柑橘綠霉病菌中多聚半乳糖醛酸酶基因(PdPG2)的表達(dá)分析

由書妍, 于瑞君, 劉紅霞, 李紅葉

(1. 大連市農(nóng)業(yè)科學(xué)研究院, 大連 116036; 2. 浙江大學(xué)農(nóng)業(yè)與生物技術(shù)學(xué)院, 杭州 310058)

柑橘綠霉病菌中多聚半乳糖醛酸酶基因(PdPG2)的表達(dá)分析

由書妍1, 于瑞君1, 劉紅霞1, 李紅葉2*

(1. 大連市農(nóng)業(yè)科學(xué)研究院, 大連 116036; 2. 浙江大學(xué)農(nóng)業(yè)與生物技術(shù)學(xué)院, 杭州 310058)

柑橘綠霉病菌Penicilliumdigitatum是儲(chǔ)藏期柑橘腐爛病最主要的病原之一,嚴(yán)重影響柑橘產(chǎn)業(yè)的發(fā)展。已有研究表明,柑橘綠霉病菌中多聚半乳糖醛酸酶(PdPG2)對(duì)其致病性有重要作用,PdPG2基因功能缺失突變株的致病性會(huì)下降,然而有關(guān)PdPG2基因的表達(dá)研究尚不完善。本文研究了PdPG2基因在不同條件下的表達(dá)情況,結(jié)果表明PdPG2是酸性表達(dá)基因,其表達(dá)量隨著pH的升高而降低,pH為3.0時(shí)其表達(dá)量為對(duì)照條件下的10倍,pH為8.0時(shí)其表達(dá)量為對(duì)照條件下的0.36倍。柑橘果膠能夠誘導(dǎo)PdPG2的表達(dá),其表達(dá)量為對(duì)照的3.6倍。因此,在侵染過(guò)程中PdPG2表達(dá)的升高是由于發(fā)病部位酸化以及橘皮降解物誘導(dǎo)共同引起的。

柑橘綠霉病菌; 多聚半乳糖醛酸酶; 基因表達(dá)

柑橘綠霉病菌引起的柑橘腐爛病是貯藏期柑橘的主要病害之一,其造成的損失通常占所有損失的90%以上,嚴(yán)重影響了我國(guó)柑橘的經(jīng)濟(jì)效益[1-2]。柑橘綠霉病菌不形成特定的侵染結(jié)構(gòu),主要通過(guò)采摘等農(nóng)事操作形成的傷口侵入。在侵入寄主的過(guò)程中,細(xì)胞壁水解酶起到了重要的作用[3-4]。目前,關(guān)于細(xì)胞壁水解酶對(duì)病原菌致病性的作用在許多真菌中都有報(bào)道,在灰葡萄孢Botrytiscinerea中,內(nèi)切多聚半乳糖醛酸酶基因缺失突變株對(duì)番茄的致病性降低[5],果膠甲酯酶基因缺失突變株對(duì)幾種作物的致病性均有所下降[6];紫麥角菌Clavicepspurpurea中,兩個(gè)多聚半乳糖醛酸酶基因缺失突變株對(duì)黑麥花組織致病性嚴(yán)重下降[7];在黃曲霉Aspergillusflavus中,內(nèi)切多聚半乳糖醛酸酶基因缺失突變株對(duì)棉鈴的致病性降低[8];在紅球叢赤殼Nectriahematococca中,果膠裂解酶A及果膠裂解酶D同時(shí)缺失其致病性降低,而單一突變其中任何一個(gè)基因都對(duì)致病性沒有影響[9]；在膠孢炭疽菌Colletotrichumgloeosporioides中,果膠裂解酶基因缺失突變株的致病性下降[10-11],可見細(xì)胞壁水解酶對(duì)病原菌的致病性有重要意義。

研究表明柑橘綠霉病菌中多聚半乳糖醛酸酶基因(PdPG2)對(duì)其致病性有重要的作用[12],該基因編碼378個(gè)氨基酸,屬于內(nèi)切多聚半乳糖醛酸酶(endo-PG,EC 3.2.1.15)。目前,關(guān)于PdPG2表達(dá)模式尚不清楚,本試驗(yàn)對(duì)柑橘綠霉病菌PdPG2基因的表達(dá)進(jìn)行研究,以探究該基因在侵染過(guò)程中表達(dá)量變化的原因,增加對(duì)柑橘綠霉病菌致病性分子機(jī)制的了解。

1 材料與方法

1.1 材料

柑橘綠霉病菌菌株P(guān)d01(CBS 130525)由本實(shí)驗(yàn)室從浙江衢州發(fā)生腐爛病的柑橘上分離[13],保存于本實(shí)驗(yàn)室;本試驗(yàn)使用的引物為自己設(shè)計(jì)(表1),由上海桑尼生物科技有限公司合成,其中PdPG2的擴(kuò)增片段為184 bp,Actin的擴(kuò)增片段為218 bp。

表1 本試驗(yàn)所用引物

Table 1 Primers for this study

引物名稱Primername序列(5'-3')SequencePdPG2-qFGAAGGAACCACCACTTTCGGPdPG2-qRTCAGGCTGTGAGCGTAGAAGAAActin-qFTCCACTACTGCCGAGCGTGAAATActin-qRCCGCCAGACTCAAGACCAAGAAC

1.2 方法

1.2.1PdPG2在侵染過(guò)程中的相對(duì)表達(dá)量分析

從市場(chǎng)上購(gòu)買成熟蜜橘Citrusnobilis果實(shí),在次氯酸中浸泡10 min后用無(wú)菌水沖洗并吹干備用。將PDA培養(yǎng)基上培養(yǎng)7 d的野生型菌株P(guān)d01孢子用雙蒸水洗脫,配制成1.0×106個(gè)/mL的孢子懸浮液。在蜜橘果實(shí)上用針簇(5根針圍成)刺1個(gè)1～2 mm深的傷口,取3 μL孢子懸浮液接種到傷口上,于25℃保濕培養(yǎng),每組試驗(yàn)3個(gè)重復(fù),分別在接種后12、24、48和96 h采用AxyPrepTMmultisource total RNA miniprep kit(Xygen,杭州)提取病斑組織的總RNA,并利用RNA PCR (AMV) 3.0 kit(TaKaRa,大連)反轉(zhuǎn)錄成cDNA。以cDNA為模板,PdPG2-qF/PdPG2-qR為引物,采用SYBR Premix ExTaqTM(Perfect Real Time)試劑盒配制PCR 反應(yīng)液,隨后在7300 Real-time PCR系統(tǒng)(ABI,美國(guó))上完成PCR反應(yīng)。PCR反應(yīng)以γ-Actin基因(GenBank, AB030227)為內(nèi)參,相對(duì)表達(dá)量的計(jì)算方法參考文獻(xiàn)[14]。

1.2.2 果膠對(duì)PdPG2相對(duì)表達(dá)量的影響

柑橘綠霉病菌的野生型菌株孢子懸浮液制作方法同1.2.1,吸取10 μL孢子懸浮液到50 mL PDB中培養(yǎng)3 d,過(guò)濾菌絲并用無(wú)菌水沖洗3次,將菌絲轉(zhuǎn)入以果膠為單一碳源的SM培養(yǎng)基中繼續(xù)培養(yǎng)60 h,按1.2.1的方法提取菌絲總RNA,測(cè)定PdPG2的相對(duì)表達(dá)量,以相同條件下,用葡萄糖為單一碳源的SM培養(yǎng)基培養(yǎng)的菌絲為對(duì)照。

1.2.3PdPG2在不同pH條件下的相對(duì)表達(dá)量

按1.2.2的方法收集在PDB中培養(yǎng)3 d的菌絲,將菌絲轉(zhuǎn)入pH分別為3.0、4.0、5.0、6.0、7.0 和8.0的PDB中繼續(xù)培養(yǎng)12 h,pH用磷酸檸檬酸緩沖液調(diào)節(jié)。按1.2.1的方法提取總RNA,測(cè)定PdPG2的相對(duì)表達(dá)量。

2 結(jié)果與分析

2.1PdPG2在侵染過(guò)程中的相對(duì)表達(dá)量

在柑橘發(fā)病過(guò)程中,PdPG2的表達(dá)如圖1所示,在侵染發(fā)病24 h后,PdPG2的相對(duì)表達(dá)量升高,為對(duì)照條件下的10倍,在侵染發(fā)病48 h后為對(duì)照條件下的14倍,在侵染發(fā)病72 h后,PdPG2的相對(duì)表達(dá)量最高,為對(duì)照條件下的18倍,在侵染發(fā)病96 h后,PdPG2的表達(dá)降低。這些結(jié)果表明PdPG2基因在侵染發(fā)病的過(guò)程中迅速升高,對(duì)致病性起到了重要的作用。

圖1 在侵染柑橘過(guò)程中PdPG2基因的表達(dá)Fig.1 Expression of PdPG2 during infection

2.2 果膠對(duì)PdPG2相對(duì)表達(dá)量的影響

試驗(yàn)結(jié)果(圖2)表明,柑橘綠霉病菌野生型菌株P(guān)d01在含果膠的培養(yǎng)基中培養(yǎng)60 h之后,PdPG2的相對(duì)表達(dá)量明顯升高,其表達(dá)量為對(duì)照條件下的3.6倍,可見果膠能夠誘導(dǎo)PdPG2的表達(dá)。

圖2 不同碳源下PdPG2的表達(dá)Fig.2 Expression of PdPG2 under different carbon source

2.3 不同pH條件對(duì)PdPG2表達(dá)的影響

PdPG2的表達(dá)與環(huán)境pH相關(guān),在酸性的條件下,PdPG2的表達(dá)明顯升高,在pH為3.0時(shí)其表達(dá)量為對(duì)照條件下的10倍,而在pH為8.0時(shí)其表達(dá)量為對(duì)照條件下的0.36倍(圖3),表明PdPG2的表達(dá)依賴于外界環(huán)境的酸性條件。

圖3 PdPG2基因在不同pH條件下的表達(dá)Fig.3 Expression of PdPG2 at different pH values

3 討論

柑橘綠霉病菌P.digitatum引起的柑橘腐爛病是貯藏期柑橘最重要的病害,給柑橘產(chǎn)業(yè)造成了嚴(yán)重的損失。研究柑橘綠霉病菌致病的分子機(jī)制,有助于增加對(duì)柑橘綠霉病菌防控的理論基礎(chǔ)。

本研究對(duì)PdPG2的表達(dá)模式進(jìn)行研究,在侵染過(guò)程中PdPG2的表達(dá)呈現(xiàn)出先升高后下降的趨勢(shì),表明在侵染過(guò)程中PdPG2起到了重要的作用。PdPG2的表達(dá)依賴于環(huán)境的酸性條件,在侵染的過(guò)程中,發(fā)病部位的pH會(huì)降低,特別是在接種后48 h內(nèi)[12],而這段時(shí)期也正是PdPG2相對(duì)表達(dá)量升高的時(shí)期,可見在柑橘綠霉病菌侵染過(guò)程中的酸化有利于PdPG2的表達(dá),從而提高原菌的致病性。PdPG2是一個(gè)誘導(dǎo)表達(dá)的基因,本試驗(yàn)發(fā)現(xiàn)果膠也能夠誘導(dǎo)PdPG2的表達(dá),果膠是柑橘果皮的重要組成成分[15],因此接種后PdPG2表達(dá)量的提高可能與發(fā)病部位的果皮降解物相關(guān)。在侵染過(guò)程中,PdPG2的表達(dá)呈現(xiàn)先升高后下降,而在接種后第48 h pH最低,此后,發(fā)病部位的pH基本不變[12],而PdPG2的表達(dá)卻繼續(xù)升高,因此我們推測(cè)橘皮果膠在誘導(dǎo)過(guò)程中也起了重要的作用。

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(責(zé)任編輯：楊明麗)

Expression profiles of polygalacturonase (PdPG2) inPenicilliumdigitatum

You Shuyan1, Yu Ruijun1, Liu Hongxia1, Li Hongye2

(1.DalianAcademyofAgriculturalSciences,Dalian116036,China; 2.CollegeofAgricultureandBiotechnology,ZhejiangUniversity,Hangzhou310058,China)

Penicilliumdigitatumis the most important pathogen causing green mold disease of postharvest citrus. Previous studies indicated thatPdPG2 played an important role in pathogenicity, and disruption ofPdPG2 resulted in attenuated virulence ofP.digitatum. However, the expression profiles ofPdPG2 were not well characterized. In this study, we investigated the expression profiles ofPdPG2. The results indicated thatPdPG2 was up-regulated under acidic conditions. The expression level ofPdPG2 was approximately 10 fold at pH 3.0 and 0.36 fold at pH 8.0 compared with that in the control. Pectin could induce the expression ofPdPG2 and the expression level was about 3.6 fold of that in the control. These results indicated that acidic condition and pectin could inducePdPG2 expression. The decreased pH of citrus rind during infection was suitable forPdPG2 expression, and pectin might be also helpful during infection.

Penicilliumdigitatum; polygalacturonase; gene expression

2016-04-18

2016-06-07

國(guó)家自然科學(xué)基金(31371961);國(guó)家現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系(CARS-27)

S 432.1

A

10.3969/j.issn.0529-1542.2017.02.023

* 通信作者 E-mail:hyli@zju.edu.cn