邵淑君,胡璋健,師愷
亞油酸乙醇胺誘導(dǎo)番茄對(duì)灰葡萄孢抗性的作用及機(jī)制
邵淑君,胡璋健,師愷
浙江大學(xué)農(nóng)業(yè)與生物技術(shù)學(xué)院,杭州 310058
灰葡萄孢()引起的灰霉病是危害番茄()的重要病害之一,防治不及時(shí)可造成30%—40%減產(chǎn)。目前生產(chǎn)上多以化學(xué)防治為主,但存在農(nóng)產(chǎn)品安全及環(huán)境污染的風(fēng)險(xiǎn)。-?;掖及罚∟AE)是植物體內(nèi)天然存在的一類脂質(zhì)生物活性化合物,其在哺乳動(dòng)物中具有多種免疫功能,但其在植物免疫中的作用和機(jī)制尚不清楚?!尽刻接??;掖及吩谡T導(dǎo)番茄對(duì)灰葡萄孢防御中的作用,為研發(fā)番茄灰霉病綠色防控技術(shù)提供依據(jù)。將灰葡萄孢分別接種在含有硬脂酰乙醇胺(NAE 18:0)、亞油酸乙醇胺(NAE 18:2)、廿二碳五烯酸乙醇胺(NAE 22:5)的培養(yǎng)基上,觀察灰葡萄孢的生長(zhǎng)情況。在此基礎(chǔ)上,以番茄‘Moneymaker’植株為材料,硬脂酰乙醇胺、亞油酸乙醇胺、廿二碳五烯酸乙醇胺外源處理番茄葉片后接種灰葡萄孢,統(tǒng)計(jì)病情指數(shù),測(cè)定熒光參數(shù)。采用qRT-PCR技術(shù)明確亞油酸乙酰胺處理后番茄葉片灰葡萄孢的相對(duì)表達(dá)量,進(jìn)一步測(cè)定番茄葉片中主要抗病基因、-、、、等的相對(duì)表達(dá)量及茉莉酸(jasmonic acid,JA)、水楊酸(salicylic acid,SA)、乙烯(ethylene,ETH)、脫落酸(abscisic acid,ABA)、生長(zhǎng)素(indoleacetic acid,IAA)含量。并以乙烯信號(hào)轉(zhuǎn)導(dǎo)突變體植株()及對(duì)照植株P(guān)earson(PB)為材料,在外源亞油酸乙酰胺處理后接種灰葡萄孢,測(cè)定番茄葉片葉綠素?zé)晒鈪?shù)和灰葡萄孢的相對(duì)表達(dá)量。體外培養(yǎng)試驗(yàn)結(jié)果表明灰葡萄孢生長(zhǎng)并不受外源-?;掖及返挠绊?,外源施用-酰基乙醇胺能顯著提高番茄植株對(duì)灰霉病的抗性,緩解由灰葡萄孢侵染導(dǎo)致的番茄葉片光系統(tǒng)II實(shí)際光化學(xué)效率(ΦPSII)的下降。3種-?;掖及分校瑏営退嵋掖及肥┯煤蠓讶~片灰霉病病情指數(shù)下降最為明顯,灰葡萄孢的相對(duì)表達(dá)量下調(diào)60%,其誘導(dǎo)灰霉病抗性效果最佳。番茄植株接種灰葡萄孢后抗性基因、-、、的表達(dá)水平均有不同程度上調(diào)。外源亞油酸乙醇胺處理使得番茄響應(yīng)灰葡萄孢接種后、、的表達(dá)進(jìn)一步增強(qiáng),其中乙烯合成基因的表達(dá)水平最高。番茄植株接種灰葡萄孢后葉片水楊酸、茉莉酸、生長(zhǎng)素和乙烯含量增加,但外源亞油酸乙醇胺處理并接種灰葡萄孢后只有乙烯含量顯著增加。進(jìn)一步研究發(fā)現(xiàn),番茄乙烯突變體植株中,外源亞油酸乙醇胺對(duì)灰葡萄孢的抗性誘導(dǎo)作用顯著受到抑制。外源施用亞油酸乙醇胺能夠提高番茄內(nèi)源光合效率和抗病基因的表達(dá)及內(nèi)源激素乙烯的含量,增強(qiáng)番茄植株對(duì)灰霉病的抗性,推測(cè)其誘導(dǎo)抗性作用可能與乙烯信號(hào)路徑相關(guān)。
番茄;-?;掖及罚粊営退嵋掖及?;灰霉?。换移咸焰?;乙烯
【研究意義】番茄()是世界性栽培蔬菜,作為模式作物,被廣泛用于生長(zhǎng)發(fā)育、果實(shí)品質(zhì)、逆境響應(yīng)等研究[1]。番茄產(chǎn)量和品質(zhì)很大程度上受栽培期間病蟲(chóng)害的影響[2]。其中由灰葡萄孢()引起的灰霉病是番茄生產(chǎn)中的重要病害,在我國(guó)各番茄種植地均有發(fā)生,感病后的番茄產(chǎn)量會(huì)降低30%—40%。-?;掖及罚∟AE)作為一種天然的活性物質(zhì),深入研究其在誘導(dǎo)番茄對(duì)灰霉病抗性中的作用及內(nèi)在機(jī)制,對(duì)于植物源生物農(nóng)藥的開(kāi)發(fā)具有重要意義?!厩叭搜芯窟M(jìn)展】20世紀(jì)50年代,人們最早在大豆卵磷脂和花生粕的成分中發(fā)現(xiàn)了-酰基乙醇胺[3],后續(xù)在其他植物中也發(fā)現(xiàn)-?;掖及返拇嬖赱4]。-?;掖及肥且活惞δ芏鄻拥闹愋盘?hào)物質(zhì),由一個(gè)酰胺鍵連接到乙醇胺的脂肪酸組成,根據(jù)其?;湹奶紨?shù)(C12—C18)和不飽和雙鍵數(shù)目(0—3)進(jìn)行分類[5-6]。-?;掖及菲毡榇嬖谟谥参镏?,其種類及含量因植株器官、植物種類和生理?xiàng)l件而異,長(zhǎng)鏈-?;掖及罚–16—C18)是植物中主要的-?;掖及奉愋?,主要由亞油酰乙醇胺(NAE 18:2)、NAE 18:1和NAE 16:0組成。其中亞油酰乙醇胺含量最多,在植株的生長(zhǎng)發(fā)育中發(fā)揮著重要作用[5,7-8]。-?;掖及凡粌H參與種子萌發(fā)[9-11]、幼苗生長(zhǎng)[12]、花器官衰老[13],也可能參與植物細(xì)胞防御信號(hào)轉(zhuǎn)導(dǎo)系統(tǒng)[8]。1998年,研究人員發(fā)現(xiàn)用真菌誘導(dǎo)子木聚糖處理,會(huì)引起煙草懸浮細(xì)胞培養(yǎng)液中-?;掖及泛糠e累[14]。之后-?;掖及费芯恐械囊粋€(gè)重大突破是成功在擬南芥植株中克隆到一種-酰基乙醇胺的水解酶——脂肪酸酰胺水解酶(FAAH),過(guò)表達(dá)擬南芥植株中的病害防御基因、激素合成及信號(hào)轉(zhuǎn)導(dǎo)基因的表達(dá)量均低于野生型植株,降低了對(duì)細(xì)菌性病原體的抗性[15]。-?;呓z氨酸內(nèi)酯(AHL)是革蘭氏陰性菌群信號(hào)感應(yīng)家族中的一員,是-?;掖及奉愃莆?。研究發(fā)現(xiàn)AHL能夠增強(qiáng)植株對(duì)生物與非生物逆境的抗性[16],其中-癸?;呓z氨酸內(nèi)酯(DHL)作為AHL的一員,能夠誘導(dǎo)番茄植株的免疫系統(tǒng)來(lái)保護(hù)植物免受灰葡萄孢的侵染[17]?!颈狙芯壳腥朦c(diǎn)】越來(lái)越多的研究證明環(huán)境友好型物質(zhì)-?;掖及吩谥仓晟L(zhǎng)發(fā)育及抗病性中發(fā)揮著重要作用,但外源施用-?;掖及肥欠衲軌蛴行岣叻阎仓陮?duì)灰霉病的抗性及機(jī)制尚不清楚。【擬解決的關(guān)鍵問(wèn)題】通過(guò)對(duì)灰葡萄孢接種植株表型及光合特性的測(cè)定,探究-?;掖及穼?duì)番茄灰霉病抗性的影響。進(jìn)一步利用qRT-PCR以及HPLC技術(shù)分析外源施用亞油酰乙醇胺對(duì)番茄植株內(nèi)源抗病基因表達(dá)、激素含量的影響,并結(jié)合激素突變體植株外源施用亞油酰乙醇胺對(duì)其灰霉病抗性的影響,探討亞油酰乙醇胺提高灰霉病抗性的內(nèi)在機(jī)制,為番茄作物病害綠色防控提供參考。
試驗(yàn)于2018—2020年在浙江大學(xué)蔬菜研究所植物生長(zhǎng)室及實(shí)驗(yàn)室完成。
1.1.1 試驗(yàn)材料 供試番茄材料為野生型植株Moneymaker、乙烯信號(hào)轉(zhuǎn)導(dǎo)突變體植株()及野生型植株P(guān)earson(PB)。利用這3種番茄材料分別開(kāi)展兩組試驗(yàn):第1組利用不同-?;掖及诽幚硪吧椭仓闙oneymaker后接種灰葡萄孢;第2組用亞油酰乙醇胺處理和PB植株后接種灰葡萄孢。以上3種番茄材料均在種子溫湯浸種后,播種在含有基質(zhì)(草炭、蛭石和珍珠巖比例為6﹕3﹕1)的栽培缽中,并置于浙江大學(xué)蔬菜研究所植物生長(zhǎng)室中生長(zhǎng),其生長(zhǎng)條件為晝夜溫度24℃/21℃,相對(duì)濕度70%—80%,光周期12 h/12 h,平均光強(qiáng)600 μmol·m-2·s-1。
1.1.2-酰基乙醇胺處理 硬脂酰乙醇胺(NAE 18:0)、亞油酰乙醇胺(NAE 18:2)、廿二碳五烯酸乙醇胺(NAE 22:5)均購(gòu)自SIGMA公司,原藥在配置過(guò)程中直接用ddH2O稀釋至80 μmol·L-1。待番茄長(zhǎng)至五葉一心時(shí),選取大小、長(zhǎng)勢(shì)一致的植株進(jìn)行-?;掖及啡陣娛┨幚怼L幚斫M分別用上述3種-?;掖及罚?0 μmol·L-1)噴施兩次,間隔24 h,對(duì)照組用ddH2O處理。不同-?;掖及?、ddH2O處理48 h后接種濃度為2×105孢子/mL灰葡萄孢,接種采用噴施法。
1.2.1 灰葡萄孢孢子培養(yǎng)及接種 灰葡萄孢培養(yǎng)用V8培養(yǎng)基(36% V8果汁(Campbell Soup Co.,Camden,NJ,USA),0.2 g·L-1CaCO3,15 g·L-1瓊脂)、PDA培養(yǎng)基(4 g·L-1馬鈴薯淀粉,20 g·L-1葡萄糖,15 g·L-1瓊脂)和對(duì)照培養(yǎng)基(ddH2O,15 g·L-1瓊脂)在25℃避光條件下培養(yǎng)15 d左右。待培養(yǎng)皿內(nèi)長(zhǎng)滿菌絲后,將菌絲塊放入重懸液(2%胰蛋白胨,4%麥芽糖)分離過(guò)濾得到孢子,并稀釋濃度至2×105孢子/mL[18]。將孢子懸浮液均勻噴施在整株葉片上,以噴施重懸液作為空白對(duì)照。接種后的番茄植株置于相對(duì)濕度100%、光周期12 h/12 h、光強(qiáng)600 μmol·m-2s-1、溫度25℃的環(huán)境下3—4 d后觀察發(fā)病情況。
1.2.2 番茄植株病情調(diào)查方法 番茄葉片接種灰葡萄孢3 d后,觀察不同處理下番茄植株灰霉病的病情,病害分級(jí)標(biāo)準(zhǔn)參照蔡銀杰等[19]的方法,根據(jù)病斑面積占葉面積百分比分為5級(jí)。0級(jí):0;1級(jí):0.1%—10%;2級(jí):10.1%—30%;3級(jí):30.1%—70%;4級(jí):>70%。病情指數(shù)計(jì)算公式:病情指數(shù)=[∑(各級(jí)葉數(shù)×該級(jí)指數(shù))/(調(diào)查總?cè)~數(shù)×最高級(jí)別數(shù))]×100。
1.2.3 葉綠素?zé)晒獬上穹治?葉綠素?zé)晒馐褂肐maging-PAM調(diào)制熒光成像系統(tǒng)(IMAG-MAXI;Heinz Walz,Germany)進(jìn)行測(cè)定。植株暗適應(yīng)20 min后,光化光強(qiáng)度和飽和光強(qiáng)度分別設(shè)為280和4 000 μmol·m-2·s-1,光系統(tǒng)Ⅱ?qū)嶋H光化學(xué)效率計(jì)算公式為ΦPSII=(Fm’-F)/ Fm’[20]。
1.2.4 植物葉片總RNA提取和qRT-PCR檢測(cè) 使用RNA提取試劑盒(Tiangen,China)提取接種灰葡萄孢后24 h的葉片總RNA,使用Fermentas RevertAidTMfirst Strand cDNA Synthesis kit合成cDNA第一鏈。使用iCycler iQ Multicolor實(shí)時(shí)定量PCR檢測(cè)系統(tǒng)儀器(LightCycler 480 II,SwissConfederation)完成qRT-PCR,參照說(shuō)明書(shū)進(jìn)行操作。
PCR反應(yīng)體系:10 μL iQTMSYBR Green Supermix,2 μL cDNA,0.4 μL前、后引物(10 μmol·L-1),7.2 μL ddH2O。PCR反應(yīng)程序:95℃預(yù)變性5 min;95℃變性15 s,58℃退火45 s,40個(gè)循環(huán)。使用Primer 5.0根據(jù)SGN上的番茄基因序列設(shè)計(jì)引物(表1),數(shù)據(jù)采用2-??CT法進(jìn)行分析[21]。
1.2.5 植物內(nèi)源激素含量測(cè)定 茉莉酸(jasmonic acid,JA)、水楊酸(salicylic acid,SA)、脫落酸(abscisic acid,ABA)和生長(zhǎng)素(indoleacetic acid,IAA)定量測(cè)定參照WU等[22]的方法,采用液相色譜質(zhì)譜串聯(lián)系統(tǒng)進(jìn)行分析(Agilent 6460,Agilent Technologies,USA)。在葉片提取過(guò)程中,樣品加入D5-JA、D4-SA、D6-ABA和 D6-IAA(OlChemlm,Czechia)作為內(nèi)參。
乙烯(ethylene,ETH)含量測(cè)定方法:每個(gè)處理取4棵長(zhǎng)勢(shì)一致植株的葉片密封在1 L橡膠平頂燒瓶中,室溫放置1 h后用注射器從每個(gè)燒瓶中抽取出1 mL頂部氣體,使用配備1.5 m×4 mm的氧化鋁玻璃柱的氣相色譜儀(Philips,UNICAM pro. GC)測(cè)定。進(jìn)樣器、檢測(cè)器和過(guò)柱溫度分別設(shè)定為130、130和200℃。
表1 qRT-PCR特異性引物
試驗(yàn)結(jié)果均為3次重復(fù)的平均值,利用Microsoft Excel 2016整理數(shù)據(jù);運(yùn)用SAS 9.1 Tukey法進(jìn)行差異顯著性分析;運(yùn)用Origin 2018進(jìn)行圖形繪制。
將相同大小的灰葡萄孢菌絲塊分別接種在含有80 μmol·L-1硬脂酰乙醇胺(NAE 18:0)、亞油酰乙醇胺(NAE 18:2)、廿二碳五烯酸乙醇胺(NAE 22:5)的V8培養(yǎng)基、PDA培養(yǎng)基以及對(duì)照培養(yǎng)基上,于25℃避光培養(yǎng)15 d。結(jié)果表明,在避光條件下培養(yǎng)9 d,添加3種-?;掖及凡⑽磳?duì)V8培養(yǎng)基和PDA培養(yǎng)基上的灰葡萄孢生長(zhǎng)有明顯的影響。在V8培養(yǎng)基上培養(yǎng)的菌落緊致均一,而在PDA培養(yǎng)基上培養(yǎng)的菌落整體表現(xiàn)疏松,分塊不均一(圖1)。因此,-酰基乙醇胺本身并不影響灰葡萄孢的生長(zhǎng),且V8培養(yǎng)基最適宜灰葡萄孢生長(zhǎng)。
為了探究-?;掖及吩谡T導(dǎo)番茄對(duì)灰霉病抗性中的作用,進(jìn)行了-?;掖及奉A(yù)處理番茄植株葉片后接種病原菌試驗(yàn)。一般情況下,植株遭到病原菌侵染會(huì)對(duì)其光合系統(tǒng)造成不可逆的損害,因此通過(guò)測(cè)定葉片ФPSII來(lái)評(píng)估病害的嚴(yán)重程度[23]。由圖2可以看出,接種灰葡萄孢的葉片ΦPSII值降低,而外源施用-?;掖及纺軌蛎黠@緩解因病原菌侵染導(dǎo)致的ΦPSII下降,這與灰葡萄孢的相對(duì)表達(dá)量一致。同時(shí)外源施用-?;掖及泛螅仓瓴∏橹笖?shù)降低至1.2—3。因此,在硬脂酰乙醇胺(NAE 18:0)、亞油酰乙醇胺(NAE 18:2)、廿二碳五烯酸乙醇胺(NAE 22:5)處理下,番茄植株對(duì)灰霉病的抗性增強(qiáng),尤以亞油酰乙醇胺處理對(duì)番茄灰霉病的抗性最強(qiáng)。
圖1 外源N-?;掖及诽幚韺?duì)體外培養(yǎng)中灰葡萄孢菌絲生長(zhǎng)的影響
A:番茄葉片光系統(tǒng)II實(shí)際光化學(xué)效率Photosystem II photochemical efficiency (ΦPSII);B:番茄葉片灰葡萄孢Actin表達(dá)量B. cinerea Actin relative expression of tomato leaves;C:病情指數(shù)Disease index;D:ΦPSII成像圖Imaging of ΦPSII
為了探究亞油酰乙醇胺誘導(dǎo)的抗病性是否與內(nèi)源激素信號(hào)路徑有關(guān),檢測(cè)了接種灰葡萄孢24 h后番茄植株中主要抗病信號(hào)路徑茉莉酸信號(hào)相關(guān)基因、水楊酸信號(hào)相關(guān)基因-和、脫落酸信號(hào)路徑相關(guān)基因信號(hào)路徑相關(guān)基因和的表達(dá)量。結(jié)果表明,植株接種灰葡萄孢24 h后、-、、、相對(duì)表達(dá)量表現(xiàn)不同程度上調(diào),但在外源亞油酰乙醇胺處理后接種灰葡萄孢的番茄植株中-、的表達(dá)量與對(duì)照組相比無(wú)顯著差異,而、和的表達(dá)量則有所增強(qiáng),尤以乙烯合成基因的表達(dá)量增幅最大(圖3-A)。
接種灰葡萄孢36 h后番茄植株葉片中激素含量測(cè)定結(jié)果表明,茉莉酸、水楊酸和生長(zhǎng)素的含量均顯著上升,但在亞油酰乙醇胺處理下茉莉酸、水楊酸、生長(zhǎng)素的含量反而受到不同程度的抑制,脫落酸含量無(wú)顯著變化,乙烯含量顯著增加(圖 3-B、3-C)。
鑒于亞油酸乙醇胺能提高乙烯途徑基因的轉(zhuǎn)錄表達(dá)和乙烯含量,進(jìn)一步利用亞油酸乙醇胺預(yù)處理乙烯信號(hào)轉(zhuǎn)導(dǎo)突變株及野生型植株P(guān)B后接種灰葡萄孢,接種24 h后對(duì)其表型進(jìn)行分析。結(jié)果表明,野生型植株P(guān)B葉片的發(fā)病情況得到了有效緩解,ΦPSII值顯著提高,灰葡萄孢的相對(duì)表達(dá)量降低了60%。而突變體植株葉片的發(fā)病情況與對(duì)照無(wú)明顯差異,ΦPSII值及灰葡萄孢的相對(duì)表達(dá)量均無(wú)明顯變化(圖4),表明亞油酸乙醇胺誘導(dǎo)的抗病性依賴于乙烯信號(hào)通路。
圖3 外源施用亞油酸乙醇胺對(duì)番茄葉片中抗病基因表達(dá)(A)和內(nèi)源激素含量(B、C)的影響
-酰基乙醇胺的功能以及代謝在動(dòng)物中已有大量的研究,研究最為廣泛的-花生四烯乙醇胺(NAE 20:4)能夠與受體結(jié)合,觸發(fā)一系列信號(hào)轉(zhuǎn)導(dǎo)事件,從而調(diào)節(jié)動(dòng)物的痛覺(jué)、能量平衡、恐懼、食欲、記憶等多種生理和行為過(guò)程[24]。進(jìn)一步研究發(fā)現(xiàn),這些脂肪酸酰胺也是植物體的內(nèi)源成分,其積累和代謝在植株生長(zhǎng)發(fā)育及免疫等方面發(fā)揮著重要的作用。例如,當(dāng)煙草葉片受到真菌激發(fā)子刺激時(shí),-酰基乙醇胺的水平上升10—50倍,暗示其可能在植株免疫中發(fā)揮一定的調(diào)控作用[15]。植物中-?;掖及纺軌騾⑴c種子萌發(fā)、葉綠體發(fā)育等生長(zhǎng)發(fā)育調(diào)控[25-26],但對(duì)病原菌的響應(yīng)及調(diào)控機(jī)制尚不清楚。本研究發(fā)現(xiàn),3種-酰基乙醇胺均能提高番茄植株對(duì)灰霉病的抗性,其中亞油酸乙醇胺的抗性誘導(dǎo)效果最好,亞油酸乙醇胺處理下的番茄接種灰葡萄孢后,其灰葡萄孢的生長(zhǎng)量與對(duì)照組ddH2O處理相比下降了60%(圖2)。目前生產(chǎn)上防治灰霉病主要依靠化學(xué)藥劑,已陸續(xù)檢測(cè)到番茄灰霉病對(duì)腐霉利等藥劑產(chǎn)生不同程度的抗藥性,防治效果顯著下降[27]。因此,尋找化學(xué)合成農(nóng)藥的新替代品已經(jīng)成為科學(xué)研究的一個(gè)重要方向。本研究發(fā)現(xiàn)亞油酸乙醇胺對(duì)番茄灰霉病誘導(dǎo)抗性效果最佳,其類似物DHL已被證實(shí)能夠誘導(dǎo)番茄植株的系統(tǒng)抗性來(lái)保護(hù)植物免受灰葡萄孢的侵染[17]。這些活性物質(zhì)的發(fā)現(xiàn)以及未來(lái)的開(kāi)發(fā)應(yīng)用將有助于病害的綠色防控。
A:番茄葉片光系統(tǒng)II實(shí)際光化學(xué)效率Photosystem II photochemical efficiency (ΦPSII);B:番茄葉片灰葡萄孢Actin表達(dá)量B. cinerea Actin relative expression of tomato leaves;C:番茄葉片ΦPSII成像圖 Imaging of ΦPSII
植物免疫調(diào)控離不開(kāi)植物激素,灰葡萄孢的侵染提高了植物體內(nèi)乙烯、茉莉酸、水楊酸、脫落酸含量,保持激素調(diào)控網(wǎng)絡(luò)的內(nèi)在平衡是植株免疫的關(guān)鍵[28]。本研究中,番茄植株受灰葡萄孢侵染后,其乙烯、茉莉酸、水楊酸含量均有一定程度提高,與前人研究結(jié)果一致。但在亞油酸乙醇胺處理下,番茄葉片中乙烯含量顯著升高,茉莉酸、水楊酸、生長(zhǎng)素的含量反而受到了不同程度的抑制,而脫落酸含量無(wú)顯著差異(圖3)。有研究報(bào)道,植株提高對(duì)死體營(yíng)養(yǎng)型病原菌抗性主要依賴于茉莉酸和乙烯信號(hào)路徑[29-30],水楊酸和脫落酸在植物對(duì)灰霉病的抗性中作用復(fù)雜,其可能和植物的種類、灰葡萄孢侵染的階段、受侵染的組織等相關(guān)[31]。大量研究已證明,乙烯合成途徑關(guān)鍵酶ACO基因表達(dá)量提高是乙烯產(chǎn)生增加的重要指標(biāo)[32-33],植株遭受病原菌侵染后,乙烯合成途徑被激活,產(chǎn)生大量乙烯[34-35],隨著乙烯水平的上升,下游抗性相關(guān)基因的表達(dá)也增強(qiáng)[36-37]。本研究中番茄植株在外源施用亞油酸乙醇胺并接種病原菌后,乙烯信號(hào)路徑相關(guān)基因大量表達(dá)(圖 3-A),與已經(jīng)報(bào)道的免疫反應(yīng)模式一致。已有研究表明擬南芥乙烯不敏感(、)和乙烯受體()突變體因改變了乙烯信號(hào)而導(dǎo)致其對(duì)灰葡萄孢的敏感性,過(guò)表達(dá)則增強(qiáng)了擬南芥植株對(duì)灰葡萄孢的抗性[38],番茄蛋白激酶TPK1b介導(dǎo)了乙烯信號(hào)路徑參與的防御作用[39],這些結(jié)果均表明,乙烯信號(hào)通路在植株對(duì)灰霉病的防御中發(fā)揮著重要作用。本研究發(fā)現(xiàn),在番茄乙烯信號(hào)轉(zhuǎn)導(dǎo)突變體植株上外源施用亞油酸乙醇胺后再接種病原菌,削弱了亞油酸乙醇胺對(duì)灰葡萄孢的抗性作用(圖4),推測(cè)亞油酸乙醇胺誘導(dǎo)的抗病性依賴于乙烯信號(hào)通路。
研究表明,乙烯和茉莉酸信號(hào)通路在灰霉病抗性中具有協(xié)同作用,乙烯和茉莉酸信號(hào)通路是相互關(guān)聯(lián)的,茉莉酸甲酯(MeJA)能夠誘導(dǎo)的表達(dá),導(dǎo)致乙烯生成增加[28]。本試驗(yàn)中番茄植株在外源施用亞油酸乙醇胺后接種灰葡萄孢,葉片中乙烯含量顯著增加,茉莉酸含量反而受到了一定程度的抑制,但較未接種灰葡萄孢的對(duì)照組相比,乙烯、茉莉酸含量均顯著升高,且茉莉酸信號(hào)通路基因的轉(zhuǎn)錄表達(dá)上調(diào)(圖3),可能也是乙烯/茉莉酸協(xié)同防御的一種表現(xiàn)。因此,在未來(lái)的研究中,還需要對(duì)茉莉酸信號(hào)通路及兩者的調(diào)控網(wǎng)絡(luò)展開(kāi)進(jìn)一步研究。
外源施用亞油酸乙醇胺能夠顯著提高番茄光合作用和抗病基因的表達(dá)及內(nèi)源激素乙烯的含量,有效誘導(dǎo)番茄植株對(duì)灰霉病的抗性。
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The role and mechanism of Linoleyl ethanolamide in Plant resistance againstin tomato
SHAO Shujun, HU Zhangjian, SHI Kai
College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058
【】Gray mold caused byis one of the important diseases of tomato and causes significant yield losses up to 30%-40%. Nowadays, chemical pesticide is usually used in tomato production, which is effective but increases the risk of food safety and results in environmental pollution.-acylethanolamines (NAEs) are a kind of naturally lipid bioactive compounds in plants, which have been identified to have a variety of immune functions in mammals, however, its function and the underlying mechanism in plant immunity are still unclear.】The objective of this study is to investigate the effects of NAEs on tomato plant defense againstinfection, and to provide a basis for the development of green control technology of tomato gray mold.】Thewas cultured in medium containing NAE 18:0, NAE 18:2, NAE 22:5, respectively, to evaluate their effects ongrowth. Tomato ‘Moneymaker’ plants were infected bywith or without exogenous NAE 18:0, NAE 18:2, NAE 22:5, and disease index and fluorescence parameters of tomato leaves were measured. qRT-PCR was used to analyze the relative gene expression ofin tomato leaves that infected bywith or without NAE 18:2 treatment. Transcript abundance of defense-related genes (e.g.,-,,,,), and contents of plant hormones (e.g.JA, SA, ETH, ABA, IAA) were measured. Fluorescence parameters of tomato leaves and the relative gene expression ofwere analyzed in ethylene-insensitive mutant infected bywith NAE 18:2.【】The growth ofwas not affected by exogenous NAEs treatment duringculture. Exogenous application of NAEs could significantly improve the resistance of tomato plants to, and alleviate the decrease of photosystem II photochemical efficiency (ΦPSII) caused byinfection. NAE 18:2 had the best effect on tomato plant defense againstamong the NAEs, which obviously reduced the disease index and thetranscript level ofby 60%. The expression levels of,-,,andcould be induced byinfection but not by NAE 18:2 treament. The expression levels of,andwere up-regulated when plants were pre-treated by NAE 18:2 beforeinfection, and the expression level ofwas the highest. Compared to the control, the contents of SA , JA, IAA and ETH in the leaves were increased significantly afterinfection, while only the contents of ETH were further increased when pre-treated by NAE 18:2. Moreover, exogenous NAE 18:2 pre-treatment could not improve the defense againstin the ETH-insensitive.【】Exogenous NAE18:2 treatment can increase leaf photosynthesis, transcript abundance of defense-related genes, and the content of plant hormone ETH. It induce the resistance of tomato plants to gray mold, which may depend on the ETH signaling pathway.
tomato;-acylethanolamines (NAE); linoleyl ethanolamide (NAE 18:2);gray mold;; ethylene (ETH)
10.3864/j.issn.0578-1752.2022.09.007
2021-11-06;
2021-12-10
國(guó)家自然科學(xué)基金(32172650)、浙江省重點(diǎn)研發(fā)計(jì)劃(2021C02040)
邵淑君,E-mail:ssjun@zju.edu.cn。通信作者師愷,E-mail:kaishi@zju.edu.cn
(責(zé)任編輯 岳梅)