龍琴,杜美霞,龍俊宏,何永睿,鄒修平,陳善春
轉(zhuǎn)錄因子CsWRKY61對(duì)柑橘潰瘍病抗性的影響
龍琴,杜美霞,龍俊宏,何永睿,鄒修平,陳善春
(西南大學(xué)/中國(guó)農(nóng)業(yè)科學(xué)院柑桔研究所國(guó)家柑桔品種改良中心,重慶 400712)
【背景】柑橘潰瘍?。╟itrus bacterial canker,CBC)是世界柑橘產(chǎn)業(yè)上危害最嚴(yán)重的病害之一,由柑橘黃單胞桿菌柑橘亞種(subsp.,)引起,在國(guó)內(nèi)外被列為檢疫對(duì)象。由于柑橘分子病理研究相對(duì)滯后,導(dǎo)致可供利用的抗性基因資源相對(duì)匱乏。WRKY轉(zhuǎn)錄因子參與植物抵御生物和非生物脅迫反應(yīng),前期研究發(fā)現(xiàn)柑橘WRKY轉(zhuǎn)錄因子可能在調(diào)控寄主抗病反應(yīng)中起著重要作用?!灸康摹客ㄟ^(guò)對(duì)超量表達(dá)、和轉(zhuǎn)基因晚錦橙()的潰瘍病抗性進(jìn)行評(píng)價(jià),明確這些基因在柑橘響應(yīng)潰瘍病菌侵染中的生物學(xué)功能和抗病育種價(jià)值。進(jìn)一步利用RNA-Seq解析調(diào)控的信號(hào)通路?!痉椒ā坷棉r(nóng)桿菌介導(dǎo)法進(jìn)行柑橘遺傳轉(zhuǎn)化,獲得超量表達(dá)、和的晚錦橙;利用實(shí)時(shí)熒光定量PCR(qRT-PCR)分析轉(zhuǎn)基因植株中目的基因的表達(dá)水平以及拷貝數(shù);以非轉(zhuǎn)基因植株為對(duì)照,采用離體針刺接種評(píng)價(jià)轉(zhuǎn)基因植株對(duì)潰瘍病的抗性;通過(guò)比較超量表達(dá)和野生型植株的轉(zhuǎn)錄組測(cè)序結(jié)果,探究提高柑橘潰瘍病抗性的內(nèi)在機(jī)制?!窘Y(jié)果】分別構(gòu)建了CAMV啟動(dòng)子控制、和表達(dá)的植物表達(dá)載體,通過(guò)組織化學(xué)染色和PCR鑒定分別獲得了6、8和6株轉(zhuǎn)基因晚錦橙。轉(zhuǎn)基因植株中目的基因的表達(dá)量有不同程度的提高,大部分轉(zhuǎn)基因植株中外源基因的拷貝數(shù)為1,只有超量表達(dá)的轉(zhuǎn)基因植株潰瘍病抗性顯著增強(qiáng),其病斑面積明顯小于野生型植株,而超量表達(dá)和的轉(zhuǎn)基因植株抗病性與野生型相比無(wú)明顯差異。轉(zhuǎn)錄組分析結(jié)果顯示,超量表達(dá)的轉(zhuǎn)基因植株中生物脅迫相關(guān)途徑(包括病原入侵的感知、活性氧爆發(fā)、轉(zhuǎn)錄因子、防御基因、激素、細(xì)胞壁和次生代謝等)和信號(hào)轉(zhuǎn)導(dǎo)相關(guān)途徑(主要是激酶受體)均被顯著激活?!窘Y(jié)論】超量表達(dá)能夠激活與生物脅迫和信號(hào)轉(zhuǎn)導(dǎo)相關(guān)的途徑,增強(qiáng)柑橘對(duì)潰瘍病的抗性;在柑橘抗病育種中存在潛在的應(yīng)用價(jià)值。
柑橘黃單胞桿菌柑橘亞種;柑橘潰瘍??;CsWRKY61;超量表達(dá);抗病性;轉(zhuǎn)錄組測(cè)序
【研究意義】柑橘潰瘍病(citrus bacterial canker,CBC)是一種柑橘檢疫性細(xì)菌病害,由柑橘黃單胞桿菌柑橘亞種(subsp.,)引起[1]。其病原菌幾乎能夠危害柑橘的所有組織,嚴(yán)重時(shí)會(huì)造成落葉和落果,進(jìn)一步發(fā)展將會(huì)導(dǎo)致枝梢枯死和幼樹(shù)死亡等,嚴(yán)重威脅世界柑橘產(chǎn)業(yè)的健康發(fā)展[2-3]。目前,只能通過(guò)化學(xué)農(nóng)藥或集中銷毀苗木進(jìn)行防控,但其勞動(dòng)力和經(jīng)濟(jì)成本較高。培育抗病栽培品種是解決該問(wèn)題的根本途徑[4]。隨著CRISPR/Cas9等現(xiàn)代分子育種技術(shù)的興起,利用分子技術(shù)定向快速改良柑橘抗病性,創(chuàng)制新型抗性品種成為可能。然而,由于柑橘分子病理研究相對(duì)滯后,導(dǎo)致可供利用的抗性基因資源相對(duì)匱乏,目前柑橘抗病分子育種進(jìn)展依然緩慢。因此,迫切需要挖掘柑橘源潛在的抗病基因,通過(guò)基因工程技術(shù)獲得抗?jié)儾〉姆N質(zhì),為育種提供材料?!厩叭搜芯窟M(jìn)展】WRKY轉(zhuǎn)錄因子是植物中最大的轉(zhuǎn)錄調(diào)控因子家族之一,是調(diào)控植物多方面過(guò)程的信號(hào)網(wǎng)絡(luò)的組成部分[5],含有一個(gè)或兩個(gè)WRKY結(jié)構(gòu)域是其最顯著的特征。WRKY結(jié)構(gòu)域大約由60個(gè)氨基酸殘基組成,其N端是絕對(duì)保守的,氨基酸序列為WRKYGQK,在C端則含有一個(gè)C2H2或C2HC型鋅指結(jié)構(gòu)。根據(jù)WRKY結(jié)構(gòu)域的數(shù)量和鋅指結(jié)構(gòu)的類型,可將WRKY轉(zhuǎn)錄因子分為3個(gè)家族。Ⅰ類含兩個(gè)WRKY結(jié)構(gòu)域;Ⅱ類和Ⅲ類含一個(gè)WRKY結(jié)構(gòu)域。其中Ⅰ類和Ⅱ類WRKY轉(zhuǎn)錄因子均為C2H2型鋅指結(jié)構(gòu):C-X4-5-C-X22-23-H-X1-H;Ⅲ類WRKY轉(zhuǎn)錄因子的鋅指結(jié)構(gòu)基序跟前兩類有所不同,為C2HC型:C-X7-C-X23-H-X1-C。此外,第Ⅱ類WRKY轉(zhuǎn)錄因子又可進(jìn)一步分為5個(gè)亞類:Ⅱ(a)、Ⅱ(b)、Ⅱ(c)、Ⅱ(d)、Ⅱ(e)[6]。WRKY轉(zhuǎn)錄因子家族在擬南芥中有74個(gè)成員[7],水稻中有109個(gè)成員[8],毛白楊中有100個(gè)WRKY基因[9],在辣椒的基因組中有71個(gè)WRKY成員[10],在蘋果中鑒定出了116個(gè)WRKY基因[11]。在甜橙基因組中成功注釋了348個(gè)WRKY基因或片段。WRKY轉(zhuǎn)錄因子特異性識(shí)別并結(jié)合W-box“TTGACC/T”順式作用元件,通過(guò)調(diào)控下游基因的表達(dá)從而調(diào)節(jié)植物的衰老、形態(tài)建成、生物和非生物脅迫響應(yīng)等多方面的進(jìn)程[12-16]。目前,已有大量關(guān)于WRKY轉(zhuǎn)錄因子調(diào)控寄主植物抗病方面的研究,主要集中在擬南芥和水稻中[17-25]。如擬南芥中WRKY31、WRKY22、WRKY50、WRKY72、WRKY70、WRKY18、WRKY40和WRKY60參與寄主多種免疫應(yīng)答調(diào)節(jié),其中WRKY70是水楊酸(SA)和茉莉酸(JA)信號(hào)途徑相互拮抗的核心調(diào)節(jié)點(diǎn),在植物抗病、抗蟲(chóng)、抗逆防御中起著重要作用[26-28]。在柑橘中,一些WRKY轉(zhuǎn)錄因子(WRKY22、WRKY45和WRKY31等)在調(diào)控寄主抗病反應(yīng)中的可能功能已有報(bào)道,比如可能是柑橘潰瘍病效應(yīng)子flg22的一個(gè)靶標(biāo)基因,參與柑橘的抗病反應(yīng)[29]。然而,更多的研究是關(guān)于WRKY轉(zhuǎn)錄因子參與激素或非生物脅迫的調(diào)節(jié)[30-32]?!颈狙芯壳腥朦c(diǎn)】WRKY家族基因在植物抗病育種中具有廣泛的應(yīng)用前景,但在柑橘中鮮有研究。前期在高抗品種四季橘()和高感品種紐荷爾甜橙()的比較研究中發(fā)現(xiàn),3個(gè)WRKY家族轉(zhuǎn)錄因子基因、和與柑橘潰瘍病抗性相關(guān)。在前期研究基礎(chǔ)上,本研究以高感品種晚錦橙()為材料,利用轉(zhuǎn)基因技術(shù)探究、和在柑橘潰瘍病菌侵染中的生物學(xué)功能。【擬解決的關(guān)鍵問(wèn)題】探明在柑橘中超量表達(dá)、和對(duì)柑橘潰瘍病抗性的影響,明確這些基因在柑橘響應(yīng)潰瘍病菌侵染中的生物學(xué)功能和抗病育種價(jià)值。
試驗(yàn)于2016年7月至2018年9月在西南大學(xué)/中國(guó)農(nóng)業(yè)科學(xué)院柑桔研究所國(guó)家柑桔品種改良中心完成。
柑橘遺傳轉(zhuǎn)化所用的材料為晚錦橙上胚軸,其種子采自中國(guó)農(nóng)業(yè)科學(xué)院柑桔研究所國(guó)家柑桔品種改良中心資源圃,其無(wú)菌上胚軸的準(zhǔn)備參考文獻(xiàn)[33]。超量表達(dá)目的基因的植物表達(dá)載體為pLGN(本實(shí)驗(yàn)室改造),含抗性和報(bào)告融合基因,以方便轉(zhuǎn)基因植株的篩選,同時(shí)含有多克隆位點(diǎn)以利于外源基因的插入。植物遺傳轉(zhuǎn)化用農(nóng)桿菌為EHA105菌株(本實(shí)驗(yàn)室凍存)。
植物基本培養(yǎng)基為MS(Murashige and Skoog),購(gòu)自Phyto Technology Laboratories?;常規(guī)酶制品購(gòu)自大連TaKaRa公司;質(zhì)粒提取和膠回收試劑盒購(gòu)自O(shè)mega公司;植物總DNA和總RNA提取試劑盒購(gòu)自Aidlab公司;實(shí)時(shí)熒光染料購(gòu)自BIORAD公司;PGEM-T克隆載體購(gòu)自Promega公司;其他常規(guī)試劑均為分析純。
、和的編碼序列參見(jiàn)文獻(xiàn)[34]。設(shè)計(jì)引物(表1),利用PCR在目的基因的5′和3′端分別添加合適的酶切位點(diǎn),T-克隆到PGEM-T上,陽(yáng)性克隆經(jīng)PCR和測(cè)序驗(yàn)證。然后,通過(guò)酶切連接技術(shù)將、、插入pLGN載體中CaMV啟動(dòng)子的下游,分別構(gòu)建植物表達(dá)載體p35S:CsWRKY50、p35S:CsWRKY61和p35S:CsWRKY72。將構(gòu)建好的植物表達(dá)載體通過(guò)電激法轉(zhuǎn)入農(nóng)桿菌EHA105中用于柑橘的遺傳轉(zhuǎn)化。
表1 基因克隆和轉(zhuǎn)基因植株鑒定所用引物
晚錦橙上胚軸遺傳轉(zhuǎn)化方法參照王軍政[35]的方法進(jìn)行。經(jīng)農(nóng)桿菌浸染后外植體置于共培養(yǎng)基(添加2 mg·L-1BA、0.5 mg·L-1IAA、1 mg·L-12,4-D、100 mg·mL-1AS的MS培養(yǎng)基)培養(yǎng)2—3 d;然后轉(zhuǎn)到篩選培養(yǎng)基(添加2 mg·L-1BA、0.5 mg·L-1IAA、500 mg·L-1carb,50 mg·L-1km的MS培養(yǎng)基)上進(jìn)行抗性芽再生和篩選培養(yǎng)。待抗性芽長(zhǎng)到一定程度(1 cm以上),取葉片進(jìn)行組織化學(xué)染色及插入基因的PCR鑒定,篩選陽(yáng)性植株,將幼芽嫁接到晚錦橙實(shí)生苗上,于MS液體培養(yǎng)基中培養(yǎng)到接穗長(zhǎng)至5 cm左右,再嫁接到枳橙實(shí)生苗上,于溫室中培養(yǎng)。
參照J(rèn)efferson等[36]的方法進(jìn)行組織化學(xué)染色。具體操作如下:取抗性芽的葉片,放入已加入5-溴-4-氯-3-吲哚葡糖苷酸(X-Gluc)染色液的試管中,于37℃避光保溫過(guò)夜。染色完成后用95%的乙醇脫色,直至非轉(zhuǎn)基因植株葉片的葉綠素完全褪去呈現(xiàn)白色,統(tǒng)計(jì)染色情況,并拍照記錄。
按照Aidlab公司DNA提取試劑盒(cat.No.DN15)方法提取陽(yáng)性植株的基因組DNA。為了避免植株內(nèi)源目的基因的干擾,根據(jù)序列和目的基因序列設(shè)計(jì)目的基因表達(dá)盒的擴(kuò)增(圖1、表1),用于目的基因在轉(zhuǎn)基因植株中整合的鑒定。PCR反應(yīng)體積25 μL,反應(yīng)條件:98℃ 1 min;98℃ 10 s,58℃ 10 s,72℃ 20 s,30次循環(huán);72℃ 5 min。擴(kuò)增完成后將PCR產(chǎn)物于瓊脂糖凝膠(1.0%,W/V)中進(jìn)行電泳分析。
參照許蘭珍等[37]定量PCR方法,分析轉(zhuǎn)基因植株外源基因拷貝數(shù)。以柑橘內(nèi)源單拷貝基因脂質(zhì)轉(zhuǎn)移蛋白基因?yàn)閮?nèi)參基因[38],以前期確認(rèn)的單拷貝轉(zhuǎn)基因植株GA-5為對(duì)照,利用qRT-PCR分析轉(zhuǎn)基因拷貝數(shù)。按照BIORAD公司的定量PCR試劑盒(cat.No.170-8882AP)說(shuō)明書(shū)進(jìn)行目的基因的拷貝數(shù)分析。反應(yīng)體積20 μL,反應(yīng)條件:95℃ 3 min;94℃ 10 s,58℃ 10 s,72℃ 10 s,40次循環(huán);72℃10 min。以GA-5植株為對(duì)照,采用2-ΔΔCt法計(jì)算轉(zhuǎn)基因植株中目的基因的拷貝數(shù)。
用Aidlab公司的 EASYspin植物RNA快速提取試劑盒(cat.No.RN09)提取柑橘葉片的總RNA。嚴(yán)格參照BIORAD公司的iScriptTMcDNA合成試劑盒(cat.No.170-8891)說(shuō)明書(shū)合成cDNA第一鏈。使用BIORAD公司的定量PCR試劑盒(cat.No.170- 8882AP)進(jìn)行目的基因的相對(duì)定量分析。所用定量PCR引物見(jiàn)表2[34],使用20 μL反應(yīng)體系,反應(yīng)條件:95℃ 3 min;94℃ 10 s,56℃ 10 s,72℃ 10 s,共40次循環(huán);72℃ 10 min。選用柑橘為內(nèi)參基因,以非轉(zhuǎn)基因柑橘植株為對(duì)照,采用2-ΔΔCt法計(jì)算轉(zhuǎn)基因柑橘植株中目的基因的相對(duì)表達(dá)量。
表2 實(shí)時(shí)熒光定量PCR所用引物序列
借鑒李云鋒等[39]的方法分離、純化和培養(yǎng)柑橘潰瘍病菌。選取成熟的新葉(每個(gè)株系選取6片),洗凈表面灰塵,用75%酒精消毒表面,再用無(wú)菌水沖洗3—5遍,于無(wú)菌培養(yǎng)皿中保濕備用。將潰瘍病菌培養(yǎng)至OD600為0.5,再用無(wú)菌水稀釋1 000倍至濃度為5×105cfu/mL,備用。采用離體針刺法接種[40],用直徑0.5 mm無(wú)菌針頭沿葉片中脈兩邊針刺24孔,每邊12孔。每孔添加1 μL潰瘍病菌菌液,接種后用含有無(wú)菌水棉球覆蓋葉柄保濕,石蠟帶封嚴(yán)培養(yǎng)皿,于28℃,16 h·d-1光照培養(yǎng)10 d。每天觀察發(fā)病情況并照相記錄。以野生型(wild-type,WT)為對(duì)照,利用ImageJ 2.0軟件計(jì)算接種10 d后病斑面積。根據(jù)病斑面積大小,將病情分為6個(gè)級(jí)別:0級(jí)(S<0.25 mm2);1級(jí)(0.25 mm2≤S<0.75 mm2);2級(jí)(0.75 mm2≤S<1.25 mm2);3級(jí)(1.25 mm2≤S<1.75 mm2);4級(jí)(1.75 mm2≤S<2.25 mm2);5級(jí)(S≥2.25 mm2)。分別對(duì)每級(jí)所包含的病斑數(shù)目進(jìn)行統(tǒng)計(jì),然后計(jì)算各株系的病情指數(shù)(disease index,DI),DI=100×Σ(各級(jí)病斑數(shù)×相應(yīng)級(jí)數(shù)值)/(病斑總數(shù)×最大級(jí)數(shù))。
取轉(zhuǎn)基因和野生型植株的葉片,液氮速凍,干冰運(yùn)輸送上海美吉公司進(jìn)行轉(zhuǎn)錄組測(cè)序和信息學(xué)分析。以甜橙基因組序列(http://citrus.hzau.edu.cn/orange/ index.php)為參考,利用TopHat2軟件對(duì)測(cè)序數(shù)據(jù)進(jìn)行比對(duì)分析。使用FPKM計(jì)算基因的表達(dá)水平。以非轉(zhuǎn)基因植株為對(duì)照,采用DESeq2軟件進(jìn)行表達(dá)差異的顯著性分析,以錯(cuò)誤發(fā)現(xiàn)率FDR<0.01,差異倍數(shù)|log2fold change|>1作為差異表達(dá)基因(DEG)的篩選標(biāo)準(zhǔn)。利用非冗余蛋白數(shù)據(jù)庫(kù)(non-redundant protein database,Nr)、非冗余核苷酸數(shù)據(jù)庫(kù)(NCBI non- redundant nucleotide database,Nt)、蛋白質(zhì)序列數(shù)據(jù)庫(kù)(SwissProt protein database,SwissProt)、蛋白質(zhì)直系同源數(shù)據(jù)庫(kù)(Cluster of Orthologous Groups,COG)、蛋白質(zhì)家族域數(shù)據(jù)庫(kù)(Protein families database,Pfam)、基因本體論數(shù)據(jù)庫(kù)(Gene Ontology,GO)和東京基因與基金組百科全書(shū)(Kyoto Encyclopedia of Genes and Genomes,KEGG)數(shù)據(jù)庫(kù)對(duì)基因進(jìn)行功能注釋。
為了詳細(xì)分析調(diào)控的代謝途徑和基因,對(duì)獲得的轉(zhuǎn)錄組數(shù)據(jù)進(jìn)一步進(jìn)行MapMan功能注釋(http://mapman.gabipd.org/web/guest/mercator)。以|log2fold change|>1且-value校正值padj<0.05為顯著性標(biāo)準(zhǔn)可視化MapMan途徑和功能,并使用Benjamin-Hochberg方法(FDR≤0.05)對(duì)MapMan途徑和功能進(jìn)行Wilcoxon雙尾檢測(cè),篩選顯著富集的途徑、功能和基因[41]。
試驗(yàn)結(jié)果均為3次重復(fù)的平均值,采用Excel 2016進(jìn)行數(shù)據(jù)整理、標(biāo)準(zhǔn)偏差計(jì)算及圖表繪制,差異顯著性分析采用SPSS 20.0軟件完成。
用組成型表達(dá)的CaMV啟動(dòng)子控制、和的表達(dá),分別構(gòu)建植物表達(dá)載體p35S:WRKY50(W50)、p35S:WRKY61(W61)、p35S:WRKY72(W72)(圖1),利用農(nóng)桿菌介導(dǎo)法進(jìn)行晚錦橙的遺傳轉(zhuǎn)化。首先通過(guò)組織化學(xué)染色及外源的PCR擴(kuò)增對(duì)卡那霉素抗性植株進(jìn)行篩選(圖2-A、2-B)。然后通過(guò)對(duì)目的基因的PCR擴(kuò)增進(jìn)一步鑒定陽(yáng)性植株(圖2-C)。為了避免內(nèi)源目的基因的干擾,PCR擴(kuò)增的上下游引物分別設(shè)計(jì)在啟動(dòng)子和目的基因上(表1、圖1)。經(jīng)鑒定共獲得W50、W61和W72轉(zhuǎn)基因植株各6、8和6株,具體見(jiàn)圖2-A。
以為內(nèi)參基因,轉(zhuǎn)基因植株GA-5為對(duì)照,對(duì)組織化學(xué)染色和PCR檢測(cè)呈陽(yáng)性的轉(zhuǎn)基因植株進(jìn)行qRT-PCR分析。結(jié)果表明,W50-1、W50-3,W61-1、W61-3、W61-5、W61-6、W61-7、W61-9、W61-11、W61-12和W72-5、W72-7、W72-10株系為單拷貝;W50-5和W72-1、W72-2株系為雙拷貝(表3)。
以野生型植株為對(duì)照,利用qRT-PCR分析、、在轉(zhuǎn)基因植株中的表達(dá)水平。結(jié)果顯示,所有W50轉(zhuǎn)基因植株中表達(dá)水平均顯著高于野生型,其中W50-3和W50-5表達(dá)水平相對(duì)較高;同樣,所有W61轉(zhuǎn)基因植株中表達(dá)水平均顯著高于野生型,其中W61-5、W61-6、W61-9和W61-11的表達(dá)相對(duì)較高;5株W72轉(zhuǎn)基因植株中表達(dá)水平顯著高于野生型,1株W72轉(zhuǎn)基因植株中表達(dá)低于野生型,其中W72-2和W72-5的表達(dá)相對(duì)較高(圖3)。結(jié)合外源基因插入的拷貝數(shù)分析結(jié)果,選擇表達(dá)水平相對(duì)較高的W50-5,W61-5、W61-9、W61-11和W72-5進(jìn)行重點(diǎn)研究。
表3 轉(zhuǎn)基因植株外源基因拷貝數(shù)實(shí)時(shí)熒光定量PCR分析
對(duì)目的基因表達(dá)水平顯著提高的轉(zhuǎn)基因株系進(jìn)行潰瘍病抗性評(píng)價(jià)。結(jié)果表明,在所獲得的轉(zhuǎn)基因植株中,只有超量表達(dá)的轉(zhuǎn)基因植株表現(xiàn)明顯的抗性增強(qiáng),其病斑明顯小于野生型植株,而超量表達(dá)和的轉(zhuǎn)基因植株病斑大小與野生型相比無(wú)明顯差異(圖4-A)。
35S:花椰菜花葉病毒CaMV 35S啟動(dòng)子Cauliflower mosaic virus 35S promoter (CaMV 35S);GUS:NPTII:β-葡萄糖醛酸酶GUS報(bào)告基因與卡那霉素NPTII抗性基因的融合基因The fusion gene of β-glucuronidase and neomycin phosphotransferase genes;nos:胭脂堿合酶基因的轉(zhuǎn)錄終止序列The terminator of the nopaline synthase gene。單箭頭表示轉(zhuǎn)基因植株鑒定用引物 Arrow indicates the primer used to determine transgenic plant
A:轉(zhuǎn)基因植株的GUS組織化學(xué)染色圖GUS histochemical staining of transgenic plants;B:轉(zhuǎn)基因植株NPTII擴(kuò)增結(jié)果Amplification results of NPTII in transgenic plants;C:部分轉(zhuǎn)基因植株目的基因的PCR擴(kuò)增PCR amplification of target genes in some transgenic plants。M:DNA marker;P:質(zhì)粒模板plasmid template;+:已鑒定的陽(yáng)性植株對(duì)照Identified positive plant control
采用雙尾t檢驗(yàn)確定與WT對(duì)照相關(guān)的統(tǒng)計(jì)學(xué)意義(*:P<0.05;**:P<0.01)。圖4同
為了對(duì)超量表達(dá)轉(zhuǎn)基因植株的抗性水平進(jìn)行定量比較,進(jìn)一步對(duì)其病斑面積和病情指數(shù)進(jìn)行了統(tǒng)計(jì)分析。結(jié)果顯示,針刺接種10 d,W61-3和W61-7株系的病斑面積與野生型無(wú)明顯差異,其余株系的病斑面積均顯著小于野生型,其中W61-5、W61-6、W61-9和W61-11株系的病斑面積相對(duì)較?。▓D4-B)。此外,病情指數(shù)的統(tǒng)計(jì)結(jié)果顯示,W61-3和W61-7株系的病情指數(shù)與野生型無(wú)明顯差異,而其余株系的病情指數(shù)均顯著降低(圖4-C)。結(jié)果表明,與野生型相比轉(zhuǎn)基因植株對(duì)潰瘍病的抗性顯著提高,其中W61-5、W61-6、W61-9和W61-11株系的抗性水平相對(duì)較高。
對(duì)抗性水平最高的轉(zhuǎn)基因株系W61-5和W61-9進(jìn)行了轉(zhuǎn)錄組測(cè)序分析。聚類熱圖分析表明,W61-5和W61-9株系中基因表達(dá)譜與野生型相比有明顯的差異(圖5-A)。此外,與野生型相比,W61-5和W61-9株系分別有1 671和2 933個(gè)差異表達(dá)基因。在W61-5中有1 116個(gè)基因上調(diào)表達(dá),555個(gè)基因下調(diào)表達(dá),在W61-9中有2 010個(gè)基因上調(diào)表達(dá),923個(gè)下調(diào)表達(dá),其中有1 469個(gè)差異表達(dá)基因在兩株轉(zhuǎn)基因植株中具有相似的表達(dá)譜(圖5-B、5-C)。MapMan pathway富集分析顯示,兩株轉(zhuǎn)基因植株中生物脅迫和信號(hào)轉(zhuǎn)導(dǎo)相關(guān)途徑均被顯著激活,以W61-9株系變化更加明顯(圖5-D)。結(jié)果表明,超量表達(dá)正調(diào)控植物應(yīng)答生物脅迫和信號(hào)轉(zhuǎn)導(dǎo)途徑。
進(jìn)一步分析W61-9株系中與生物脅迫相關(guān)的差異基因情況。結(jié)果顯示,有85個(gè)差異基因直接與生物脅迫相關(guān),且有75個(gè)基因顯著上調(diào)表達(dá)。這些基因包括病原入侵的感知、活性氧爆發(fā)、信號(hào)轉(zhuǎn)導(dǎo)、轉(zhuǎn)錄因子和防御基因。另外,許多與脅迫相關(guān)的激素信號(hào)、細(xì)胞壁和次生代謝等基因也顯著上調(diào)表達(dá)(圖6、表4)。
對(duì)W61-9株系中與信號(hào)轉(zhuǎn)導(dǎo)途徑相關(guān)的差異基因情況進(jìn)一步分析。結(jié)果顯示,信號(hào)轉(zhuǎn)導(dǎo)途徑中主要是激酶受體基因受到影響,其中有10類激酶受體基因受到顯著影響,且絕大部分都是上調(diào)表達(dá),包括富亮氨酸重復(fù)、奇異果甜蛋白、未知功能域(DUF 26)、植物凝集素、葉銹病LRK10 like、S-基因座、胞壁相關(guān)激酶、賴氨酸基序、褶皺樣和受體樣胞漿激酶(圖7-A、表4)。此外,與翻譯后修飾及蛋白質(zhì)降解相關(guān)的基因也絕大部分上調(diào)表達(dá)(圖7-B、表4)。
圖4 轉(zhuǎn)基因植株的潰瘍病抗性評(píng)價(jià)
A:差異基因表達(dá)分析聚類熱圖。紅色表示上調(diào),藍(lán)色表示下調(diào)Cluster heat map for differential gene expression analysis. Red and blue indicate up-regulated and down-regulated, respectively。B:差異基因表達(dá)分析火山圖。紅色表示上調(diào),綠色表示下調(diào)Volcano map of differential gene expression analysis. Red and green indicate up-regulated and down-regulated, respectively。C:差異基因表達(dá)分析維恩圖。中間重疊部分代表共有的差異基因Venn diagram of differential gene expression analysis. The middle overlap represents the shared genes。D:差異表達(dá)基因的MapMan可視化分析。紅色表示上調(diào),藍(lán)色表示下調(diào)MapMan visual analysis of differential genes. Red and blue indicate up-regulated and down-regulated, respectively
表4 轉(zhuǎn)基因植株中生物脅迫相關(guān)的差異基因情況
該圖為MapMan注釋的轉(zhuǎn)基因株系W61-9中與生物脅迫相關(guān)的差異基因情況。每個(gè)方塊表示一個(gè)基因,紅色表示上調(diào)表達(dá),綠色表示下調(diào)表達(dá)The figure shows the differential genes involved in biotic stress of W61-9 transgenic line by MapMan. Each square represents one gene. Red and green indicate up-regulated expression and down-regulated expression, respectively
A:W61-9中受體樣激酶相關(guān)基因的表達(dá)情況Differential expression of receptor-like kinase-related genes of W61-9 line;B:W61-9中蛋白質(zhì)代謝相關(guān)基因的表達(dá)情況Differential expression of genes related to protein metabolism of W61-9 line
WRKY轉(zhuǎn)錄因子家族成員眾多,參與調(diào)節(jié)植物對(duì)生物、非生物脅迫的響應(yīng)以及生長(zhǎng)發(fā)育等多方面進(jìn)程[12]。在柑橘中,Ayadi等發(fā)現(xiàn)和在所有非生物脅迫條件下都可能上調(diào),但是唯一被病原菌誘導(dǎo)上調(diào)表達(dá)的基因,表明其在柑橘抗病性方面有潛在價(jià)值;此外還發(fā)現(xiàn)可能與黃龍病的防御反應(yīng)相關(guān)[31]。在水稻中,的過(guò)表達(dá)增強(qiáng)了水稻對(duì)紫外線的耐受性和抗病性[42];過(guò)表達(dá)和增加了水稻對(duì)稻瘟病和白葉枯的敏感性,而抑制表達(dá)能夠增強(qiáng)其抗病性[21]。在擬南芥中,超量表達(dá)能夠提高對(duì)灰霉病的抗性,敲除該基因增加了對(duì)灰霉菌的敏感性[43];異源表達(dá)來(lái)自葡萄的提高了轉(zhuǎn)基因植株對(duì)白粉病的抗性[44]。在楊樹(shù)中,轉(zhuǎn)的毛白楊對(duì)黑斑病的耐受性增強(qiáng);過(guò)表達(dá)的毛果楊對(duì)潰瘍病的抗性提高[45]。而在本研究中,過(guò)表達(dá)增強(qiáng)了轉(zhuǎn)基因柑橘對(duì)潰瘍病的抗性,表明WRKY轉(zhuǎn)錄因子在植物抗病性方面有很大的應(yīng)用價(jià)值。
本研究發(fā)現(xiàn)超量表達(dá)通過(guò)激活轉(zhuǎn)基因植株中生物脅迫和信號(hào)轉(zhuǎn)導(dǎo)相關(guān)途徑來(lái)提高潰瘍病抗性。這些基因主要涉及病原入侵的感知、活性氧爆發(fā)、轉(zhuǎn)錄因子和防御基因,與脅迫相關(guān)的激素信號(hào)、細(xì)胞壁和次生代謝等基因,以及受體樣激酶基因。有研究表明,病原菌侵染后感病部位會(huì)出現(xiàn)活性氧的急劇上升,從而誘導(dǎo)植物發(fā)生細(xì)胞程序性死亡,這種“活性氧爆發(fā)”被稱作是細(xì)胞水平上寄主植物對(duì)病原菌侵染的最早應(yīng)答之一,對(duì)寄主抗病性有積極作用[46-47]。大量研究顯示轉(zhuǎn)錄因子、激素信號(hào)、細(xì)胞壁和次生代謝相關(guān)基因在植物抗病性中發(fā)揮重要作用[48-52]。Gao等研究表明對(duì)蘿卜皺紋病毒有負(fù)調(diào)控作用,可能參與植物免疫信號(hào)通路[53]。在水稻中,被鑒定為稻瘟病抗性基因[54]。此外,有研究發(fā)現(xiàn)AtWRKY61對(duì)多種脅迫都有響應(yīng),且這種響應(yīng)很可能通過(guò)其抑制相關(guān)基因表達(dá)而實(shí)現(xiàn);同時(shí)AtWRKY61分別與AtWRKY9和AtWRKY72存在蛋白互作,這種互作可能對(duì)其參與調(diào)節(jié)多種脅迫的應(yīng)答反應(yīng)有重要作用[55]。本研究中轉(zhuǎn)錄組測(cè)序揭示,有20個(gè)WRKY轉(zhuǎn)錄因子受到超量表達(dá)的顯著影響,這些WRKY轉(zhuǎn)錄因子與CsWRKY61的互作關(guān)系有待深入研究。此外,病程相關(guān)蛋白(PR蛋白)是寄主植物在受到病原物侵染后誘導(dǎo)表達(dá)豐度最高的一類蛋白,是系統(tǒng)獲得性抗性(SAR)的分子標(biāo)記。Qiu等[16]研究表明,WRKY轉(zhuǎn)錄因子與PR蛋白存在相互調(diào)節(jié)關(guān)系。課題組前期研究發(fā)現(xiàn),CsWRKY與CsPR-1相互影響,和的轉(zhuǎn)錄水平在過(guò)表達(dá)的轉(zhuǎn)基因柑橘中明顯上調(diào)[34]。在本研究中,轉(zhuǎn)錄組測(cè)序結(jié)果顯示超量表達(dá)的轉(zhuǎn)基因植株中絕大部分PR蛋白的表達(dá)顯著提高。這些結(jié)果與超量表達(dá)顯著提高了轉(zhuǎn)基因植株對(duì)潰瘍病的抗性相吻合,但其具體的調(diào)節(jié)機(jī)制有待進(jìn)一步研究。
Gao等[56]研究表明,可能通過(guò)正調(diào)控水楊酸信號(hào)途徑,負(fù)調(diào)控茉莉酸信號(hào)途徑從而增強(qiáng)對(duì)灰霉病的抗性;周鵬飛[34]研究表明,受潰瘍病菌誘導(dǎo)后,的轉(zhuǎn)錄水平在抗病品種四季橘中大幅度提高,而在感病品種紐荷爾臍橙中變化不明顯,表明可能對(duì)潰瘍病抗性有積極作用;此外,Bhattarai等[57]發(fā)現(xiàn)WRKY72型轉(zhuǎn)錄因子參與了番茄和擬南芥的基礎(chǔ)免疫以及番茄R基因介導(dǎo)的基因?qū)虻目剐?;Xu等[27]研究表明,接種潰瘍病菌后,隨著接種時(shí)間的增加在感病品種紐荷爾臍橙中的表達(dá)量逐漸下降,而在抗病品種四季橘中無(wú)明顯變化,推測(cè)潰瘍病菌侵染通過(guò)下調(diào)的表達(dá),可能對(duì)介導(dǎo)的抗病過(guò)程有抑制作用。然而,本研究中抗病性評(píng)價(jià)結(jié)果顯示超量表達(dá)和的植株病斑面積與野生型對(duì)照無(wú)明顯差異,推測(cè)可能是轉(zhuǎn)基因植株太少未篩選到抗性株系,或者轉(zhuǎn)基因植株通過(guò)其他途徑抵消了和發(fā)揮的作用。
能夠激活與生物脅迫和信號(hào)轉(zhuǎn)導(dǎo)相關(guān)的途徑,是柑橘抗病育種中有潛在應(yīng)用價(jià)值的抗性基因。研究結(jié)果為柑橘潰瘍病抗性遺傳改良提供了重要基因資源。
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Effect of Transcription Factor CsWRKY61 on Citrusbacterial Canker Resistance
LONG Qin, Dumeixia, Long JunHong, Heyongrui, Zou XiuPing, Chen ShanChun
(National Center for Citrus Variety Improvement, Citrus Research Institute, Southwest University/Chinese Academy of Agricultural Sciences, Chongqing 400712)
【Background】Citrus bacterial canker (CBC) caused bysubsp.() is one of the most serious citrus diseases in the world, which is a quarantine disease. Due to the relatively backward research of citrus molecular pathology, the available resistance gene resources are relatively scarce. WRKY transcription factor is involved in plant responses to biotic and abiotic stress. The previous study has found that citrus WRKY transcription factor may play an important role in regulating host disease resistance response.【Objective】The objective of this study is to evaluate the canker resistance of transgenic citrus () with over-expression of,and, clarify the biological function and disease resistance breeding value of these genes in citrus in response to. RNA-seq was further used to analyze the signaling pathway regulated by.【Method】-mediated method was used to obtain transgenic citrus plants with over-expression of,and. Real-time quantitative PCR (qRT-PCR) was used to analyze the expression level and copy number of the target genes.pinprick inoculation was used to evaluate the resistance of transgenic plants to canker disease. The molecular mechanism ofimproving citrus bacterial canker resistance was investigated by transcriptome sequencing analysis of over-expressionand wild-type (WT) plants.【Result】The plant expression vectors of CAMVpromoter controlling the expression of,andwere constructed, and 6, 8 and 6 transgenic lines were obtained bystaining and PCR identification, respectively. The expression of the target gene in transgenic plants increased in different degrees. The copy number of exogenous genes in most transgenic plants was 1. Only the transgenic plants with over-expression ofhad significantly enhanced canker disease resistance, and the lesion area was significantly smaller than that of WT plants, while over-expression ofandtransgenic plants had no significant difference in disease resistance compared with WT. Transcriptomics analysis showed that biotic stress related pathways (including pathogen recognition, respiratory burst, transcription factors, defense genes, hormones, cell wall and secondary metabolism, etc.) and signal transduction-related pathways (mainly kinase receptors) were significantly activated in over-expression oftransgenic plants. 【Conclusion】Over-expression ofcan activate pathways related to biotic stress and signal transduction, enhance citrus bacterial canker resistance. It is suggested thathas potential application value in citrus disease resistance breeding.
subsp.(); citrus bacterial canker; CsWRKY61; over-expression; disease resistance; transcriptome sequencing
2019-09-26;
2019-11-13
國(guó)家重點(diǎn)研發(fā)計(jì)劃(2018YFD1000300)、重慶市自然科學(xué)基金-博士后基金(cstc2019jcyj-bshX0024)、中央高?;究蒲袠I(yè)務(wù)費(fèi)(XDJK2019C027)、國(guó)家現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系建設(shè)專項(xiàng)資金(CARS-26)
龍琴,E-mail:longlong860923@126.com。通信作者鄒修平,E-mail:zouxiuping@cric.cn。通信作者陳善春,E-mail:scchen@cric.cn
(責(zé)任編輯 岳梅)