鄒修平,龍俊宏,彭愛(ài)紅,陳敏,龍琴,陳善春
超量表達(dá)通過(guò)抑制生長(zhǎng)素信號(hào)轉(zhuǎn)導(dǎo)增強(qiáng)柑橘潰瘍病抗性
鄒修平,龍俊宏,彭愛(ài)紅,陳敏,龍琴,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)
由柑橘黃單胞桿菌柑橘亞種(subsp,)引起的柑橘潰瘍病是柑橘生產(chǎn)上最具毀滅性的一種病害。植物生長(zhǎng)素在調(diào)控柑橘潰瘍病菌引起的寄主侵染部位膿皰形成中起重要作用。生長(zhǎng)素早期響應(yīng)基因通過(guò)?;胚?3-乙酸(indole-3-acetic acid,IAA)調(diào)控植物激素動(dòng)態(tài)平衡。前期研究發(fā)現(xiàn)柑橘在調(diào)控生長(zhǎng)素響應(yīng)潰瘍病侵染中起著重要作用?!尽客ㄟ^(guò)對(duì)超量表達(dá)轉(zhuǎn)基因晚錦橙的抗病性、植株表型、細(xì)胞和激素變化進(jìn)行分析,利用RNA-Seq解析調(diào)控的信號(hào)通路,探明調(diào)控激素動(dòng)態(tài)平衡影響柑橘潰瘍病抗性的內(nèi)在機(jī)制。利用針刺法對(duì)離體轉(zhuǎn)基因葉片接種潰瘍病菌,統(tǒng)計(jì)接種第10 天時(shí)病斑面積和病情指數(shù),以野生型為對(duì)照,評(píng)價(jià)轉(zhuǎn)基因植株的抗性水平;提取感病前后葉片內(nèi)源激素,利用高效液相色譜技術(shù)(high performance liquid chromatography,HPLC )檢測(cè)轉(zhuǎn)基因植株中激素含量變化;溫室中觀察轉(zhuǎn)基因植株表型變化;通過(guò)測(cè)量葉片縱徑、橫徑和厚度分析轉(zhuǎn)基因植株葉型變化特征;制備葉片表皮切片,顯微觀察表皮細(xì)胞和氣孔,并統(tǒng)計(jì)轉(zhuǎn)基因植株表皮細(xì)胞長(zhǎng)度和氣孔密度;采用RNA-Seq測(cè)序技術(shù)研究轉(zhuǎn)基因植株轉(zhuǎn)錄組變化情況,并利用Nr、Nt、Pfam、COG、SwissProt和gene ontology (GO)數(shù)據(jù)庫(kù)注釋基因功能,進(jìn)一步利用KEEG數(shù)據(jù)庫(kù)和MapMan軟件解析超量表達(dá)影響的重要基因、功能和途徑,闡明調(diào)控柑橘潰瘍病抗性的分子機(jī)制。超量表達(dá)顯著增強(qiáng)轉(zhuǎn)基因植株的潰瘍病抗性;轉(zhuǎn)基因植株分枝增多且下垂,葉片向上卷曲,變小,顏色淺;轉(zhuǎn)基因植株氣孔密度增加,表皮細(xì)胞變短;激素含量分析顯示,轉(zhuǎn)基因植株自由生長(zhǎng)素(IAA)和茉莉酸(jasmonic acid,JA)含量顯著降低,而水楊酸(salicylic acid,SA)含量顯著增加;轉(zhuǎn)錄組測(cè)序分析表明,超量表達(dá)顯著抑制生長(zhǎng)素信號(hào)轉(zhuǎn)導(dǎo)相關(guān)基因表達(dá),特別是所有注釋的Aux/IAA家族基因均下調(diào)表達(dá),相反,與生物脅迫相關(guān)基因的表達(dá)為上調(diào),其中絕大部分基因?yàn)椴〕滔嚓P(guān)蛋白基因。超量表達(dá)通過(guò)?;杂蒊AA抑制生長(zhǎng)素信號(hào)轉(zhuǎn)導(dǎo),調(diào)控JA和SA的動(dòng)態(tài)平衡,改變細(xì)胞和植株的形態(tài)建成,從而增強(qiáng)柑橘對(duì)潰瘍病的抗性。研究結(jié)果暗示調(diào)控激素動(dòng)態(tài)平衡在柑橘抗病育種中具有潛在價(jià)值。
柑橘黃單胞桿菌柑橘亞種;柑橘潰瘍?。簧L(zhǎng)素;;抗病性
【研究意義】柑橘潰瘍病(citrus bacterial canker,CBC)是一種危害極嚴(yán)重的世界性病害,其病原菌為柑橘黃單胞桿菌柑橘亞種(subsp.,)。感染柑橘潰瘍病的植株在葉、枝和果實(shí)上出現(xiàn)火山狀病斑,嚴(yán)重時(shí)落葉、枯枝、落果,產(chǎn)量降低、果實(shí)品質(zhì)變劣,一旦疫情在柑橘產(chǎn)區(qū)傳播蔓延,對(duì)柑橘產(chǎn)業(yè)具有毀滅性、災(zāi)難性打擊[1-2]。柑橘產(chǎn)業(yè)中大部分栽培品種屬于潰瘍病易感品種。因此,大力開(kāi)展柑橘潰瘍病抗(感)機(jī)理和重要基因資源挖掘的研究,對(duì)柑橘優(yōu)良抗性新品種的選育和柑橘產(chǎn)業(yè)的健康穩(wěn)定發(fā)展具有重要意義,也將進(jìn)一步深化對(duì)病原菌致病機(jī)理、植物與病原互作機(jī)制的理解?!厩叭搜芯窟M(jìn)展】植物生長(zhǎng)素在調(diào)控柑橘潰瘍病菌侵染引起的膿皰形成中起重要作用。Costacurta等[3]研究發(fā)現(xiàn),柑橘潰瘍病菌能分泌吲哚-3-乙酸(indole-3-acetic acid,IAA),而且甜橙葉片提取液能促進(jìn)病原菌繁殖和IAA合成;Cernadas等[4]研究表明,柑橘潰瘍病菌侵染促進(jìn)甜橙生長(zhǎng)素合成、運(yùn)輸和信號(hào)轉(zhuǎn)導(dǎo)相關(guān)基因的轉(zhuǎn)錄。進(jìn)一步研究發(fā)現(xiàn),萘乙酸(NAA)處理促進(jìn)甜橙感病部位水浸狀膿皰的形成,而生長(zhǎng)素抑制劑1-N-萘基鄰氨甲酰苯甲酸(NPA)抑制潰瘍病癥狀的發(fā)展[5-6],顯示生長(zhǎng)素具有促進(jìn)柑橘潰瘍病癥狀發(fā)展的作用。目前,關(guān)于植物生長(zhǎng)素介導(dǎo)寄主感病的機(jī)制研究主要集中在擬南芥、水稻等植物中[7]。病原菌通過(guò)向寄主侵染部位細(xì)胞分泌IAA或增強(qiáng)寄主IAA的合成,致使寄主細(xì)胞內(nèi)IAA的水平急劇上升,高濃度的IAA使植物細(xì)胞壁pH下降,胞壁結(jié)構(gòu)蛋白酸化,細(xì)胞壁發(fā)生重排,從而引起細(xì)胞壁松弛,細(xì)胞擴(kuò)張和膨大,以利于病原菌的入侵和擴(kuò)散[8-9]。而且,侵染部位高濃度的IAA抑制水楊酸(salicylic acid,SA)介導(dǎo)的抗病反應(yīng)[7,10-11]。面對(duì)病原菌對(duì)生長(zhǎng)素的操控,植物必然啟動(dòng)相應(yīng)的對(duì)抗機(jī)制來(lái)維持感病部位細(xì)胞激素的動(dòng)態(tài)平衡,努力消除病原菌的危害。其中,生長(zhǎng)素響應(yīng)因子GH3起著重要作用[7-8]。編碼蛋白具有植物激素酰胺合成酶活性,催化自由IAA與氨基酸結(jié)合,從而使其失活,當(dāng)植物需要IAA時(shí),通過(guò)IAA酰胺水解酶水解作用釋放出IAA,以此調(diào)控細(xì)胞內(nèi)IAA動(dòng)態(tài)平衡[12-13]。另外,一些GH3也具有酰胺化SA和茉莉酸(jasmonic acid,JA)等激素的活性[14]。的功能研究主要集中在擬南芥和水稻中,在調(diào)控植物抗病性反應(yīng)中起重要作用[12,15]。目前認(rèn)為參與植物抗病反應(yīng)的機(jī)制為:病原菌侵染導(dǎo)致寄主感病部位細(xì)胞IAA水平急劇上升,寄主通過(guò)生長(zhǎng)素受體TIR1感知上升的IAA水平并觸發(fā)生長(zhǎng)素信號(hào)途徑,迅速激活表達(dá),GH3酶將過(guò)量的IAA酰胺化,使活性IAA水平下降,進(jìn)而限制植物細(xì)胞壁增生和松弛,從而正向增強(qiáng)寄主的抗性[9,16-17]。比如超量表達(dá)和能增強(qiáng)水稻對(duì)白葉枯病和稻瘟病抗性[16-17],而能賦予植物廣譜抗性[9]。隨著基因組、轉(zhuǎn)錄組、蛋白組等大規(guī)模組學(xué)技術(shù)的應(yīng)用,GH3家族基因在豆類(lèi)、蘋(píng)果、玉米、苜蓿、番茄、棉花等作物中的功能研究已在開(kāi)展,其在生物和非生物抗性、激素動(dòng)態(tài)平衡、信號(hào)途徑中的作用備受關(guān)注[18-23]。但在柑橘中,鮮有關(guān)于GH3家族基因參與調(diào)控植物生長(zhǎng)發(fā)育和抗性的研究報(bào)道?!颈狙芯壳腥朦c(diǎn)】前期研究發(fā)現(xiàn)[24],柑橘生長(zhǎng)素早期響應(yīng)基因在調(diào)控生長(zhǎng)素響應(yīng)潰瘍病菌侵染中起著重要作用,超量表達(dá)顯著降低柑橘對(duì)潰瘍病的感病性,但其機(jī)制尚待解析?!緮M解決的關(guān)鍵問(wèn)題】通過(guò)分析超量表達(dá)對(duì)轉(zhuǎn)基因植株潰瘍病抗性、激素水平、生長(zhǎng)發(fā)育和轉(zhuǎn)錄組的影響,闡明調(diào)控柑橘潰瘍病抗性的機(jī)制,為進(jìn)一步解析柑橘潰瘍病菌侵染中生長(zhǎng)素途徑調(diào)控寄主抗性的機(jī)理打下基礎(chǔ),為柑橘抗病育種提供新思路和新材料。
試驗(yàn)于2015—2018年在中國(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。
供試植物材料為超量表達(dá)轉(zhuǎn)基因晚錦橙()和野生型晚錦橙[24]。轉(zhuǎn)基因植株以及野生型晚錦橙種植于中國(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育種圃。育種圃的溫度控制在25—28℃,相對(duì)濕度控制在60%—70%,光周期L﹕D= 16 h﹕8 h。
轉(zhuǎn)基因植株種植在田間2年后,觀察其表型。選取完全成熟的春稍,從頂部開(kāi)始選取第3—5節(jié)間的葉片,使用直尺測(cè)量葉片的縱徑和橫徑,利用游標(biāo)卡尺測(cè)量葉片的厚度。
上午采摘完全伸展的成熟葉片,將離主脈0.5 cm處的葉片剪成0.5 cm×1 cm長(zhǎng)方形,迅速放入3 mol·L-1的NaOH溶液中70℃水浴15 min,去除溶液,無(wú)菌水沖洗兩次,再用蒸餾水沖洗4次。將葉片下表皮粘在透明膠帶上,用手術(shù)刀片去盡殘余葉肉組織。將粘有表皮細(xì)胞的透明膠帶剪下,置于載玻片上制成臨時(shí)切片,于光學(xué)顯微鏡下觀察,并在40×物鏡和10×目鏡(放大倍數(shù)為400倍)條件下成像。試驗(yàn)重復(fù)3次,每次每個(gè)株系檢測(cè)3片葉。使用ImageJ1.47軟件統(tǒng)計(jì)氣孔的數(shù)目和表皮細(xì)胞長(zhǎng)度。氣孔密度=視野范圍內(nèi)的氣孔數(shù)/視野范圍面積(個(gè)/mm2)。
柑橘生長(zhǎng)素IAA、水楊酸、茉莉酸含量測(cè)定參照Marques等[25]的方法。采摘1 g鮮重葉片,液氮速凍,研磨成粉;加入5 ml 80%甲醇浸提過(guò)夜,然后13 000×離心10 min。倒掉上清液,加入1ml 1%乙酸重懸沉淀。重懸液按照Oasis cartridges(Waters,美國(guó)馬薩諸塞州)的方法進(jìn)行純化,純化后的激素溶解于100 μL 10%的甲醇。提取的激素送鐘鼎生物公司(南京)進(jìn)行HPLC檢測(cè)。
以轉(zhuǎn)基因晚錦橙和野生型晚錦橙的成熟葉片為材料,利用針刺接種法[26]接種潰瘍病菌。具體方法:采集轉(zhuǎn)基因和野生型晚錦橙的成熟葉片,清水洗凈,70%乙醇?xì)⒕?—5 s,再用無(wú)菌水清洗干凈。將葉片背面向上平鋪在無(wú)菌培養(yǎng)皿中,培養(yǎng)皿底部鋪上濕潤(rùn)濾紙保濕,葉柄處用濕棉花覆蓋保濕,每一片葉背面針刺12個(gè)小孔,用移液槍吸取1 μL(5×104CFU/mL)潰瘍病菌懸浮液滴加到小孔處。培養(yǎng)皿用Parafilm封口膜密封,置于28℃光照培養(yǎng)箱培養(yǎng)。接種后第10天拍照,用軟件Image J 1.47 統(tǒng)計(jì)病斑面積(mm2)。病斑面積的大小分為8個(gè)級(jí)別,分級(jí)方法如下:以字母R表示病斑面積,0級(jí)(R≤0.25 mm2),1級(jí)(0.25 mm2<R≤0.5 mm2),2級(jí)(0.5 mm2<R≤0.75 mm2),3級(jí)(0.75 mm2<R≤1 mm2),4級(jí)(1 mm2<R≤1.25 mm2),5級(jí)(1.25 mm2<R≤1.5 mm2),6級(jí)(1.5 mm2<R≤1.75 mm2),7級(jí)(R>1.75 mm2)。病斑面積分級(jí)后,根據(jù)以下公式統(tǒng)計(jì)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))。根據(jù)病情指數(shù),分析轉(zhuǎn)基因植株抗?jié)儾∏闆r。
取轉(zhuǎn)基因和野生型植株的葉片,液氮速凍,送北京百邁克科技股份有限公司進(jìn)行轉(zhuǎn)錄組測(cè)序和信息學(xué)分析。試驗(yàn)設(shè)置3個(gè)生物學(xué)重復(fù)。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。以野生型為對(duì)照,采用DESeq2軟件進(jìn)行表達(dá)差異顯著性分析,差異表達(dá)基因(differential expressed gene,DEG)的篩選標(biāo)準(zhǔn)為錯(cuò)誤發(fā)現(xiàn)率(false discovery rate,F(xiàn)DR)<0.01 且差異倍數(shù) |log2fold change|>1。
利用Nr(non-redundant protein database,非冗余蛋白數(shù)據(jù)庫(kù))、Nt(NCBI non-redundant nucleotidedatabase,非冗余核苷酸數(shù)據(jù)庫(kù))、SwissProt(SwissProt protein database,蛋白質(zhì)序列數(shù)據(jù)庫(kù))、COG(Cluster of Orthologous Groups,蛋白質(zhì)直系同源數(shù)據(jù)庫(kù))、Pfam(Protein families database,蛋白質(zhì)家族域數(shù)據(jù)庫(kù))、GO(Gene Ontology,基因本體論數(shù)據(jù)庫(kù))、KEGG(Kyoto Encyclopedia of Genes and Genomes,東京基因與基金組百科全書(shū))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?zhuān)╤ttp://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[27]。
試驗(yàn)結(jié)果均為3次重復(fù)的平均值,用Excel 2016進(jìn)行數(shù)據(jù)整理、標(biāo)準(zhǔn)偏差計(jì)算及圖表的繪制,差異顯著性用SPSS 20.0統(tǒng)計(jì)軟件進(jìn)行分析。
利用葉片離體接種技術(shù)對(duì)兩年生轉(zhuǎn)基因晚錦橙植株進(jìn)行潰瘍病抗性評(píng)價(jià)。接種潰瘍病菌10 d時(shí),統(tǒng)計(jì)病斑面積和病情指數(shù)(圖1)。結(jié)果顯示,野生型植株病斑面積為2.67 mm2,4株(b7、b13、b19和b20)轉(zhuǎn)基因株系病斑面積分別為1.52、2.48、1.21、2.22 mm2,其中,b7和b19的病斑面積顯著小于野生型(圖1-A)。b7、b13、b19和b20株系的病情指數(shù)分別為45.55、89.12、44.48、77.02,而野生型植株的病情指數(shù)為83.48,轉(zhuǎn)基因株系b7和b19的病情指數(shù)顯著低于野生型對(duì)照(圖1-B)。結(jié)果表明超量表達(dá)能顯著提高柑橘抗?jié)儾〉哪芰Α8鶕?jù)抗性評(píng)價(jià)結(jié)果,以下研究主要以b7和b19株系為材料開(kāi)展。
WT:野生型Wild type;b7、b13、b19和b20:轉(zhuǎn)基因植株Transgenic plant。下同 The same as below
柱上不同小寫(xiě)字母表示差異顯著(Tukey’s檢驗(yàn),<0.05)。圖2同 Different lowercases on the bars represent significant differences (Tukey’s test,<0.05). The same as Fig. 2
圖1轉(zhuǎn)基因植株潰瘍病抗性評(píng)價(jià)
Fig. 1 Resistance evaluation oftransgenic plants to citrus bacterial canker
為了分析超量表達(dá)對(duì)自由IAA含量的影響,利用HPLC檢測(cè)在正常情況和潰瘍病誘導(dǎo)條件下轉(zhuǎn)基因株系 b7和b19的IAA含量。結(jié)果顯示,病原菌誘導(dǎo)前轉(zhuǎn)基因植株中自由IAA濃度顯著低于野生型植株(圖2)。接種潰瘍病菌3 d時(shí),野生型與轉(zhuǎn)基因植株中自由IAA含量均顯著下降,轉(zhuǎn)基因植株自由IAA含量依然顯著低于野生型植株。結(jié)果表明,超量表達(dá)不但抑制自由IAA的積累,而且促進(jìn)病原菌侵染引起的自由IAA的下降。
為了分析對(duì)植株體內(nèi)SA和JA含量變化影響,檢測(cè)了野生型與轉(zhuǎn)基因植株中SA和JA的含量。結(jié)果表明,與野生型相比,轉(zhuǎn)基因株系b7和b19中SA的含量顯著升高,而JA的含量顯著降低(圖3)。
Control:潰瘍病菌侵染前自由IAA含量檢測(cè)Free IAA content detection before Xcc inoculation;Xcc:潰瘍病菌侵染3 d時(shí)自由IAA含量檢測(cè)Free IAA content detection at 3 d after Xcc inoculation
*表示與野生型(WT)相比差異顯著(Tukey’s檢驗(yàn),p<0.05)。圖4、圖6同
兩年溫室表型觀察發(fā)現(xiàn),與野生型相比,b7轉(zhuǎn)基因株系出現(xiàn)輕微卷葉;b19轉(zhuǎn)基因株系葉片明顯向上卷曲,葉片變小,顏色淺,整片葉子較野生型葉片明顯下垂,莖較軟易彎曲,植株呈枯萎狀態(tài)(圖4-A)。葉型指數(shù)統(tǒng)計(jì)分析表明,轉(zhuǎn)基因植株葉片的縱徑(圖4-B)和橫徑(圖4-C)以及葉片厚度(圖4-D)均顯著小于野生型。
進(jìn)一步分析了轉(zhuǎn)基因株系b7和b19表皮細(xì)胞和氣孔的變化(圖5)。結(jié)果顯示,轉(zhuǎn)基因植株的表皮細(xì)胞長(zhǎng)度顯著降低(圖6-A),氣孔密度顯著增加(圖6-B)。
為了進(jìn)一步探討與柑橘潰瘍病抗性的關(guān)系,對(duì)抗性水平最高的轉(zhuǎn)基因株系b19進(jìn)行了轉(zhuǎn)錄組測(cè)序分析。聚類(lèi)熱圖分析表明,b19株系中基因表達(dá)譜與野生型相比有明顯的差異(圖7-A)。GO等功能注釋共獲得31 035個(gè)Unigene基因。與野生型相比,b19株系中有1 456個(gè)差異表達(dá)基因(DEG),940個(gè)DEG上調(diào)表達(dá),516個(gè)DEG下調(diào)表達(dá)(圖7-B)。KEGG富集分析顯示(圖7-C),148個(gè)DEG明顯富集于20個(gè)途徑和功能組,其中29個(gè)基因顯著富集于植物激素信號(hào)轉(zhuǎn)導(dǎo)途徑、17個(gè)基因富集于植物-病原菌互作途徑、15個(gè)基因富集于苯丙酸生物合成途徑、13個(gè)基因富集于氨基糖和核苷酸的糖代謝途徑。差異表達(dá)基因MapMan可視化分析進(jìn)一步表明,超量表達(dá)顯著影響細(xì)胞壁、壓力和信號(hào)傳遞功能組(圖7-D)。特別地,超量表達(dá)顯著上調(diào)生物脅迫功能組,而顯著下調(diào)生長(zhǎng)素信號(hào)轉(zhuǎn)導(dǎo)Aux/IAA 家族功能組(圖7-D)。
圖4 CsGH3.6轉(zhuǎn)基因植株的表型分析
箭頭指示氣孔arrows indicate stomata
圖6 CsGH3.6轉(zhuǎn)基因植株表皮細(xì)胞長(zhǎng)度(A)和氣孔密度(B)統(tǒng)計(jì)分析
2.6.1 生長(zhǎng)素途徑相關(guān)基因 進(jìn)一步利用MapMan詳細(xì)調(diào)查了轉(zhuǎn)錄組數(shù)據(jù)庫(kù)中與生長(zhǎng)素合成和降解、生長(zhǎng)素運(yùn)輸和信號(hào)轉(zhuǎn)導(dǎo)相關(guān)的差異表達(dá)基因(表1)。在生長(zhǎng)素合成-降解中有4個(gè)基因差異表達(dá),其中3個(gè)上調(diào)表達(dá),1個(gè)下調(diào)表達(dá);生長(zhǎng)素運(yùn)輸?shù)幕蛴?個(gè),均下調(diào)表達(dá);而在生長(zhǎng)素信號(hào)通路中差異表達(dá)的基因有19個(gè),其中有17個(gè)基因下調(diào)表達(dá),注釋的13個(gè)AUX/IAA家族基因均下調(diào)表達(dá)。這些結(jié)果表明超量表達(dá)明顯抑制生長(zhǎng)素的運(yùn)輸和信號(hào)轉(zhuǎn)導(dǎo)相關(guān)基因表達(dá)。
2.6.2 生物脅迫相關(guān)基因 圖8顯示MapMan注釋的轉(zhuǎn)基因株系b19中與生物脅迫相關(guān)的差異基因情況。有60個(gè)基因展現(xiàn)差異表達(dá)水平,這些基因涉及到壓力感知、活性氧爆發(fā)、信號(hào)轉(zhuǎn)導(dǎo)、基因轉(zhuǎn)錄和防御基因表達(dá)(圖8)。除4個(gè)防御基因下調(diào)表達(dá)外,所有與生物脅迫相關(guān)的差異基因均上調(diào)表達(dá),特別是42個(gè)防御基因中有38個(gè)上調(diào)表達(dá)?;蚬δ茏⑨尡砻?,所有防御基因均為病程相關(guān)蛋白PR基因。這些結(jié)果暗示,超量表達(dá)激活了植株的防御反應(yīng)。
2.6.3 植物生長(zhǎng)發(fā)育相關(guān)基因 MapMan分析顯示,與植物生長(zhǎng)發(fā)育相關(guān)的差異基因富集于細(xì)胞分裂與細(xì)胞周期、細(xì)胞組織、發(fā)育功能組和細(xì)胞壁。上調(diào)表達(dá)基因數(shù)目(60個(gè))明顯多于下調(diào)表達(dá)基因數(shù)目(50個(gè))。細(xì)胞壁在植物生長(zhǎng)發(fā)育中起著重要作用,轉(zhuǎn)錄組數(shù)據(jù)顯示15個(gè)細(xì)胞壁相關(guān)基因上調(diào)表達(dá),26個(gè)基因下調(diào)表達(dá)(圖9)。
表2展示了差異倍數(shù)≥2的細(xì)胞壁相關(guān)基因功能注釋。參與細(xì)胞壁合成的兩個(gè)重要基因纖維素合成酶基因和細(xì)胞壁蛋白基因差異表達(dá)水平在5倍以上。3個(gè)參與細(xì)胞壁修飾的木葡聚糖內(nèi)糖基轉(zhuǎn)移酶基因也展現(xiàn)出較高的表達(dá)水平。角質(zhì)合成相關(guān)基因和蠟質(zhì)合成相關(guān)基因表達(dá)水平上調(diào),而與細(xì)胞壁疏松相關(guān)的延展蛋白基因下調(diào)表達(dá)。
生長(zhǎng)素響應(yīng)因子在調(diào)控植物的生長(zhǎng)發(fā)育、激素動(dòng)態(tài)平衡、生物和非生物脅迫相關(guān)抗性中起著重要作用。本研究中,超量表達(dá)轉(zhuǎn)基因植株中自由IAA水平顯著降低,植株表型出現(xiàn)過(guò)表達(dá)導(dǎo)致自由IAA缺失引起的典型表型變化[16],證明CsGH3.6具有酰基化IAA的生物學(xué)功能。另外,轉(zhuǎn)基因植株JA含量也顯著下降,暗示CsGH3.6也可能具有?;疛A的功能[28]。潰瘍病抗性評(píng)價(jià)表明,超量表達(dá)顯著增強(qiáng)植株對(duì)潰瘍病的抗性[24]。而且,本研究發(fā)現(xiàn),超量表達(dá)不僅抑制活性IAA的積累,且促進(jìn)病原菌侵染后活性IAA水平的顯著下降。生長(zhǎng)素IAA被認(rèn)為是一種致病因子,具有促進(jìn)柑橘潰瘍病菌致病的作用[5,24]。這些結(jié)果暗示,通過(guò)?;秩静课换钚訧AA來(lái)正向調(diào)控柑橘對(duì)潰瘍病的抗性。
A:基因表達(dá)量聚類(lèi)熱圖 Clustering heat map of gene expression quantity;B:差異基因表達(dá)分析火山圖Volcano map of differentially expressed genes;C:KEGG富集圖 KEGG enrichment;D:MapMan可視化分析,紅色表示上調(diào),藍(lán)色表示下調(diào) MapMan visualization, red and blue indicate up-regulated and down-regulated, respectively
表1 b19轉(zhuǎn)基因植株中生長(zhǎng)素相關(guān)差異表達(dá)基因統(tǒng)計(jì)分析
*僅展示差異倍數(shù)|log2fold change|≥1的差異基因Differentially expressed genes with |log2fold change|≥1 are presented
圖8 生物脅迫相關(guān)差異基因統(tǒng)計(jì)分析
圖9 生長(zhǎng)發(fā)育相關(guān)差異基因統(tǒng)計(jì)分析
表2 b19轉(zhuǎn)基因植株中部分細(xì)胞壁相關(guān)的差異表達(dá)基因
病原菌侵染中膿皰形成是柑橘潰瘍病癥狀發(fā)展的關(guān)鍵[29]。病原菌通過(guò)分泌IAA或促進(jìn)寄主IAA的合成,調(diào)控寄主細(xì)胞的膨大和增生,進(jìn)而促進(jìn)膿皰的形成[5,24],以利于病原菌突出寄主表面向周?chē)乔秩窘M織擴(kuò)散。因此,植物表皮細(xì)胞在柑橘調(diào)控潰瘍病抗性中起著重要作用[30]。本研究對(duì)轉(zhuǎn)基因植株表皮細(xì)胞分析發(fā)現(xiàn),表皮細(xì)胞變短。表皮細(xì)胞變短暗示活性IAA的下調(diào)抑制了細(xì)胞的正常生長(zhǎng),這種細(xì)胞生長(zhǎng)的鈍化可能不利于病原菌侵染中膿皰的形成和擴(kuò)散。細(xì)胞壁是植物防御病原菌侵染的第一道物理屏障。轉(zhuǎn)錄組數(shù)據(jù)顯示,細(xì)胞壁合成相關(guān)基因表達(dá)水平明顯升高,特別是與增強(qiáng)細(xì)胞壁強(qiáng)度相關(guān)的角質(zhì)和蠟質(zhì)合成基因和表達(dá)水平上調(diào);相反,與細(xì)胞壁疏松相關(guān)的延展蛋白基因表達(dá)水平下調(diào)。結(jié)果暗示轉(zhuǎn)基因植株細(xì)胞壁強(qiáng)度增強(qiáng)。細(xì)胞壁的這種變化同樣有利于抑制膿皰的形成,增強(qiáng)植株的抗性[29]。綜上所述,超量表達(dá)抑制侵染部位活性IAA的積累,IAA含量的下降改變了細(xì)胞壁重構(gòu)相關(guān)基因的表達(dá)或蛋白質(zhì)的活性,進(jìn)而抑制寄主細(xì)胞壁松弛和細(xì)胞膨大,阻礙病原菌的入侵和擴(kuò)散,這可能是調(diào)控侵染部位膿皰形成的內(nèi)在機(jī)制。另外,本研究發(fā)現(xiàn)轉(zhuǎn)基因植株氣孔密度顯著增大。IAA負(fù)調(diào)控氣孔發(fā)育[31],抑制IAA的積累和極性運(yùn)輸會(huì)誘發(fā)氣孔簇生,比如擬南芥和突變體展現(xiàn)氣孔簇生狀[32-33]。轉(zhuǎn)錄組測(cè)序表明,轉(zhuǎn)基因植株中和表達(dá)水平顯著下降,與轉(zhuǎn)基因植株氣孔密度增大相關(guān)。氣孔是潰瘍病菌入侵柑橘的自然入口,有研究表明氣孔密度與柑橘品種潰瘍病抗性呈一定的負(fù)相關(guān)性,即氣孔密度越大,品種的抗病性越低[34-35]。因此,氣孔密度的增大可能會(huì)削弱超量表達(dá)對(duì)膿皰形成的抑制作用,影響轉(zhuǎn)基因植株抗性水平的進(jìn)一步提升。是如何從細(xì)胞發(fā)育水平影響病原菌侵染中膿皰的形成有待進(jìn)一步深入研究。
本研究顯示,超量表達(dá)增強(qiáng)了柑橘的抗病反應(yīng)。首先,激素分析表明,轉(zhuǎn)基因植株SA含量顯著增加,暗示SA介導(dǎo)的抗性反應(yīng)增強(qiáng)。在溫室和田間中,外施SA及其類(lèi)似物能有效增強(qiáng)柑橘對(duì)潰瘍病的抗性,已廣泛用于柑橘潰瘍病防治中[36]。同樣,超量表達(dá)SA信號(hào)下游的中心調(diào)節(jié)子基因顯著增強(qiáng)轉(zhuǎn)基因柑橘對(duì)潰瘍病的抗性,這種抗性增強(qiáng)與PR基因的表達(dá)增強(qiáng)緊密相關(guān)[37]。因此,轉(zhuǎn)基因植株中大量PR基因的上調(diào)表達(dá)暗示超量表達(dá)增強(qiáng)了SA介導(dǎo)的抗病反應(yīng)。其次,JA通常拮抗SA介導(dǎo)的抗病反應(yīng)[38],因此,超量表達(dá)轉(zhuǎn)基因植株JA含量降低有利于增強(qiáng)植物抗病性。有研究表明,侵染部位高濃度的IAA能抑制SA介導(dǎo)的抗病反應(yīng)[7,10-11],說(shuō)明轉(zhuǎn)基因植株中IAA積累的下降能促進(jìn)SA介導(dǎo)的抗病反應(yīng)。綜上,超量表達(dá)可能通過(guò)削弱或解除IAA和JA對(duì)SA抗病途徑的抑制,進(jìn)而增強(qiáng)植株的防御反應(yīng)。
超量表達(dá)對(duì)植株的形態(tài)發(fā)育造成了嚴(yán)重影響。轉(zhuǎn)基因植株呈現(xiàn)葉片向上卷曲,分枝增多,植株萎蔫,喪失頂端優(yōu)勢(shì)的表型,與擬南芥超量表達(dá)及其獲得性突變體植株表型相似[39]。生長(zhǎng)素是頂端優(yōu)勢(shì)形成的重要調(diào)控物質(zhì)。當(dāng)生長(zhǎng)素運(yùn)輸受阻或其信號(hào)通路受抑制,生長(zhǎng)素對(duì)側(cè)芽活動(dòng)的抑制解除,進(jìn)而導(dǎo)致植株頂端優(yōu)勢(shì)喪失,分枝增多,葉小而卷曲[40]。轉(zhuǎn)錄組測(cè)序顯示,轉(zhuǎn)基因植株中減少的自由IAA顯著抑制生長(zhǎng)素的運(yùn)輸和信號(hào)轉(zhuǎn)導(dǎo)途徑相關(guān)基因表達(dá),特別是所有注釋的AUX/IAA家族基因均顯著下調(diào)表達(dá)。AUX/IAA是生長(zhǎng)素早期響應(yīng)因子,通過(guò)泛素化途徑調(diào)控轉(zhuǎn)錄因子的表達(dá),進(jìn)而影響細(xì)胞大小和植株生長(zhǎng)發(fā)育[41]。當(dāng)細(xì)胞中IAA降低,AUX/IAA抑制ARF激活下游基因、、的表達(dá),最終抑制IAA的信號(hào)轉(zhuǎn)導(dǎo),影響植株的正常生長(zhǎng)[40]。這種抑制作用會(huì)進(jìn)一步抑制轉(zhuǎn)錄[41]。而和的表達(dá)下調(diào)說(shuō)明生長(zhǎng)素在植物體內(nèi)的分配受阻。另外,超量表達(dá)導(dǎo)致的自由IAA減少進(jìn)一步上調(diào)IAA糖基化和?;虮磉_(dá),這會(huì)進(jìn)一步抑制自由IAA的積累。這些結(jié)果清楚說(shuō)明,超量表達(dá)顯著抑制了IAA的積累和信號(hào)轉(zhuǎn)導(dǎo),進(jìn)而影響許多與細(xì)胞、組織和個(gè)體發(fā)育相關(guān)基因的表達(dá),改變植株的形態(tài)特征。
由于超量表達(dá)轉(zhuǎn)基因植株生長(zhǎng)發(fā)育受到影響,盡管已經(jīng)獲得了潰瘍病抗性提高的轉(zhuǎn)基因株系,但在柑橘潰瘍病抗性育種方面依然受到限制。一種解決策略是利用病原菌誘導(dǎo)型啟動(dòng)子驅(qū)動(dòng)的表達(dá),當(dāng)植物感染病原病菌時(shí)才會(huì)啟動(dòng)表達(dá),從而有利于獲得表型正常且具有潰瘍病抗性的轉(zhuǎn)基因柑橘[42]。
超量表達(dá)通過(guò)?;杂蒊AA來(lái)抑制生長(zhǎng)素信號(hào)轉(zhuǎn)導(dǎo),改變植物細(xì)胞的形態(tài)建成以利于寄主抗性的提升,同時(shí),解除IAA和JA對(duì)SA的拮抗,促進(jìn)SA介導(dǎo)的抗病反應(yīng),增強(qiáng)柑橘對(duì)潰瘍病的抗性,研究結(jié)果為利用生長(zhǎng)素信號(hào)轉(zhuǎn)導(dǎo)改良柑橘潰瘍病抗性提供了依據(jù)。
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Overexpression ofenhanced resistance to citrus canker disease by inhibiting auxin signaling transduction
Zou XiuPing, Long JunHong, Peng AiHong, Chen Min, Long Qin, Chen ShanChun
(National Center for Citrus Variety Improvement, Citrus Research Institute, Southwest University/Chinese Academy of Agricultural Sciences, Chongqing 400712)
【】Citrus canker, induced bysubsp(), is one of the most destructive disease in citrus production. Auxin plays an important role in regulating-induced pustule formation in citrus., an early auxin response gene, regulates plant hormone homeostasis through acylating indole-3-acetic acid (IAA). The previous study found thathad a vital role in response toinfection. 【】Here, to explore the internal mechanism of) overexpressingwere performed in this study.【】To evaluate resistance to citrus canker in transgenic plants overexpressing,fully expanded intact leaves gathered from transgenic plants were infected withby the pin-prick inoculation, and the diseased areas and disease severity index were investigated 10 d after inoculation, the wild type (WT) plant was used as the control. To detect hormone levels in transgenic plants, different hormones were isolated from leaves before and afterinfection and their contents were determined through high performance liquid chromatography (HPLC). Compared with WT plant, changes of phenotypes (plant types and leaf longitudinal diameter, transverse diameter and thickness) in transgenic plants were investigated in the greenhouse, and the length of epidermal cell and stomata density were further analyzed using optical microscopy. RNA sequencing was performed to investigate transcript changes in transgenic plant, and gene functions were annotated based on Nr, Nt, Pfam, COG, SwissProt and gene ontology (GO) databases. To elucidate the molecular mechanism ofwere investigated using the KEGG database and MapMan software.【】Overexpression ofsignificantly enhanced citrus canker resistance in transgenic plants. The branches of transgenic plants increased and drooped, the leaves curled upward, became smaller and lighter in color. The stomata density of transgenic plants increased and the length of epidermal cells became shorter. Hormone analyses showed that the contents of free auxin IAA and jasmonic acid (JA) in transgenic plants decreased significantly, while the content of salicylic acid (SA) increased significantly. Transcriptome sequencing showed that overexpression ofsignificantly inhibited the expression of auxin signal transduction related genes, especially the expression level of all the predicted Aux/IAA genes was down-regulated in transgenic plant. Conversely, the expression level of genes related to biological stress was up-regulated, most of which were pathogenesis-related genes. 【】Overexpression ofcan inhibit auxin signal transduction through acylating free IAA, regulate the homeostasis of JA and SA, change the morphogenesis of cells and plants, and finally enhance the plant resistance to citrus canker. The results suggest that the regulation of hormone homeostasis has potential value in citrus disease resistance breeding.
subsp(); citrus canker; auxin;; resistance
10.3864/j.issn.0578-1752.2019.21.009
2019-05-23;
2019-07-15
國(guó)家重點(diǎn)研發(fā)計(jì)劃(2018YFD1000300)、國(guó)家現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系建設(shè)專(zhuān)項(xiàng)資金(CARS-27)、重慶市自然科學(xué)基金(cstc2017jcyjBX0020)、廣東省科技創(chuàng)新戰(zhàn)略專(zhuān)項(xiàng)資金(2018B020202009)
鄒修平(通信作者),E-mail:zouxiuping@cric.cn
(責(zé)任編輯 岳梅)