胡安華，祁靜靜，張慶雯，陳善春，鄒修平，許蘭珍，彭愛(ài)紅，雷天剛，姚利曉，龍琴，何永睿，李強(qiáng)

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柑橘潰瘍病相關(guān)基因的克隆與表達(dá)

胡安華，祁靜靜，張慶雯，陳善春，鄒修平，許蘭珍，彭愛(ài)紅，雷天剛，姚利曉，龍琴，何永睿，李強(qiáng)

（西南大學(xué)/中國(guó)農(nóng)業(yè)科學(xué)院柑桔研究所，重慶 400712）

【目的】克隆并分析其表達(dá)特性，轉(zhuǎn)化柑橘得到超表達(dá)轉(zhuǎn)基因株系，并進(jìn)行柑橘潰瘍病抗性評(píng)價(jià)，為柑橘潰瘍病分子育種提供理論依據(jù)?！痉椒ā繌耐礤\橙和四季橘中克隆柑橘，使用Mega6進(jìn)行多序列比對(duì)并構(gòu)建系統(tǒng)發(fā)育樹(shù)；采用在線軟件BaCelLo和SignalP 4.0進(jìn)行亞細(xì)胞定位和信號(hào)肽預(yù)測(cè)并用GFP瞬時(shí)表達(dá)確定CsPGIP在細(xì)胞內(nèi)的定位；利用實(shí)時(shí)熒光定量PCR（qRT-PCR）比較接種潰瘍病菌前后高感品種和高抗品種中柑橘的表達(dá)特性，分析潰瘍病菌侵染與表達(dá)的相關(guān)性；農(nóng)桿菌介導(dǎo)遺傳轉(zhuǎn)化晚錦橙，采用GUS染色初篩、PCR鑒定和qRT-PCR相結(jié)合的方法鑒定超表達(dá)轉(zhuǎn)基因株系；觀察轉(zhuǎn)基因和野生型株系表型變化，分析其株高、葉片表型；離體針刺法對(duì)超表達(dá)轉(zhuǎn)基因株系和野生型株系進(jìn)行柑橘潰瘍病抗性評(píng)價(jià)，統(tǒng)計(jì)病斑面積和病情指數(shù)，分析表達(dá)對(duì)柑橘抗、感潰瘍病的影響?！窘Y(jié)果】晚錦橙和四季橘均編碼328個(gè)氨基酸，與已報(bào)道的柑橘中的同源性高達(dá)99.39%，都包含2個(gè)基因典型的LRR結(jié)構(gòu)域（LRR_1和LRR_2）；構(gòu)建系統(tǒng)進(jìn)化樹(shù)發(fā)現(xiàn)甜橙中的CsPGIP與葡萄中的PGIP（GSVIVT01033370001）遺傳距離最近，相似度達(dá)到62.97%，推測(cè)CsPGIP與葡萄中的PGIP具有類(lèi)似的抗病效果。亞細(xì)胞定位和信號(hào)肽預(yù)測(cè)結(jié)果表明CsPGIP屬于分泌蛋白，GFP洋蔥瞬時(shí)表達(dá)證明柑橘CsPGIP定位在細(xì)胞膜和細(xì)胞壁，與預(yù)測(cè)結(jié)果一致。高感品種晚錦橙和高抗品種四季橘接種潰瘍病菌后的表達(dá)特性不同，在高感品種中表達(dá)顯著下調(diào)，而高抗品種中表達(dá)顯著上調(diào)且維持在較高水平，推測(cè)與柑橘潰瘍病的抗性相關(guān)。構(gòu)建超表達(dá)載體并轉(zhuǎn)化晚錦橙，通過(guò)PCR鑒定和qRT-PCR確定其中9個(gè)（OE1、OE3、OE4、OE5、OE6、OE9、OE10、OE12和OE14）為超表達(dá)陽(yáng)性株系。通過(guò)對(duì)轉(zhuǎn)基因株系的表型觀察發(fā)現(xiàn)OE3、OE14株系表型與野生型株系相比差異明顯，植株表現(xiàn)為較矮小，其中OE14出現(xiàn)葉片卷曲、增厚的表型變化。對(duì)超表達(dá)轉(zhuǎn)基因株系（8個(gè)株系）進(jìn)行離體抗?jié)儾≡u(píng)價(jià)，結(jié)果顯示超表達(dá)轉(zhuǎn)基因株系可以使柑橘潰瘍病病斑面積降至野生型的24.11%—83.88%，其中OE1株系的病斑面積最??；從病情指數(shù)來(lái)看，除OE3株系外，其余株系的病情指數(shù)均比野生型顯著下降（為野生型的23.12%—75.49%），其中OE1下降最顯著，綜上結(jié)果可知超表達(dá)可以有效抑制柑橘潰瘍病菌的生長(zhǎng)?！窘Y(jié)論】是柑橘響應(yīng)潰瘍病菌侵染的重要基因，可抑制或減輕柑橘潰瘍病的發(fā)病程度，在柑橘抗?jié)儾C(jī)理研究方面具有較大的應(yīng)用價(jià)值，也可作為柑橘抗?jié)儾》肿佑N的一個(gè)候選基因。

柑橘潰瘍??；多聚半乳糖醛酸酶抑制蛋白；；超表達(dá)；潰瘍病抗性

0 引言

【研究意義】柑橘是我國(guó)南方最重要的果樹(shù)作物，其中柑橘潰瘍?。╟itrus bacterial canker，CBC）是影響柑橘產(chǎn)業(yè)發(fā)展最為嚴(yán)重的病害之一。柑橘潰瘍病是由地毯黃單胞柑橘致病變種（subsp.，）引起的世界性檢疫病害[1-3]。目前為控制柑橘潰瘍病危害通常采取化學(xué)防治為主，生物防治為輔的綜合防治策略[4]。由于以上防治措施對(duì)環(huán)境不友好，需要投入大量的人力、物力，因此培育抗病新品種是減少柑橘潰瘍病危害的根本途徑。近年來(lái)日趨成熟的分子育種技術(shù)具有效率高、周期短、可對(duì)性狀進(jìn)行定向改良等優(yōu)點(diǎn)，愈來(lái)愈受到關(guān)注。通過(guò)分子育種挖掘潰瘍病相關(guān)的候選基因?qū)τ诟涕佼a(chǎn)業(yè)的發(fā)展具有重要意義。【前人研究進(jìn)展】多聚半乳糖醛酸酶抑制蛋白（polygalacturonase inhibitor protein，PGIP）基因是一個(gè)常用的抗病基因，陳波等通過(guò)挖掘、分析柑橘中的（登錄號(hào)：BAA31841.1）編碼蛋白質(zhì)序列，證明是一個(gè)編碼327個(gè)氨基酸并包含兩個(gè)富含亮氨酸重復(fù)序列（leucine-rich repeat，LRR）LRR-2、LRR-1的基因[5]。LRR結(jié)構(gòu)域在植物生長(zhǎng)發(fā)育和抗病反應(yīng)等方面發(fā)揮著重要作用[6]，與識(shí)別病原體的特異性有一定關(guān)系，且決定與配體結(jié)合的專(zhuān)一性[7]，PGIP通過(guò)抑制病原菌多聚半乳糖醛酸酶（polygalacturonase，PGs）的活性防止病原菌侵染植物組織[8-15]。大量的研究證明PGIP可提高植物對(duì)真菌病害的抗性，例如棉花[16]、煙草[9,17-18]、小麥[19]、擬南芥[8,20]、谷子[21-22]等。但是越來(lái)越多的研究發(fā)現(xiàn)PGIP在抗細(xì)菌病方面也發(fā)揮重要的作用。組成型表達(dá)梨后發(fā)現(xiàn)PcPGIP對(duì)細(xì)菌葉緣焦枯菌（）有明顯的抗性[23]；Hwang等在煙草和結(jié)球甘藍(lán)中轉(zhuǎn)入蕪菁的后，發(fā)現(xiàn)該基因增強(qiáng)了對(duì)細(xì)菌性病害軟腐病菌（）的抗性[14]；青枯菌（）中PGs的活性可被番茄莖中提取的PGIP強(qiáng)烈抑制[24]；紋枯病菌（）中PGs活性可以被水稻的原核表達(dá)產(chǎn)物抑制[25]；FENG等[26]的研究則發(fā)現(xiàn)超表達(dá)可增強(qiáng)水稻對(duì)條斑病菌（）的抗性，而抑制表達(dá)使水稻對(duì)條斑病更加敏感?！颈狙芯壳腥朦c(diǎn)】前期轉(zhuǎn)錄組研究發(fā)現(xiàn)，潰瘍病高感品種晚錦橙（）和高抗品種四季橘（）在感染潰瘍病菌前后表達(dá)差異顯著，推測(cè)可能與柑橘潰瘍病的抗性相關(guān)。【擬解決的關(guān)鍵問(wèn)題】以柑橘潰瘍病抗性品種四季橘和感性品種晚錦橙為材料，通過(guò)生物信息學(xué)分析、亞細(xì)胞定位、表達(dá)分析和轉(zhuǎn)基因功能驗(yàn)證等研究，探索與柑橘潰瘍病抗、感性的關(guān)系，為柑橘抗?jié)儾》肿佑N提供理論依據(jù)。

1 材料與方法

試驗(yàn)于2016年12月至2018年8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)家柑桔工程技術(shù)研究中心完成。

1.1 植物材料與病原菌

選取4年生晚錦橙和四季橘葉片（完全展開(kāi)的3個(gè)月葉齡的春稍葉片）、2年生資陽(yáng)香橙（）砧木為供試材料。材料取自西南大學(xué)溫網(wǎng)室和國(guó)家柑桔品種改良中心育種圃（19° 51′ N，106° 37′ E）。晚錦橙種子取自成熟果實(shí)，消毒后無(wú)菌條件下播種于MS培養(yǎng)基，3周后取上胚軸切成1 cm莖段作為轉(zhuǎn)化外植體；潰瘍病菌是由西南大學(xué)柑桔研究所保存的亞洲種A株系。

1.2 CsPGIP的克隆與分析

晚錦橙和四季橘RNA提取采用RNA快速提取試劑盒（Aidlab），并反轉(zhuǎn)錄為cDNA（TaKaRa）；根據(jù)Phytozome甜橙基因組[27]中基因序列（ID: orange1.1g020203m）設(shè)計(jì)特異引物OE-CsPGIP-f/r（表1）并分別以晚錦橙和四季橘cDNA為模板PCR擴(kuò)增；PCR產(chǎn)物連接pGEM-T easy載體（Promega）并轉(zhuǎn)化感受態(tài)菌株DH5（TaKaRa），陽(yáng)性克隆委托擎科生物有限公司測(cè)序；利用Mega6[28]進(jìn)行氨基酸多序列比對(duì)分析并繪制NJ系統(tǒng)發(fā)育樹(shù)。

1.3 CsPGIP的亞細(xì)胞定位

利用BaCelLo[29]和SignalP4.0[30]進(jìn)行CsPGIP的亞細(xì)胞定位和信號(hào)肽預(yù)測(cè)；根據(jù)序列設(shè)計(jì)不含終止密碼子的特異引物SCL-CsPGIP-f/r（表1）并以晚錦橙cDNA為模板進(jìn)行PCR擴(kuò)增，產(chǎn)物與pSAT6- mGFP-N1載體連接，構(gòu)建CsPGIP::mGFP融合基因，再將融合基因連接到pLGN-2x35s載體，最終得到pLGN::CsPGIP::mGFP載體；將含有pLGN::CsPGIP:: mGFP載體和pLGN::mGFP載體的EHA105農(nóng)桿菌（OD=0.1）注射洋蔥下表皮，28℃暗培養(yǎng)36 h，制片并用熒光顯微鏡（OLYMPUS：BX51）觀察明、暗視野下的表達(dá)情況。

下劃線標(biāo)注的為酶切位點(diǎn)The enzyme sites are marked with underline

1.4 柑橘潰瘍病菌對(duì)CsPGIP的誘導(dǎo)表達(dá)分析

將高感品種晚錦橙和高抗品種四季橘葉片用自來(lái)水清洗干凈并用75%的乙醇擦拭消毒，無(wú)菌水沖凈后置于無(wú)菌培養(yǎng)皿。將OD600=0.5的潰瘍病菌菌懸液注射到晚錦橙和四季橘葉片下表皮，對(duì)照組注射無(wú)菌LB液體培養(yǎng)基，于28℃光照培養(yǎng)。分別于0、12、24、36、48 h取樣，切取葉片的接種部位提取總RNA并反轉(zhuǎn)錄。根據(jù)基因特異性區(qū)域和柑橘內(nèi)參基因設(shè)計(jì)定量PCR引物qPCR-CsPGIP-f/r and qPCR-Actin-f/r（表1）。利用實(shí)時(shí)熒光定量PCR（quantitative real-time PCR，qRT-PCR）分析的相對(duì)表達(dá)量。每個(gè)處理進(jìn)行3次生物學(xué)重復(fù)和3次技術(shù)重復(fù)。

1.5 CsPGIP超表達(dá)載體構(gòu)建與轉(zhuǎn)化

將克隆的具有I和I酶切位點(diǎn)的片段和pLGNe-2×35S-MCS-nos超表達(dá)載體用I和I雙酶切，酶切后的基因片段和載體片段連接構(gòu)建pLGNe-CsPGIP-2×35S-MCS-nos超表達(dá)載體并轉(zhuǎn)化農(nóng)桿菌（EHA105）。柑橘轉(zhuǎn)化參照PENG等[31]的方法。含有重組質(zhì)粒的農(nóng)桿菌于LB液體培養(yǎng)基28℃培養(yǎng)至OD600=0.5，侵染晚錦橙外植體（1 cm上胚軸莖段）15 min，外植體用滅菌濾紙擦干后均勻擺放到共培養(yǎng)基（含2 mg·L-1IP、1 mg·L-1IAA和2,4-D、0.1 mg·L-1AS的MS培養(yǎng)基）于28℃暗培養(yǎng)，72 h后轉(zhuǎn)移到篩選培養(yǎng)基（含2 mg·L-1BA、1 mg·L-1IAA、50 mg·L-1Kana的MS培養(yǎng)基）于28℃暗培養(yǎng)，7 d后轉(zhuǎn)移到28℃光照培養(yǎng)。

1.6 轉(zhuǎn)基因株系鑒定

光照培養(yǎng)約50 d后，待不定芽生長(zhǎng)到1 cm左右，切取少量芽進(jìn)行GUS染色，顯色為藍(lán)色的芽初步認(rèn)定為擬轉(zhuǎn)化芽。初篩得到的擬轉(zhuǎn)化芽嫁接到砧木，待長(zhǎng)大后取其葉片提取基因組DNA，以此為模板用特異基因驗(yàn)證引物OE-f（35s）/OE-r（CsPGIP）（表1）進(jìn)行PCR鑒定。陽(yáng)性轉(zhuǎn)基因株系提取RNA并反轉(zhuǎn)錄為cDNA，利用qRT-PCR分析各株系中的表達(dá)量。

1.7 轉(zhuǎn)基因株系的抗性評(píng)價(jià)

轉(zhuǎn)基因株系的抗病性評(píng)價(jià)參照PENG等[32]的方法進(jìn)行。選取完全展開(kāi)的3個(gè)月葉齡轉(zhuǎn)基因株系及野生型晚錦橙葉片，進(jìn)行離體抗性評(píng)價(jià)。用接種針在每片葉片的背面刺4—6組孔，每組6個(gè)，每個(gè)針孔接種潰瘍病菌1 μL（OD600=0.5），同時(shí)對(duì)照組接種無(wú)菌LB培養(yǎng)基。28℃光照培養(yǎng)10 d，拍照記錄病斑。用軟件Image J V1.47（National Institutes of Health，Bethesda，MD）統(tǒng)計(jì)病斑面積（lesion area，LA，mm2）。按照病斑面積大小將病情分為8個(gè)級(jí)別，用字母LA表示病斑面積，0級(jí)（LA≤0.5 mm2），1級(jí)（0.5 mm2＜LA≤1.0 mm2），2級(jí)（1.0 mm2＜LA≤1.5 mm2），3級(jí)（1.5 mm2＜LA≤2.0 mm2），4級(jí)（2.0 mm2＜LA≤2.5 mm2），5級(jí)（2.5 mm2＜LA≤3.0 mm2），6級(jí)（3.0 mm2＜LA≤3.5 mm2），7級(jí)（LA＞3.5 mm2）。根據(jù)以下公式計(jì)算病情指數(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)）。

1.8 qRT-PCR與統(tǒng)計(jì)分析

相對(duì)表達(dá)量采用2-ΔΔCt法（ΔCt = CtCsPGIP-CtActin）計(jì)算，使用Excel進(jìn)行數(shù)據(jù)統(tǒng)計(jì)分析并繪圖。＜0.05表示差異顯著，＜0.01表示差異極顯著。

2 結(jié)果

2.1 CsPGIP生物信息學(xué)分析

晚錦橙和四季橘的Cs編碼的CsPGIP均含有328個(gè)氨基酸，與已報(bào)道柑橘PGIP（BAA31841.1）[5]同源性為99.39%，3個(gè)基因編碼的PGIP均含有PGIP關(guān)鍵結(jié)構(gòu)域LRR_1和LRR_2，屬于同源基因（圖1）。

通過(guò)對(duì)CsPGIP與其他8個(gè)物種（擬南芥、高粱、水稻、亞麻屬、苜蓿、楊樹(shù)、谷子和葡萄）共38條PGIP序列進(jìn)行系統(tǒng)發(fā)育分析，結(jié)果顯示不同物種間PGIP序列具有很強(qiáng)的保守性，相同物種具有較高的相似度；單子葉和雙子葉植物單獨(dú)聚在一起，分成兩個(gè)大組；柑橘CsPGIP與葡萄PGIP（GSVIVT01033370001）遺傳距離最近，相似度達(dá)到62.97%（圖2）。

2.2 CsPGIP亞細(xì)胞定位

利用BaCelLo進(jìn)行CsPGIP亞細(xì)胞定位預(yù)測(cè)，定位于細(xì)胞膜上的預(yù)測(cè)分值（2.272）顯著高于其他部位（≤1.494），CsPGIP可能定位在細(xì)胞膜上。信號(hào)肽預(yù)測(cè)結(jié)果顯示其N(xiāo)端有含23個(gè)氨基酸的信號(hào)肽：MSNTSLLSLFFFLCLCISPSLSD，表明CsPGIP為分泌蛋白。為驗(yàn)證亞細(xì)胞定位和信號(hào)肽的預(yù)測(cè)，以柑橘與構(gòu)建融合表達(dá)載體，通過(guò)洋蔥表皮瞬時(shí)表達(dá)進(jìn)行亞細(xì)胞定位，顯微觀察顯示融合蛋白定位在細(xì)胞膜和細(xì)胞壁結(jié)合部（圖3 A1-A3），進(jìn)一步進(jìn)行質(zhì)壁分離后觀察顯示融合蛋白在細(xì)胞膜和細(xì)胞壁中都有積累（圖3 B1-B3），而對(duì)照組定位在整個(gè)細(xì)胞中（圖3 C1-C3、D1-D3）。CsPGIP定位在細(xì)胞膜和細(xì)胞壁中的觀察結(jié)果與預(yù)測(cè)一致。

深藍(lán)色為相同氨基酸序列，淺藍(lán)色為不同的氨基酸序列，LRR_1和LRR_2為L(zhǎng)RR結(jié)構(gòu)域

以上基因均來(lái)自Phytozome（http://www.phytozome.com/）基因組數(shù)據(jù)庫(kù)Genes in this study are all from Phytozome (http://www.phytozome.com/)

A1：明視野觀察CsPGIP-GFP融合蛋白Image of CsPGIP-GFP under bright field；A2：暗視野觀察CsPGIP-GFP融合蛋白Image of CsPGIP-GFP under dark field；A3：A1、A2視野疊加Overlap of A1 and A2；B1：明視野觀察CsPGIP-GFP融合蛋白質(zhì)壁分離Image of CsPGIP-GFP under bright field (plasmolysis)；B2：暗視野觀察CsPGIP-GFP融合蛋白質(zhì)壁分離Image of CsPGIP-GFP under dark field (plasmolysis)；B3：B1、B2視野疊加Overlap of B1 and B2；C1：明視野觀察GFP表達(dá)Image of GFP under bright field；C2：暗視野觀察GFP表達(dá)Image of GFP under dark field；C3：C1、C2視野疊加Overlap of C1 and C2；D1：明視野觀察GFP質(zhì)壁分離GFP of plasmolysis under bright field；D2：暗視野觀察GFP質(zhì)壁分離GFP of plasmolysis under dark field；D3：D1、D2視野疊加Overlap of D1 and D2；標(biāo)尺Scale：100 μm

2.3 柑橘潰瘍病菌對(duì)CsPGIP的誘導(dǎo)表達(dá)分析

實(shí)時(shí)熒光定量PCR結(jié)果分析顯示柑橘在5個(gè)時(shí)間點(diǎn)（0、12、24、36和48 h）表達(dá)水平存在不同程度的差異（圖4），其中高感品種晚錦橙在接種潰瘍病菌12 h后的表達(dá)出現(xiàn)顯著下調(diào)并維持在較低的水平。而高抗品種四季橘在接種潰瘍病菌后表達(dá)出現(xiàn)不同程度上調(diào)，在12 h時(shí)表達(dá)量最高，為0 h的2.91倍，12 h后仍維持在較高水平。結(jié)果表明表達(dá)與潰瘍病菌的侵染具有密切關(guān)系，經(jīng)潰瘍病菌誘導(dǎo)而顯著上調(diào)可能是四季橘抗?jié)儾〉脑蛑弧?/p>

2.4 轉(zhuǎn)基因株系的鑒定及CsPGIP表達(dá)分析

經(jīng)GUS染色初篩（結(jié)果未顯示）結(jié)合PCR鑒定，共獲得9個(gè)轉(zhuǎn)基因株系，分別為OE1、OE3、OE4、OE5、OE6、OE9、OE10、OE12和OE14（圖5-A）。以轉(zhuǎn)基因株系和野生型對(duì)照同期葉片提取RNA，qRT-PCR進(jìn)行表達(dá)量測(cè)定，相對(duì)于野生型對(duì)照，以上9個(gè)株系表達(dá)量均出現(xiàn)不同程度的上調(diào)表達(dá)，其中OE10上調(diào)表達(dá)最高（圖5-B）。

不同小寫(xiě)字母表示差異顯著（P＜0.05）Different lowercases indicate significant difference (P＜0.05)

A：CsPGIP轉(zhuǎn)基因株系的PCR鑒定PCR amplification of CsPGIP in over-expression transgenic lines；B：轉(zhuǎn)基因株系中CsPGIP的相對(duì)表達(dá)量檢測(cè)The relative expression level of CsPGIP in over-expression transgenic lines。 M：分子量標(biāo)準(zhǔn)Marker；OE1—OE14：Gus初篩的轉(zhuǎn)基因材料lines verified from Gus staining；WT：野生型wild-type；陽(yáng)性株系特異擴(kuò)增條帶為1 530 bp PCR product size of positive lines is 1 530 bp

2.5 轉(zhuǎn)基因株系的表型分析

觀察分析9株轉(zhuǎn)基因株系表型，3個(gè)樹(shù)齡一年的株系OE1、OE3和OE4中，OE3與野生型對(duì)照差異明顯，植株較矮?。▓D6-A、6-D）。7個(gè)樹(shù)齡6個(gè)月的株系OE5、OE6、OE9、OE10、OE12和OE14與野生型比較，OE14株系出現(xiàn)了異常，植株矮?。▓D6-B、6-E）、葉片卷曲、增厚（圖6-C）。

A：樹(shù)齡1年的轉(zhuǎn)基因株系（OE1、OE3、OE4）和野生型對(duì)照（WT1）植株The phenotype of 1-year-old transgenic lines (OE1, OE3, OE4) and the wild-type control (WT1)；B：樹(shù)齡6個(gè)月的轉(zhuǎn)基因株系（OE5、OE6、OE9、OE10、OE12、OE14）和野生型對(duì)照（WT2）植株P(guān)henotype of 6-month-old transgenic lines (OE5, OE6, OE9, OE10, OE12, OE14) and the wild-type control (WT2)；C：野生型對(duì)照WT2和轉(zhuǎn)基因株系OE14的葉片Leaves of WT2 and OE14；D：樹(shù)齡1年的轉(zhuǎn)基因株系（OE1、OE3、OE4）和野生型對(duì)照（WT1）的株高（測(cè)量方法：從嫁接口到頂梢的距離）Height of 1-year-old transgenic lines (OE1, OE3, OE4) and the wild-type control (measurement method: distance from the graft to the top tip)；E：樹(shù)齡6個(gè)月的轉(zhuǎn)基因株系（OE5、OE6、OE9、OE10、OE12、OE14）和野生型對(duì)照（WT2）的株高Height of 6-month-old transgenic lines (OE5, OE6, OE9, OE10, OE12, OE14) and the wild-type control

2.6 轉(zhuǎn)基因株系的潰瘍病抗性評(píng)價(jià)

對(duì)8個(gè)轉(zhuǎn)基因株系（OE1、OE3、OE4、OE5、OE6、OE9、OE10和OE12）進(jìn)行了抗病性評(píng)價(jià)。采用針刺法離體接種潰瘍病菌，以接種LB培養(yǎng)基的葉片作為對(duì)照。10 d后，接種LB培養(yǎng)基的葉片均未發(fā)病（圖7-A），而接種潰瘍病菌的葉片均不同程度發(fā)病，病斑大小存在一定的差異（圖7-B）；經(jīng)過(guò)統(tǒng)計(jì)分析，轉(zhuǎn)基因株系病斑面積顯著小于野生型對(duì)照（圖7-C），僅為野生型對(duì)照病斑面積的24.11%—83.88%；轉(zhuǎn)基因株系病情指數(shù)僅為野生型對(duì)照的23.12%—86.52%（圖7-D）。從轉(zhuǎn)基因株系接種潰瘍病菌抗性評(píng)價(jià)結(jié)果得出株系OE1、OE4、OE5、OE6、OE9、OE10和OE12可顯著減小葉片潰瘍病病情指數(shù)，其中OE1株系對(duì)柑橘潰瘍病抗性得到極顯著提高。

3 討論

植物細(xì)胞壁是抵御病菌入侵的第一道防線，病原細(xì)菌和真菌必須通過(guò)植物細(xì)胞壁在植物體內(nèi)建立生物營(yíng)養(yǎng)感染的定殖位點(diǎn)后進(jìn)行擴(kuò)大感染[33]。PGIP是植物細(xì)胞壁產(chǎn)生的LRR類(lèi)防御蛋白，能特異性的抑制病原菌分泌的PGs，從而抑制病原菌對(duì)植株的侵染。LRR基序是參與蛋白質(zhì)之間互作的結(jié)構(gòu)域[34]，PGIP通過(guò)LRR基序抑制PGs的活性[35]。有研究表明PGIP在多種物種中對(duì)提高病害的抗性有顯著作用，棉花可增強(qiáng)植株對(duì)黃萎病和鐮孢菌枯萎病的抗性[16]；過(guò)表達(dá)增強(qiáng)了結(jié)球甘藍(lán)對(duì)細(xì)菌性軟腐病的抗性[14]；CaPGIPs在植物的抗病方面起著重要作用[36]；葡萄VvPGIP1可以降低轉(zhuǎn)基因煙草對(duì)灰霉病菌的敏感性，并對(duì)病原菌的PGs有不同程度的抑制作用[17]。本研究結(jié)果表明，晚錦橙中的超量表達(dá)可增強(qiáng)柑橘對(duì)潰瘍病菌的抗性。

A：接種LB培養(yǎng)基的轉(zhuǎn)基因株系和野生型對(duì)照葉片Disease spots of transgenic lines and the wild-type inoculated with LB；B：接種潰瘍病菌的轉(zhuǎn)基因株系和野生型對(duì)照葉片Disease spots of transgenic lines and the wild-type inoculated with Xcc；C：接種潰瘍病菌的轉(zhuǎn)基因株系和野生型對(duì)照病斑面積Lesion area of transgenic lines and the wild-type inoculated with Xcc；D：接種潰瘍病菌的轉(zhuǎn)基因株系和野生型對(duì)照病情指數(shù)Disease index of transgenic lines and the wild-type inoculated with Xcc。WT：野生型對(duì)照wild-type control；OE1—OE12：轉(zhuǎn)基因株系transgenic lines。*表示差異顯著（P＜0.05），**表示差異極顯著（P＜0.01）* represents significant difference (P＜0.05), ** represents extremely significant difference (P＜0.01)

在潰瘍病菌的誘導(dǎo)下，在高感品種晚錦橙中下調(diào)表達(dá)而在高抗品種四季橘中顯著上調(diào)表達(dá)。晚錦橙和四季橘中的CsPGIP蛋白僅存在3個(gè)氨基酸的差異，但它們具有相同的LRR類(lèi)防御蛋白特有的結(jié)構(gòu)域LRR_1和LRR_2（圖1），因而這兩種蛋白本身對(duì)病原菌的抵抗能力可能差異不大。導(dǎo)致在不同潰瘍病抗性的柑橘品種中差異表達(dá)的原因可能是調(diào)控機(jī)制的差異。柑橘抵抗?jié)儾【娜肭质且粋€(gè)復(fù)雜的調(diào)控網(wǎng)絡(luò)。潰瘍病病原菌主要的效應(yīng)因子pthA4通過(guò)III型分泌系統(tǒng)進(jìn)行柑橘基因組后，與柑橘體內(nèi)的潰瘍病感病基因結(jié)合[37]。研究表明，不同潰瘍病抗性的柑橘品種中都含有，但在不同抗性的柑橘品種中存在啟動(dòng)子序列的差異[31]，這種啟動(dòng)子序列的差異可能會(huì)引起基因表達(dá)的差異。在不同的抗、感潰瘍病柑橘品種中，雖然相同，但其轉(zhuǎn)錄后可能存在轉(zhuǎn)錄后修飾現(xiàn)象，轉(zhuǎn)錄呈現(xiàn)多態(tài)性，這種轉(zhuǎn)錄后的修飾也會(huì)導(dǎo)致在不同抗性的柑橘品種中表達(dá)的差異。因而進(jìn)一步克隆晚錦橙和四季橘中的啟動(dòng)子，分析啟動(dòng)子序列差異；同時(shí)對(duì)不同潰瘍病抗性的柑橘品種中轉(zhuǎn)錄的結(jié)構(gòu)多態(tài)性進(jìn)行研究，有望闡明在不同潰瘍病抗性的柑橘品種中差異表達(dá)的原因。

本研究對(duì)9個(gè)轉(zhuǎn)基因株系進(jìn)行表型分析，發(fā)現(xiàn)僅有兩個(gè)轉(zhuǎn)基因株系出現(xiàn)了植株矮小的現(xiàn)象，其中一個(gè)株系（OE14）的葉片卷曲增厚。多個(gè)物種的已在不同的植株中進(jìn)行超表達(dá)，但轉(zhuǎn)基因并未引起植株表型的差異[16-26]。本研究中轉(zhuǎn)基因植株表型變化可能是隨機(jī)整合到柑橘基因組中時(shí)引起某些基因或調(diào)控序列失活造成的。由于僅有兩個(gè)株系出現(xiàn)了表型變化且其中一株過(guò)于矮小無(wú)法進(jìn)行表型相關(guān)研究，后期將會(huì)對(duì)潰瘍病抗性評(píng)價(jià)、插入位點(diǎn)、基因表達(dá)和細(xì)胞組織結(jié)構(gòu)進(jìn)行綜合研究，以探究對(duì)植物生長(zhǎng)發(fā)育的影響。

潰瘍病抗性評(píng)價(jià)結(jié)果顯示不同的轉(zhuǎn)基因株系可不同程度顯著減小葉片病情指數(shù)，其中OE1株系對(duì)柑橘潰瘍病抗性極顯著提高且表型正常。目前對(duì)柑橘潰瘍病抗性機(jī)理尚未清楚，因而篩選出的抗?jié)儾〉霓D(zhuǎn)基因柑橘可以作為材料進(jìn)一步研究的作用機(jī)理。

4 結(jié)論

CsPGIP為定位于細(xì)胞壁和細(xì)胞膜的蛋白，受潰瘍病菌誘導(dǎo)表達(dá)。的表達(dá)特性表明該基因是柑橘響應(yīng)潰瘍病侵染的重要基因，超表達(dá)該基因可以提高柑橘對(duì)潰瘍病的抗性，該基因在柑橘抗?jié)儾C(jī)理研究方面具有較大的應(yīng)用價(jià)值，可作為柑橘抗?jié)儾》肿佑N的一個(gè)候選基因。

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（責(zé)任編輯 岳梅）

Cloning and Expression Analysis of the Citrus Bacterial Canker-Related Genein Citrus

HU AnHua, QI JingJing, ZHANG QingWen, CHEN ShanChun, ZOU XiuPing, XU LanZhen, PENG AiHong, LEI TianGang, YAO LiXiao, LONG Qin, HE YongRui, LI Qiang

(Citrus Research Institute, Southwest University/Chinese Academy of Agricultural Sciences, Chongqing 400712)

【Objective】The objective of this study is to cloneand analyze its expression characteristics, constructtransgenic citrus and evaluate the resistance to citrus bacterial canker (CBC), and to provide a theoretical basis for molecular breeding of citrus bacterial canker.【Method】was annotated from the genomic databases and cloned from Wanjincheng and Calamondin. Mega6 was used for multiple sequence alignment and phylogenetic tree was constructed. Two online softwares BaCelLo and SignalP 4.0 were used for the prediction of subcellular localization and signal peptide. The predicted result was then demonstrated by GFP transient expression. The expression profile ofinduced bysubsp.() was also analyzed in Wanjincheng and Calamondin by using qRT-PCR method. The correlation betweeninfection andexpression was analyzed. Genetic transformation of Wanjincheng was conducted by-mediated method. The over-expressed lines were identified by Gus staining, PCR and qRT-PCR. The phenotypic changes of transgenic and wild-type lines were observed, plant height and leaf phenotype were analyzed.acupuncture was used to evaluate the resistance of transgenic lines and wild-type lines to citrus bacterial canker. The effect ofexpression on resistance and susceptibility to citrus bacterial canker was analyzed by statistical analysis of lesion area (LA) and disease index (DI). 【Result】Thecloned from Wanjincheng and Calamondin encodes 328 amino acids, which is 99.39% homology with the reportedfrom Clementina, and contains two typical LRR domains (LRR_1 and LRR_2). In the phylogenetic tree, the genetic distance between CsPGIP and grape PGIP (GSVIVT01033370001) was the closest, and the similarity was 62.97%. It is inferred that CsPGIP and grape PGIP have similar resistance to disease. The prediction of subcellular localization and signal peptide indicated that CsPGIP was a secretory protein, and GFP transient expression proved that CsPGIP located on cell membrane and cell wall, which was consistent with the predicted results. The expression ofin canker sensitive plant Wanjincheng and canker resistant plant Calamondin was different after inoculated with. the expression ofwas significantly down-regulated in Wanjincheng, but significantly up-regulated and maintained at a high level in Calamondin. It is speculated thatwas related to resistance to citrus bacterial canker.over-expression vector was constructed and transformed into Wanjincheng, and nineover-expression lines (OE1, OE3, OE4, OE5, OE6, OE9, OE10, OE12 and OE14) were identified asover-expression positive lines by PCR identification and qRT-PCR. Through the phenotypic observation of transgenic lines, it was found that the phenotypes of OE3 and OE14 lines were significantly different from those of wild-type lines. The plant was short, in which OE14 was also abnormal with curly property and greater thickness. Thecanker resistance of eightover-expression lines was evaluated. The results showed that the lesion area on the eightover-expression lines was smaller compared to that on the wild-type (24.11%-83.88%), and the lesion area of OE1 was the smallest. In terms of disease index, the disease index ofover-expression lines (except OE3) was significantly lower than that of wild-type (23.12%-75.49%), and the decrease of OE1 was the most significant. The above results showed that over-expression ofcould effectively inhibit the growth of citrus bacterial canker.【Conclusion】is an important gene which can inhibit or reduce the incidence of citrus bacterial canker, and has a great application value in the mechanism study of citrus resistance to bacterial canker. In the same time, it can be used as a candidate gene for molecular breeding of citrus bacterial cankerresistance.

citrus bacterial canker (CBC); polygalacturonase inhibitor protein;; over-expression; CBC resistance

10.3864/j.issn.0578-1752.2019.04.006

2018-10-13；

2018-11-26

國(guó)家現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系建設(shè)資金（CARS-26）、重慶市社會(huì)事業(yè)與民生體系保障科技創(chuàng)新專(zhuān)項(xiàng)（cstc2016shms-ztzx80001，cstc2017shms-xdny80051）、廣西科技重大專(zhuān)項(xiàng)（桂科AA18118046）

胡安華，E-mail：782497097@qq.com。 通信作者李強(qiáng)，E-mail：liqiang@cric.cn。通信作者何永睿，E-mail：heyongrui@cric.cn