr#18的豐度與線蟲的發(fā)育階段密切相關(guān)，如成蟲和幼蟲體內(nèi)蛔甙ascr#18的含量明顯不同[142]。這些現(xiàn)象表明線蟲的植物寄主、動(dòng)物寄主及和線蟲相關(guān)的微生物都利用不同進(jìn)化策略來識(shí)別這類高度保守的小分子化合物[148]，如捕食性真菌通過識(shí)別蛔甙進(jìn)而形成特殊性捕食裝置[149]?；走癮scr#18誘導(dǎo)植物的根部和葉部產(chǎn)生保守防御反應(yīng)(如水楊酸和茉莉酸介導(dǎo)的信號(hào)途徑)，并且低濃度ascr#18處理的植物能夠提高對(duì)某些病毒、細(xì)菌、真菌、根結(jié)線蟲或孢囊線蟲的抗性，然而高濃度ascr#18不能誘導(dǎo)植物產(chǎn)生抗性；和植物地上部分器官相比，根部對(duì)低濃度ascr#18的響應(yīng)更為靈敏；分離的其它類型蛔甙也能激發(fā)植物產(chǎn)生誘導(dǎo)抗性[148]。ascr#18激發(fā)的這種抗性反應(yīng)機(jī)制與其他病原物類似，如擬南芥是依靠細(xì)菌鞭毛蛋白、脂多糖和肽苷等病原相關(guān)分子元件(Pathogen-associated molecular pattern,PAMP)低濃度時(shí)來識(shí)別外源病原菌[148]。蛔甙信息素也能影響植物線蟲早期對(duì)寄主的識(shí)別過程，Williamson等[150]報(bào)道了蛔甙asc#18浸泡過的北方根結(jié)線蟲更容易被番茄根所吸引。經(jīng)線蟲分泌物浸泡的線蟲更容易加速集聚成團(tuán)，說明了蛔甙可能參與了線蟲的集聚過程。然而，具體是哪些線蟲組分在該過程中起作用，需要對(duì)線蟲分泌物的成分作進(jìn)一步的分離鑒定。

4結(jié)論和展望

文章綜述了植物寄生線蟲對(duì)寄主及其土壤中潛在化學(xué)物質(zhì)趨性的研究進(jìn)展，詳細(xì)介紹了模擬土壤三維空間透明的Pluronic膠是目前研究線蟲對(duì)植物趨性的良好介質(zhì)。通過該凝膠系統(tǒng)，明確了根結(jié)線蟲趨于低酸性(pH 4.5~5.4)；CO2對(duì)根結(jié)線蟲的吸引是由于CO2改變了介質(zhì)的pH值所致；根結(jié)線蟲對(duì)低濃度的HCN有集聚現(xiàn)象，這個(gè)聚團(tuán)基因被標(biāo)記到根結(jié)線蟲的基因組上；線蟲在有無植物根存在時(shí)都會(huì)聚集在一起，說明可能存在諸如蛔甙類的小分子物質(zhì)作為媒介參與了線蟲間的群體感應(yīng)；寄主的植物激素信號(hào)途徑同樣也介導(dǎo)了線蟲對(duì)寄主的早期識(shí)別過程。具體是哪些寄主植物或者線蟲自身體內(nèi)的活性物質(zhì)參與介導(dǎo)線蟲的趨化性可能是將來的研究熱點(diǎn)，鑒定這些化學(xué)物質(zhì)將為線蟲的安全有效防治提供有價(jià)值的理論和應(yīng)用基礎(chǔ)。

從分子生物學(xué)角度來看，植物釋放的化學(xué)信號(hào)導(dǎo)致線蟲的行為發(fā)生變化，而行為的變化亦可能伴隨著基因表達(dá)的變化，像基因的從頭轉(zhuǎn)錄(De novo transcription)、預(yù)先存在的mRNA或預(yù)先存在的蛋白發(fā)生了修飾等，因此鑒定這些參與早期信號(hào)識(shí)別的基因及研究這些基因的功能將是另外一個(gè)研究重點(diǎn)。Pluronic膠系統(tǒng)是目前用來建立一套線蟲反應(yīng)寄主根系信號(hào)梯度的cDNA文庫的最理想系統(tǒng)，此系統(tǒng)能顯示信號(hào)梯度并控制收集線蟲的時(shí)間，且避免了土壤中的微生物污染。因此通過這個(gè)系統(tǒng)可以研究線蟲在識(shí)別寄主植物前后基因表達(dá)變化的轉(zhuǎn)錄組分析(Transcriptome analysis，RNA-seq)和蛋白組分析(Proteome analysis)，從而在全基因組范圍內(nèi)來預(yù)測(cè)所參與識(shí)別信號(hào)的線蟲基因并解析其功能，對(duì)探索新的防治策略具有重要的理論價(jià)值。

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Pluronic Gel System: An Approach to Investigate Chemotaxis of Plant Parasitic Nematodes

WANG Cong-li,LI Chun-jie,HU Yan-feng

(KeyLaboratoryofMollisolsAgroecology,NortheastInstituteofGeographyandAgroecology,CAS,Harbin150081,China)

Abstract：Cheomtaxis is the most important mechanism for host-localization of plant-parasitic nematodes.In this review, we discuss the research advances of chemotaxis of plant-parasitic nematodes to plant and potential chemicals from root rhizosphere. We introduce that one transparent thermos-reversible Pluronic gel simulates soil three-dimensional way to allow nematodes move freely in the gel and currently the gel is the best medium to study nematode behavior and chemotaxis. Through this gel system, root-knot nematodes were found to prefer to gather together at low pH (pH range 4.5~5.4); the attraction of CO2 to nematodes was not due to CO2 itself but due to acidification of solutions by dissolved CO2 in the gel; nematodes formed clumps at low concentration of HCN (15 μM~22 μM) and the clumping gene was mapped to nematode genome; ethylene signal pathway modulated nematode attraction. In addition, ascarosides as conserved nematode signaling molecules eliciting plant defenses and pathogen resistance also involved in nematode aggregation and clumping. Current research hotspot is to identify semiochemicals from root or nematode exuduates which modify or regulate nematode chemotaxis.

Key words：plant parasitic nematodes; chemotaxis; Pluronic gel; attraction

中圖分類號(hào)：S432.4+5

文獻(xiàn)標(biāo)識(shí)碼：A

作者簡(jiǎn)介：第一作者及通訊王從麗(1973-)，女，河南唐河人，博士，研究員，主要從事線蟲和植物互作研究.

基金項(xiàng)目：國家自然科學(xué)基金項(xiàng)目(31471749)和中國科學(xué)院“百人計(jì)劃”項(xiàng)目.

收稿日期：2015-01-05.

文章編號(hào)：2095-2961(2016)01 -0001 -13

doi:10.11689/j.issn.2095-2961.2016.01.001