姜紅，畢陽，李昌健，王毅，李生娥，劉耀娜，王斌

（甘肅農(nóng)業(yè)大學(xué)食品科學(xué)與工程學(xué)院，蘭州 730070）

馬鈴薯品種‘青薯168’和‘隴薯3號’塊莖愈傷能力的比較

姜紅，畢陽，李昌健，王毅，李生娥，劉耀娜，王斌

（甘肅農(nóng)業(yè)大學(xué)食品科學(xué)與工程學(xué)院，蘭州 730070）

【目的】研究抗/感干腐病的馬鈴薯品種‘青薯168’和‘隴薯3號’塊莖間愈傷能力的差異，從木質(zhì)素與軟木脂積累以及苯丙烷代謝角度探討導(dǎo)致差異的原因?！痉椒ā恳钥共∑贩N‘青薯168’和感病品種‘隴薯3號’塊莖為試材，人工模擬損傷后置于常溫下進(jìn)行愈傷，通過測定損傷塊莖愈傷期間失重率的變化和塊莖在不同愈傷時期接種硫色鐮孢(Fusarium sulphureum)后病情指數(shù)的變化評價愈傷效果，利用間苯三酚-HCl和甲苯胺藍(lán)-中性紅染色法分別觀察塊莖損傷部位木質(zhì)素與軟木脂的形成,并量化木質(zhì)化細(xì)胞層厚度和軟木脂熒光強(qiáng)度以說明木質(zhì)素和軟木脂的沉積狀況，通過測定苯丙烷代謝途徑的關(guān)鍵酶和產(chǎn)物即苯丙氨酸解氨酶活性和木質(zhì)素、總酚與類黃酮含量的變化，比較兩個品種塊莖愈傷能力的不同。【結(jié)果】愈傷期間，‘青薯168’的失重率顯著低于‘隴薯3號’。第14天時，‘青薯168’和‘隴薯3號’的失重率分別為0.93%和1.96%，‘青薯168’的失重率比‘隴薯3號’低52.3%。同樣，塊莖在不同愈傷時期接種硫色鐮孢后，‘青薯168’的病情指數(shù)也顯著低于‘隴薯3號’。愈傷第3天接種，‘青薯168’和‘隴薯3號’的病情指數(shù)為20.36和71.59，前者的病情指數(shù)比后者低71.5%。愈傷期間，‘青薯168’的木質(zhì)素與軟木脂積累速率顯著高于‘隴薯3號’。第14天時，‘青薯168’的木質(zhì)化細(xì)胞層厚度和軟木脂熒光總強(qiáng)度分別高出‘隴薯3號’47.4%和60.6%。此外，‘青薯168’愈傷期間表現(xiàn)出較高的苯丙烷代謝活性，其苯丙氨酸解氨酶活性和木質(zhì)素含量在第14天時分別高出‘隴薯3號’77%和65%。兩個品種的總酚含量在愈傷后期也存在差異，第14天時，‘青薯168’的總酚含量高出‘隴薯3號’76%。但兩個品種間類黃酮含量在愈傷期間的變化不明顯?！窘Y(jié)論】抗病品種‘青薯168’的愈傷能力顯著高于感病品種‘隴薯3號’，抗病品種所具備的良好愈傷能力與其高活力的苯丙烷代謝密切相關(guān)。

馬鈴薯；品種；塊莖；愈傷；苯丙烷代謝

0 引言

【研究意義】馬鈴薯（Solanum tuberosum）在中國糧菜供給中具有重要地位[1]。但其塊莖貯藏期間的腐爛損失頗為嚴(yán)重，由各類鐮刀菌引起的干腐病是導(dǎo)致馬鈴薯腐爛的重要病害，塊莖腐爛不僅造成巨大的經(jīng)濟(jì)損失，而且會積累真菌毒素，帶來食用的安全隱患[2]。采收時在馬鈴薯塊莖表面形成的大量傷口是致腐病原物侵入的主要通道[3]，但塊莖可通過形成愈傷組織來阻止病原物的侵入[4-5]。調(diào)查發(fā)現(xiàn)，不同品種的馬鈴薯如‘隴薯3號’、‘隴薯5號’和‘隴薯6號’等在貯藏期間的感病程度不同，耐貯性也就不同[6]。因此，研究不同抗性品種馬鈴薯塊莖的愈傷能力，對于闡明不同品種塊莖在貯藏期間的病害程度差異具有重要意義，也可為塊莖貯藏提供理論依據(jù)?！厩叭搜芯窟M(jìn)展】不同品種塊根和塊莖的愈傷能力存在較大差異。例如，‘Kemb 10’和‘SPK004’兩個甘薯品種在愈傷期間不能連續(xù)形成木質(zhì)層，木質(zhì)化評分較低，抗病性不強(qiáng)。而‘Yanshu 1’和‘BP1-SP-2’兩個品種則可連續(xù)形成木質(zhì)層，木質(zhì)化評分較高，抗病性較強(qiáng)[7]。AMAND等[8]分析了18個受損甘薯品種塊根愈傷期間的乙烯釋放量，發(fā)現(xiàn)不同甘薯品種間乙烯釋放量、細(xì)胞木質(zhì)化和傷口周皮的形成均存在顯著差異，且乙烯釋放量與細(xì)胞的木質(zhì)化和周皮的形成存在一定的相關(guān)性。DASTMALCHI等[9-10]研究發(fā)現(xiàn)，不同品種馬鈴薯塊莖愈傷組織中的極性和非極性提取物存在差異，品種‘Yukon Gold’的提取物中因富含酚類和長鏈脂肪酸而有利于軟木脂的合成，而‘Norkotah Russet’和‘Atlantic’等品種的提取物中因缺乏這些物質(zhì)而阻礙了軟木脂的形成?！颈狙芯壳腥朦c】針對單一或不同品種馬鈴薯塊莖愈傷組織形成的生理生化及分子生物學(xué)研究已有報道，但對抗/感馬鈴薯品種間塊莖愈傷能力的綜合比較卻未見報道?！緮M解決的關(guān)鍵問題】以西北產(chǎn)區(qū)兩個對干腐病抗性差異較大的馬鈴薯品種‘青薯168’（抗?。┖推贩N‘隴薯3號’（感?。樵嚥模ㄟ^人工模擬創(chuàng)傷，在常溫下進(jìn)行愈傷，從組織化學(xué)和生理生化水平比較兩個品種塊莖愈傷期間的愈傷效果、木質(zhì)素和軟木脂積累以及苯丙烷代謝關(guān)鍵酶活性及其產(chǎn)物含量的差異，以期為馬鈴薯的塊莖愈傷提供理論依據(jù)。

1 材料與方法

試驗于2014—2015年在甘肅農(nóng)業(yè)大學(xué)食品學(xué)院采后生物學(xué)與技術(shù)實驗室完成。

1.1 材料與儀器

供試馬鈴薯品種‘青薯168’與‘隴薯3號’于2014年10月采自甘肅省渭源縣會川鎮(zhèn)，塊莖裝入網(wǎng)袋后當(dāng)天運抵甘肅農(nóng)業(yè)大學(xué)食品學(xué)院采后生物學(xué)與技術(shù)實驗室，于常溫（20—25℃，RH 70%—80%）下貯藏備用。供試硫色鐮孢（Fusarium sulphureum）由甘肅省農(nóng)業(yè)科學(xué)院植物保護(hù)研究所提供，PDA培養(yǎng)基上保存待用。

超凈工作臺（蘇凈集團(tuán)蘇州安泰空氣技術(shù)有限公司）；立式壓力蒸汽滅菌鍋（上海申安醫(yī)療器械廠）；恒溫培養(yǎng)箱（上海一恒科技有限公司）；正置萬能顯微鏡（OLYMPUS公司）；紫外-可見光分光光度計（日本島津）；臺式高速冷凍離心機(jī)（長沙湘儀離心機(jī)有限公司）。

1.2 方法

1.2.1 塊莖創(chuàng)傷 參考包改紅等[11]的方法并作修改。選取外觀整齊，大小一致，無病蟲害，無機(jī)械損傷的馬鈴薯塊莖，用1%的次氯酸鈉消毒清洗后，在每個塊莖中部分別切出3塊長×寬×深=20 mm×20 mm×2 mm的傷口，每處理用塊莖200個，重復(fù)3次。

1.2.2 塊莖愈傷 參考包改紅等[11]的方法。將人工損傷的塊莖裝入打孔的聚乙烯保鮮袋（25 cm×40 cm，厚度0.02 mm）中，置于常溫（20—25℃，RH 70%—80%）條件下，并用黑色塑料薄膜覆蓋避光，進(jìn)行愈傷。

1.2.3 愈傷效果評價 失重率采用重量法測定，每個處理用塊莖15個，重復(fù)3次。病情指數(shù)的測定采用孢子懸浮液的配置參照包改紅等[11]的方法，于培養(yǎng)了7 d的硫色鐮孢培養(yǎng)皿中加入適量的無菌水（含0.01% Tween-80），用涂布器刮下培養(yǎng)基表面的病原菌孢子，用4層紗布過濾至三角瓶中，振蕩20 s，采用血球計數(shù)板計數(shù)，制備成濃度為1×106CFU/mL的孢子懸浮液。在創(chuàng)傷塊莖愈傷的第0、3、5、7、14天時，將已配制好的孢子懸浮液均勻噴灑于塊莖表面，晾干后裝入打孔的聚乙烯保鮮袋中，于黑暗條件下貯藏，7 d后觀察并統(tǒng)計病情指數(shù)。每個品種處理選用塊莖30個，重復(fù)3次。

式中，發(fā)病級別評定標(biāo)準(zhǔn)：0級，創(chuàng)傷面不發(fā)生干腐??；1級，發(fā)生干腐病的面積占創(chuàng)傷面積的1/4；2級，發(fā)生干腐病的面積占創(chuàng)傷面積的1/2；3級，發(fā)生干腐病的面積占創(chuàng)傷面積的3/4；4級，創(chuàng)傷面完全發(fā)生干腐病。

1.3 木質(zhì)素及軟木脂沉積觀察

木質(zhì)素的沉積觀察參照ALBA等[12]的方法并修改。用不銹鋼刀片垂直于愈傷塊莖創(chuàng)傷表面切出0.2—0.3 mm厚的薄片，蒸餾水沖洗3次后，置于載玻片上，滴加1%（w/v）間苯三酚溶液及濃鹽酸染色1 min，置于光學(xué)顯微鏡下觀察，拍照觀察。

軟木脂（suberin poly aliphatic，SPA）的沉積觀察參照LULAI等[13-14]的方法并修改。用不銹鋼刀片垂直于塊莖愈傷表面切出0.2—0.3 mm厚的薄片，蒸餾水沖洗3次后，進(jìn)行如下染色觀察。初染：配制0.05%（w/v）的甲苯胺藍(lán)染液，染色45 min，染色過程中輕微振蕩使其染色均勻；脫染料：分別用75%酒精、蒸餾水沖洗3次；復(fù)染：吸取中性紅染液，染色1—1.5 min；除去染料：分別用75%酒精、蒸餾水沖洗3次。將染色的切片置于載玻片上，滴上清水加蓋蓋玻片，在熒光顯微鏡下觀察，拍照。

塊莖損傷表面的木質(zhì)化細(xì)胞層厚度和軟木脂總熒光強(qiáng)度分別根據(jù)文獻(xiàn)[7,15]的方法通過IS Capture與Imagine J圖像軟件進(jìn)行測量計算。

1.4 苯丙烷代謝關(guān)鍵酶活性及產(chǎn)物含量的測定

取樣參照吳覺天等[16]的方法。在創(chuàng)傷后的0、3、5、7和14 d時，取皮下2—3 mm厚的愈傷組織2 g，用錫箔紙包好，液氮冷凍，粗酶液提取之前保存在-80℃超低溫冰箱中待用。

1.4.1 苯丙氨酸解氨酶(phenylalnine ammonialyase, PAL)活性 參照YIN等[17]的方法。稱取愈傷組織2 g，用3 mL 0.05 mol·L-1的硼酸緩沖液（含1% PVPP，5 mmol·L-1β-琉基乙醇，pH 8.8）冰浴研磨成勻漿，4℃、15 000×g離心30 min，取上清液用于酶活測定。酶促反應(yīng)體系：將500 μL粗酶液加入0.05 mol·L-1的硼酸緩沖液中（pH 8.8）孵育，再加入20 mmol·L-1的L-苯丙氨酸，37℃保溫30 min，充分混合后290 nm下測定吸光值，作為反應(yīng)初始值（OD0）。再在37℃下反應(yīng)1 h，測定其OD/290nm，作為終止值（OD1）。對照組以硼酸緩沖液代替酶液。每分鐘內(nèi)OD/290 nm變化0.01為1個酶活性單位（U），以U·g-1FW表示。重復(fù)3次。

1.4.2 木質(zhì)素、總酚及類黃酮含量 木質(zhì)素含量的測定參照YIN等[17]方法。取2 g愈傷組織，在預(yù)冷的5 mL 95%乙醇中研磨成勻漿狀，然后4℃，14 000 ×g離心30 min，棄去上清液，將沉淀物用95%乙醇沖洗3次，再用乙醇∶正己烷＝1∶2（V/V）沖洗3次，再次收集沉淀在60℃烘箱中干燥24 h，將干燥物轉(zhuǎn)移至離心管中，溶于1 mL 25%溴化乙酰冰醋酸溶液，70℃恒溫水浴30 min，最后加入1 mL 2 mol·L-1NaOH中止反應(yīng)。再加2 mL冰醋酸和0.1 mL 7.5 mol·L-1羥胺鹽酸，離心，取上清液0.5 mL，用冰醋酸定容至5 mL，在280 nm下測定吸光值，樣品重復(fù)3次。木質(zhì)素含量以FW表示。

總酚及類黃酮含量的測定參照參照YIN等[17]方法。取2 g愈傷組織，于預(yù)冷的5mL 1% HCl-甲醇溶液中，冰浴研磨成勻漿進(jìn)行提取，然后于4℃下12 000×g離心30 min，收集上清液分別于280、325 nm下測定吸光值?？偡优c類黃酮含量分別以O(shè)D280·g-1FW和OD325·g-1FW表示。重復(fù)3次。

1.5 數(shù)據(jù)統(tǒng)計與分析

全部數(shù)據(jù)采用Excel 2007計算平均值和標(biāo)準(zhǔn)誤（±SE），采用SPSS 17.0進(jìn)行Duncan’s多重比較檢驗分析。

2 結(jié)果

2.1 馬鈴薯塊莖抗/感品種愈傷期間失重率和病情指數(shù)

愈傷期間，兩個品種塊莖的失重率均逐漸升高，‘隴薯3號’的失重率顯著高于‘青薯168’（圖1-A）。第14天時‘隴薯3號’的失重率是‘青薯168’的2.1倍。兩個品種塊莖在不同愈傷時期接種硫色鐮孢后的病情指數(shù)均逐漸降低。愈傷前期‘隴薯3號’的病情指數(shù)顯著高于‘青薯168’（圖1-B），第3和5天時，該品種的病情指數(shù)分別是‘青薯168’的3.5和3.6倍。第7天時，兩個品種病情指數(shù)接近，無顯著差異，之后逐漸趨于一致。

2.2 馬鈴薯塊莖抗/感品種愈傷期間木質(zhì)素和軟木脂的積累

兩個品種塊莖愈傷組織的木質(zhì)素沉積隨愈傷時間的延長而增加（圖2-A）。但‘青薯168’的木質(zhì)素沉積速度明顯快于‘隴薯3號’。前者的木質(zhì)素染色較深，細(xì)胞形成層數(shù)較多。第14天時，‘青薯168’形成愈傷組織的細(xì)胞層數(shù)約為4—5層，細(xì)胞較小且排列緊密，而‘隴薯3號’的僅為2—3層，細(xì)胞較大且排列疏松。同樣，兩個品種塊莖愈傷組織中的軟木脂沉積也在不斷增加（圖2-B）。‘青薯168’的木栓化程度明顯快于‘隴薯3號’。前者的熒光強(qiáng)度較高，細(xì)胞形成層數(shù)較多。第14天時，‘青薯168’的愈傷組織有5—6層細(xì)胞發(fā)出強(qiáng)烈熒光，而‘隴薯3號’只有2—3層細(xì)胞發(fā)出微弱熒光。

愈傷期間，兩個品種塊莖愈傷組織的木質(zhì)化細(xì)胞層厚度均不斷增加（圖3-A）?！嗍?68’的木質(zhì)層厚度顯著大于‘隴薯3號’。第14天時，‘青薯168’的木質(zhì)層厚度高出‘隴薯3號’47.4%。同樣，兩個品種愈傷組織中的軟木脂總熒光強(qiáng)度也持續(xù)增加（圖3-B），第14天時，‘青薯168’的熒光強(qiáng)度高出‘隴薯3號’60.6%。

2.3 馬鈴薯塊莖抗/感品種愈傷期間苯丙氨酸解氨酶（PAL）活性以及木質(zhì)素、總酚和類黃酮含量

愈傷期間，兩個品種塊莖愈傷組織中的PAL活性明顯存在差異（圖4-A）?！嗍?68’的PAL活性呈緩慢上升趨勢，而‘隴薯3號’呈現(xiàn)先下降，然后基本保持穩(wěn)定的趨勢。但‘青薯168’的PAL活性顯著高于‘隴薯3號’。第14天時，高出‘隴薯3號’77%。隨著愈傷時間的延長，兩個品種愈傷組織中的木質(zhì)素含量均逐漸上升，但‘青薯168’的木質(zhì)素含量顯著高于‘隴薯3號’（圖4-B）。第7天時，‘青薯168’的木質(zhì)素含量高出‘隴薯3號’65%。兩個品種愈傷組織中的總酚含量在愈傷期間也存在差異（圖4-C）?！嗍?68’的總酚含量不斷增加，而‘隴薯3號’的基本穩(wěn)定。但‘青薯168’的總酚含量在中期和后期均顯著高于‘隴薯3號’，第14天時含量高出‘隴薯3號’76%。兩個品種的類黃酮含量在愈傷期間的變化趨勢基本相同，均隨愈傷時間的延長而不斷增加（圖4-D）。但在愈傷前期差異顯著，第0和3天時，‘青薯168’的類黃酮含量分別高出‘隴薯3號’43.5%和16.3%。而第5天以后，兩個品種間的類黃酮含量基本保持一致。

圖1 馬鈴薯品種‘青薯168’和‘隴薯3號’塊莖愈傷期間失重率（A）和病情指數(shù)（B）Fig. 1 Weight loss (A) and disease index (B) of potato tuber cultivars ‘Qingshu No. 168’ and ‘Longshu No. 3’ during wound healing

圖2 馬鈴薯品種‘青薯168’和‘隴薯3號’塊莖愈傷期間木質(zhì)素（A）與軟木脂（B）的積累Fig. 2 The accumulation of lignin (A) and SPA (B) of potato tuber cultivars ‘Qingshu No. 168’ and ‘Longshu No. 3’ during wound healing

圖3 馬鈴薯品種‘青薯168’和‘隴薯3號’塊莖愈傷期間木質(zhì)化細(xì)胞層厚度（A）和軟木脂熒光總強(qiáng)度（B）Fig. 3 Thickness of lignified cell layers (A) and total fluorescent intensity of SPA (B) of potato tuber cultivars ‘Qingshu No. 168’ and ‘Longshu No. 3’ during wound healing

圖4 馬鈴薯品種‘青薯168’和‘隴薯3號’塊莖愈傷期間苯丙氨酸解氨酶活性（A）以及木質(zhì)素（B）、總酚（C）和類黃酮含量（D）Fig. 4 Activity of PAL (A), content of lignin (B), total phenolics (C) and flavonoids (D) of potato tuber cultivars ‘Qingshu No. 168’and ‘Longshu No. 3’ during wound healing

3 討論

馬鈴薯塊莖受不同程度的損傷后，傷誘導(dǎo)的栓化作用能夠使塊莖傷口自動愈合，在傷口表面新生成一層完整周皮，從而可以較好地維持塊莖品質(zhì)，抵抗病原物的侵染[18-19]。本研究發(fā)現(xiàn)不同品種間塊莖的愈合能力存在較大差異。抗病品種‘青薯168’的失重率、病情指數(shù)顯著低于感病品種‘隴薯3號’，而‘青薯168’的木質(zhì)素與軟木脂沉積速率及苯丙烷代謝活性顯著高于‘隴薯3號’，表明抗病品種比感病品種具有更強(qiáng)的愈傷能力。

受損馬鈴薯塊莖的傷口應(yīng)答和愈傷涉及許多生物過程，其中最為重要的就是傷誘導(dǎo)的栓化。該過程包括初級栓化和次級栓化兩個時期，前者是傷口處形成‘封閉層’，而后者是‘封閉層’下形成‘傷口周皮’[3]。栓化完成后，傷口處會積累大量的脂質(zhì)聚合物、軟木脂和木質(zhì)素等成分，這些物質(zhì)在阻止水分蒸騰、營養(yǎng)損失及抵抗病原物方面發(fā)揮著重要作用[20]。愈傷期間，感病品種‘隴薯3號’的失重率顯著高于抗病品種‘青薯168’（圖1-A），這可能與感病品種周皮的形成速度較慢，軟木脂的積累速度較低有關(guān)。已有報道表明，軟木脂是由沉積在細(xì)胞壁上的聚酚類物質(zhì)（SPP）和沉積在細(xì)胞壁與細(xì)胞膜之間的聚脂類物質(zhì)（SPA）通過甘油交聯(lián)、蠟質(zhì)填充而形成的雜聚物，這兩類物質(zhì)對病原物的抵抗作用存在差異，SPP可抵抗細(xì)菌性病害，而SPA只對真菌性病害表現(xiàn)抗性[21-23]。當(dāng)SPA在傷后5—7 d沉積至第一層細(xì)胞時，塊莖就具備了抵抗真菌侵染的能力[14]。本研究所觀察到的塊莖病情指數(shù)隨著愈傷時間延長而降低的結(jié)果與之相符。此外，塊莖愈傷組織積累的脂質(zhì)和木質(zhì)素以及周皮細(xì)胞層的厚度均會顯著影響塊莖對病原物侵染的抗性[24]。因此，抗病品種‘青薯168’所表現(xiàn)出的良好愈傷效果與其愈傷組織中木質(zhì)素和SPA的快速積累（圖2-A、2-B）以及較大的木質(zhì)化細(xì)胞層厚度與軟木脂熒光強(qiáng)度（圖3-A、3-B）密切相關(guān)。JARVINEN等[23]發(fā)現(xiàn)，紅皮抗病品種‘Asterix’和白皮感病品種‘Nikola’塊莖的軟木脂組織中SPA的成分與含量的變化在貯藏期間存在差異。由此表明，不同品種馬鈴薯塊莖的愈傷能力與其愈傷組織中木質(zhì)素與軟木脂的積累速度密切相關(guān)。

蛋白組學(xué)的研究結(jié)果指出，馬鈴薯塊莖的愈傷涉及細(xì)胞增大和分裂、細(xì)胞結(jié)構(gòu)、信號轉(zhuǎn)導(dǎo)、能量代謝和次生代謝等多種生化過程[25]。其中以苯丙烷途徑為代表的多種次生代謝在塊莖周皮的形成中起著重要作用[26-28]。苯丙氨酸解氨酶（PAL）作為植物體內(nèi)合成總酚、類黃酮及木質(zhì)素等物質(zhì)的關(guān)鍵酶[29-30]，在愈傷組織的形成中發(fā)揮著重要作用。該酶為軟木脂的合成提供了酚前體[31]，如阿魏酸和羥基肉桂酸衍生物等[32-34]。PAL在參與木質(zhì)素形成的同時也參與了防御機(jī)制的誘導(dǎo)[35-36]。本研究顯示，抗病品種‘青薯168’較高的PAL活性是導(dǎo)致該品種愈傷組織中木質(zhì)素與軟木脂快速積累的關(guān)鍵（圖4-A），該結(jié)果與SHAO等[37]在‘紅富士’和‘嘎啦’蘋果果實愈傷期間的觀察結(jié)果類似。愈傷組織中積累的酚類物質(zhì)和木質(zhì)素還可能會直接毒殺病原物并參與物理屏障的形成[38-39]。有報道表明，過氧化物酶和H2O2參與了愈傷結(jié)構(gòu)的形成[40]，在過氧化物酶和H2O2的介導(dǎo)下酚酸類物質(zhì)的聚合才能實現(xiàn)[41]。同樣，MAHGOUBA等[42]也發(fā)現(xiàn)馬鈴薯抗病品種可產(chǎn)生更多的次生代謝產(chǎn)物，其總酚含量和過氧化物酶活性較高，木栓層較厚，能更好地氧化羥基肉桂醇生成木質(zhì)素和交聯(lián)細(xì)胞壁，從而具備較強(qiáng)的抗病性。由此表明，活性氧和過氧化物酶在馬鈴薯塊莖愈傷組織形成中發(fā)揮了重要作用，但具體的作用機(jī)理尚有待今后進(jìn)一步研究。

4 結(jié)論

抗/感馬鈴薯品種塊莖的愈傷能力存在較大差異，抗病品種的愈傷能力要顯著高于感病品種?？共∑贩N所具備的良好愈傷能力與其高活力的苯丙烷代謝密切相關(guān)，在苯丙氨酸解氨酶的作用下，塊莖愈傷組織中快速積累酚前體，在過氧化物酶和H2O2參與下聚合為木質(zhì)素與軟木脂，促進(jìn)了愈傷結(jié)構(gòu)的形成。

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（責(zé)任編輯 岳梅）

附表1 本研究篩選用于性狀-標(biāo)記關(guān)聯(lián)分析的分子標(biāo)記

Supplementary table 1 The molecular markers used for trait-marker association analysis in this study

Comparison of Healing Ability on Potato Tuber Cultivars‘Qingshu No. 168’ and ‘Longshu No. 3’

JIANG Hong, BI Yang, LI ChangJian, WANG Yi, LI ShengE, LIU YaoNa, WANG Bin
(College of Food Science and Engineering, Gansu Agricultural University, Lanzhou 730070)

【Objective】The objective of this study is to find out the differences of tuber healing ability of resistant and susceptible potato cultivars against dry rot (‘Qingshu No. 168’ and ‘Longshu No. 3’), and explore the reasons causing differences in the levels of lignin and suberin accumulation and phenylpropanoid metabolism.【Method】The resistant cultivar ‘Qingshu No. 168’ and susceptible cultivar ‘Longshu No. 3’ were used as materials. The damage of tubers was artificially simulated and heal was conducted at ambient temperature. The healing effect was assessed by determining the weight loss and disease index of wounded tubers that were inoculated with Fusarium sulphureum at every healing time point. Phloroglucinol-HCl staining and toluidin blue O-neutral red staining were used to observe lignin and suberin deposition, and the lignified cell layers and suberin fluorescent intensity were quantified. Moreover, the key enzyme and metabolites of phenylpropanoid metabolism pathway such asphenylalanine ammonia lyase activity, and the contents of lignin, total phenol and flavonoid were determined to compare the differences of healing ability between the two cultivars.【Result】The weight loss of ‘Qingshu No. 168’ was significantly lower than that of ‘Longshu No. 3’ during healing. The weight loss of ‘Qingshu No. 168’ and ‘Longshu No. 3’ was 0.93% and 1.96% after 14 days of healing, and the weight loss of ‘Qingshu No. 168’ was 52.3% lower than that of ‘Longshu No. 3’. The disease index of ‘Qingshu No. 168’ was also significantly lower than ‘Longshu No. 3’ after inoculated withF. sulphureumduring healing. After 3 days of healing for inoculated tubers, the disease index of ‘Qingshu No. 168’ and ‘Longshu No. 3’ was 20.36 and 71.59, respectively, the former was 71.5% lower than the later. During healing, ‘Qingshu No. 168’ had higher accumulation rate of lignin and suberin than ‘Longshu No. 3’. After healing for 14 days, the thickness of lignified cell layers and total fluorescent intensity of suberin of ‘Qingshu No. 168’ were 47.4% and 60.6% higher than ‘Longshu No. 3’. In addition, ‘Qingshu No. 168’ showed more phenylpropanoid metabolism activity than ‘Longshu No. 3’. The activity of phenylalanine ammonialyase and the lignin content of‘Qingshu No. 168’ were 77% and 65% higher than ‘Longshu No. 3’after healing for 14 days, respectively. The total phenolics contents of the two cultivars showed a significant difference at late stage of healing, ‘Qingshu No. 168’ was 76% higher than‘Longshu No. 3’ after healing for 14 days. However, no significant difference was found in the flavonoids content between the two cultivars during healing.【Conclusion】The resistant cultivar ‘Qingshu No. 168’ showed stronger healing ability than susceptible cultivar ‘Longshu No. 3’. A stronger healing ability of resistant cultivar is related with its higher phenylpropanoid metabolism activity.

potato; cultivars; tuber; wound healing; phenylpropanoid metabolism

第一部分：淀粉合成相關(guān)基因等位標(biāo)記Part I: Allelic markers related to starch biosynthesizing genes

基因Gene標(biāo)記Primers序列Sequence類型Type染色體Chromosome位置（Mb）位點Locus文獻(xiàn)Reference SSIIc-3-F ATCTTTAGACGATTAGCG AAGTCACAAGTAGAAGGG STS 10 15.34 3'-UTR 6bp Del Tian et al. 2010 SSIIIa SSIIIa-F GAACTTGTGCCTTAAGCTGACTG GGAATAGTAAGCCGAAGGACTT STS 8 5.35 Intron 2 20bp Del Tian et al. 2010 SSIIIb-IF CCGTTTCTAGTTTATCTTCTGAAT TGCTCTTATGGTGGAAATCACG TGCAACGGACGGCTGCTC TSP 4 31.54 PromoterSNP(A/T) Tian et al. 2010 SSIIIb SSIIIb-F AAGAAGGGAAGGGAGTCAGC GCCATCTCCATTGCCAGC STS 4 31.54 5'UTR9bp Ins/3bp Ins/ 2bp Del Tian et al. 2010 SSIVa SSIVa-IF GACGCCGTTAACTTTTTCTAA CGAATGGCCAAACCTG ATAACCCGCTGCTATTCTCT CCGTTGACTTTTTCTCACAT ARMS 1 30.37 5’-UTR SNP(T/C) Tian et al. 2010 SSIVb SSIVb-1-IF CTGCTACAGTCCCTTTCG TGGGCCATGCTGAAT GAAAGATAGGGAAATTGTGG ACTTCGGTGAGAAACAAACT ARMS 5 26.4 Intron5SNP (A/G) Tian et al. 2010 AGPL1-IF GCAGGGTATGAGGCG GATATAGAAAGTTCATTCCAGGT GTATTTGGTCTGCTTCAAAA AAGCCTTCAGGTCAGTATCT ARMS 5 28.79 Intron 11 SNP (A/G) Tian et al. 2010 AGPL1 AGPL1-F CGTTCAGGTTCAGGCAATCA GGAAGGGTGGTGATGTGGAG STS 5 28.79 Promoter region 5bp/8bp Del Tian et al. 2010 AGPL2-1-IF GTTAACCAGCCAGAAGACTG AACTACACCACATCAATTACCA TGTCGTTTTCTTTTTATTGAA TATATACGCACTTCCTGTGC ARMS 1 25.69 Intron 1 SNP(T/G) Tian et al. 2010 AGPL2-2-IF CGTGTACCTAACGATGCGT AAGTTTATTTGTCAGTATTGAACTC TGTAAGGAAATGAAGAGCG CGTAATACTAGGAGGGGTCA ARMS 1 25.69 3'-UTR SNP(G/T) Tian et al. 2010 AGPL2 AGPL2-F CAATCGCTGCCATCGGTTG TTCCACATCGTTAGGTACACG STS 1 25.69 3'-UTR 5bp Del Tian et al. 2010 AGPS1-1-F TCTATTCTCAGCCCTCCAACC GTGTGTTTAGAGGTGCTTTTCG STS 9 7.25 Promotor 29bp/39bp Del Tian et al. 2010 AGPS1 AGPS1-2-F TACGCTATGCTCTTGAAAC TATCTTCCCAGTAACCATCA STS 9 7.25 Intron 4, 31bp Del Tian et al. 2010 AGPS2-IF ATGTAAACTGTTAGAATCGAATAC CATCCTTATCATTAAGTCTGCTA CAAAAGCACTCATTGGAAC GTCAAGAGCCTATGGGAAC ARMS 8 15.54 Intron 2 SNP(C/T) Li et al. 2008 AGPS2 AGPS2-F GACGAAAAGTGAAAGTTGC GGTTCTTGATGCTGATGTG STS 8 15.54 Intron 6 AT Del Li et al. 2008 BEI BEI-1-IF TGTTAGCTTCATGTTCTTATTTC CAATTCAGTAACTGTCGCA CCTGTAGGTTATCAAATTGC ARMS 6 30.91 Intron 6 SNP(T/C) Tian et al. 2010

基因Gene標(biāo)記Primers序列Sequence類型Type染色體Chromosome位置（Mb）位點Locus文獻(xiàn)Reference CAGTGTACATTTCCTTGTCC BEI-2-IF GGCTATCAGGCTTACTATCGT TTTCAACAACCGCCCTAACTCA CCTTTCTTGCCACAATCGTCC TSP 6 30.91 Exon 14 SNP(T/C) Han et al. 2004; Liu et al. 2004; He et al. 2006 BEI-2-F GTGGGGAAAACAAGTAAGTCTG AGTTCCATCAGAAGAATCAGGG STS 6 30.91 Intron 2, 15bp Insert Tian et al. 2010 BEI-4-F ATTTCTTTGGCCACAGGCGA CCCAGATTCGGAACAAGAAC SSR 6 30.91 Intron 2 (CT)n Bao et al. 2002b; Bao et al. 2006 BEIIa-1-F CACCAATTATATTAGCGTGCTCC CGTGGCTCTTGGCTCTCTTG STS 4 20.05 3'-UTR 22bp Del Tian et al. 2010 BEIIa BEIIa-2-F CCATCACCTCAAATACATCACTC AGACTGGAATGCCCCTTAGG STS 4 20.05 3'-UTR 13bp Del Tian et al. 2010 BEIIb-IF GAAGGAGCAAGAAAAACAAC TTCACAGATTGCTGGCTAC ACCTGCGTTGTCTATGCTC CACCTTGAAATCACTGGAA ARMS 2 19.36 3'-UTR SNP(C/G) Han et al. 2004; Liu et al. 2004; He et al. 2006; Bao et al. 2006; Lee et al. 2009 BeIIb-1-F TCGGTCCTAATATTTTGCGCTG CCTTAACTTGACACCGAATCCG STS 2 19.36 5'-UTR 13bp Indels Yan et al. 2005; Yan et al. 2007 BeIIb-2-F AAGGTTAGCATTGGTTGGTGAG TCTCCTTGAACAGCGACAGC STS 2 19.36 3'-UTR 28bp Indels Tian et al. 2010 BeIIb BeIIb-3-F GTGGGGTTCTCAACTTAGC CATCAGCATTGTTAGGCAG STS 2 19.36 Intron 6, 56bp/32bp Insert Tian et al. 2010 ISA1-IF AGAGTTTTTGAGAGTGGCAA TTTGAAAAAACAGGACGC TTTTAATCTCCCTGTCTTGC ATTTTGTGAGCAATGTTGAA ARMS 8 25.77 Promoter SNP(G/A) Tian et al. 2010 ISA1-2-F TGTGGGAATACCTTCAACTG ATAAAACCCTTACAGGCTTG STS 8 25.77 Intron 8 10bp Insert Tian et al. 2010 ISA1 ISA1-F ATAGATGCTAATGTGATGTGGC TGGTATAGGCACAACCGTAGA STS 8 25.77 Promoter 10bp Del He et al. 2006; Tian et al. 2010 ISA2 ISA2-2-IF CGTACGGGTCCAACAGTGAGA CGGTGGCATCCGTCG CCTTCCCCAGTTGTAGCTCG AATGCCAATGCCGTTTCTCT ARMS 5 19.07 Iso2_GA_Ref1712 Kharabian-Masouleh et al. 2011 PUL-1-F AGAGAAGGAGAAAGAAGTGGAGAC GTCCAAACTGAATCACTCAATCG STS 4 4.39 promoter region 13bp Del Tian et al. 2010;Yan et al. 2010 PUL-2-F GACAACCGTCCGCTTTAGTTTC GCATTTGAGAGGGTTTGGATTC STS 4 4.39 promoter region 6bp/9bp Del Tian et al. 2010 PUL-3-F CTGTATGGACTGAGTAGTCGATGG TGAGCCTCATCTGCCAGAGT STS 4 4.39 Intron 9 53bp Del Tian et al. 2010 PUL-4-F TACACCATCCTCACTACCA GCAACATCTAAAACACCAA STS 4 4.39 Intron 15 18bp Insert Tian et al. 2010 PUL-5-F ATTGGCATTTGTAAGTTTC CAATCTTGGTTTTATCCTG STS 4 4.39 Intron 24 240bp Del Tian et al. 2010 PUL PUL-6-F ATTTAACTGTATGGACTGAG GATACCAACCAAACAAGA STS 4 4.39 Intron 24 240bp Del Tian et al. 2010

第二部分：稻米籽粒表型相關(guān)連鎖分子標(biāo)記Part II: Linkage markers related to phenotype of rice grain

標(biāo)記Primers正向引物Forward Primer (5'-3')反向引物Reverse Primer (5'-3')染色體Chromosome位置（Mb）文獻(xiàn)Reference HL944 TGCCAGCCTAGCGAGCCTAA ATTGCAGCGAGCTACACG 3 35.16 RM16232 GACTCATCCGCTGAGAGTTTCG GAGGTATCCGGAGAGGAAGAGG 3 36.17 He et al. Y42 AGTGTAGAACAGCACTGGAATC AAGCTTAGCTACGCTAATGACC 4 19.37 Zhang et al. Y48 TCACCATATGGAAGCATCAAG TATGTGTTTGTTCATGTGCAC 4 19.38 Wang et al. RM5586 AGATGGCTGGCCAACAGACTGG ACAATGCCCATCCACTGCTTCC 4 19.71 Wang et al. INV1 TGGATTTAGGCTTGTTTGAGC CGTGACCTGCATGTTTCTCT 4 20.23 RM16942 CCAGTACTCTCGCTCCACTCTCC ATCGCTTTCACGTCACCAAGG 4 20.37 SSR9 CTCGTCACTCTCACCACCAC CCCACTGACAAGCACACGTA 4 21.58 RM17307 AGAGCTTGGAGGCACCAATACCG AGAAAGAACTCCGGCCACCTTCG 4 27.33 Zou et al. RM17363 GGAGCAGGCATCTGATCTAGGG CAGTTGGGCAACAGATCCATCC 4 28.54 Kobayashi et al. nkssr04-19 CTGGAATCACAAACCACGAC GCTACCTCAAGCTCCACGAC 4 30.72 Fan et al. RM3276 TCCGTCTCGACTCTTCCATC GATGAGACACCACGGACATG 4 30.72 Deshmukh et al. RM5478 GGATCCAATGCGATGCTACTCC CGAAACAGTCGCGATTAACTGG 4 33.26 Chen et al. RM3466 CAGGACGAGATCCAAGCCAAGG TCGTCCTCGCCACCACTACTCC 4 33.83 Zhou et al. b RM589 GTGGCTTAACCACATGAGAAACTACC TCACATCATTAGGTGGCAATCG 5 1.38 Tan et al. RI0399 CCTATGTCGCCACTCATCG CACAACAATGCAGTCCCATC 5 3.29 Li et al. C62 GATTGACTGATAAATTGACAGC CTAACTCCCATGGAATTAC 5 3.38 Li et al. RM574 GGCGAATTCTTTGCACTTGG ACGGTTTGGTAGGGTGTCAC 5 3.39 RM17947 GTTCTTGCCTTCTCGGCATGTGG GGTCACTGATTCCACCATTCACACC 5 3.48 RM18033 AGAGAGACGAGAGCTTGCTGTGC TACTTCCTCCATTCCTGGTCTCC 5 5.31 Wan et al. MS40671 TTTGATTGCCATTATCGAGTTAG GTGTGCGTGAAGAGAACAGT 5 5.34 RM18035 CCGTGGTGATAAACTGTTAACTGAGG CGCATATGCATCAGCATCACG 5 5.34 RMw513 GTATTTGTTTGTCGCATTC TAGGACCATAGATGTGAGTTA 5 5.36 RM18360 TCGAGACTGATCGGAGTTTAGGC CGCTCCTCCCTAACACCTCTACG 5 14.09 Ebitani et al. RM163 CGCCTTTATGAGGAGGAGATGG AAACTCTTCGACACGCCTTGC 5 19.17 Ebitani et al. PPDKBSSR1 GCTTGAAACAAGCTAAAATACCG CAGATTATTTGCTCACGCTATAAAA 5 19.65 Kang et al. RM18751 CCGTGTGTTGGCTTAGAATCAAGG GCCACTTTCCAAACATCAGAAAGC 5 21.11 RM3575 ACAGCCTCAAATTGTGAGCAAGG GCTGTATGATCTGTATCCATCCATCC 5 21.31 RM31 CGCTCCTCCACTCTTCTCCTACC CGTGCAGAAAGTCCATTACTCTCC 5 28.59 Tan et al. RM3414 TAGGGCAATTGTGCAAGTGG TTGGGAATTGGGTAGGACAG 6 2.88 Bao et al. RM204 CTAGCTAGCCATGCTCTCGTACC CTGTGACTGACTTGGTCATAGGG 6 3.17 Zhou et al. b RM253 CCATCTCTGCCTCTGACTCACC TCCTTCAATGGTCGTATCTTCTCC 6 5.43 Shirasawa et al. RM7179 CACGTGTCAGCTTAAGAGCG TTACATCATAAGCCCGCAGG 6 19.38 Guo et al. RM20201 TTAGAGGTAACGGAGGCACAACC GATGGCTTGAGAGCGTTTGTAGG 6 20.16 RM3187 TCCCCACATCGTGTCGTC TTTTTCCCCTTCTACCCTCG 6 20.58 RM162 TTGTTCCAGTTCAGGTCTTGTGC CCCTACAAACACCATAAGAAGCAACC6 24.04 Liu et al. M10 GGATTTTTCTTTCCACCTTTG AGGCATGAACTTGAAGTGAGTC 6 24.28 Wu et al. M9 AATGAATAGATTACCACATGCTA TGCCTCTTATTTTACTTTTCTTT 6 24.38

標(biāo)記Primers正向引物Forward Primer (5'-3')反向引物Reverse Primer (5'-3')染色體Chromosome位置（Mb）文獻(xiàn)Reference RM20547 CTCTTCTTCTTCTGTCCGTCTTGG CCATCTTCATTACCGACCTCTGC 6 27.03 Zhou et al. b RM30 GGTTAGGCATCGTCACGG TCACCTCACCACACGACACG 6 27.25 Qiao et al. RM6926 CGATCGGCCTATCTTTCTGTGC GCTAGCAGTGGGATCATGTTTGG 6 28.59 RM3765 ACACCATGACACCAAAGGAAGG GGATGCTTCCAATCCTCTCACC 6 29.21 MID06029 TGAAAGATAATAATCTGTAAGGCAGTTGTGATTAGCGTTGCCACTGA 6 30.28 RM481 TAGCTAGCCGATTGAATGGC CTCCACCTCCTATGTTGTTG 7 2.87 Tian et al. ID52 GTTTGGTGGTGTTCATGGTCT GATCAGCTCTCACCAATCCAG 7 2.88 RM5436.2 CAAAGGGGGTGTCCTCTATG GTTGCTCGTCCTACATGTGC 7 9.07 Xing et al. RM5499 GGACGAAAGGGTATTTGATTGG CCTCAAGGTGGTCTCCTTCTCC 7 9.99 InDel1 CCATAGTAAGACGACCTT GATATTCTGTCAGCAGTT 7 24.66 Shao et al. RM21945 CTACACAAGTGAACGCCATCAGG GTTCTAGGGTGTCCTTTCATGAGC 7 24.93 RM21964 AAGACAGCCTTCAAGGGATTTGG GTACGTGCACCGAGCAGAGC 7 25.29 Zhou et al. a RM22020 CGTTGAGACGGTCACCTAATGC GCTCAAATGTTTGACACGAAGC 7 26.28 RM6389 GACGAGGAGTTCGTCGCTAC CCTTCTCCTTCGTCTCCTCC 7 28.28 Bai et al. RID711 GCACATGCATGCTAGGACAT AGCCGGTAAATTTCTTGCAC 7 28.48 RM408 AATTGCCCAACGAGCTAACTTCC TGAGCTGTTTGTGCTCTTCTACTTCG 8 0.12 Tabata et al. RM22483 GACCATGGTGTGAGTGTGACAGG CAAGTCCTACCTCAACCGCTACC 8 4.33 RM310 CCAAAACATTTAAAATATCATG GCTTGTTGGTCATTACCATTC 8 5.11 Zhang et al. RM126 CGCGTCCGCGATAAACACAGGG TCGCACAGGTGAGGCCATGTCG 8 5.22 RM30000.CNR99 AGCTCACCTCGTTTTGCGTA CGGACAAATTCGTTCACCTC 8 21.7 Xie et al. RM23427 AGGGAGTCGGAGACCATGACG TACCGCGTATCATGTCCTTGACG 8 25.21 He et al. RM502 GCGATCGATGGCTACGAC ACAACCCAACAAGAAGGACG 8 26.36 Wang et al. PSM711 ATGACCGTCTGCTTCCTCTAA AACATCGACAGGGAGAAGTGC 8 26.37 RM15937 GGAAGAACCTGCGTATCAAGACC CCACACGGAAGCAGAATTAGCC 8 30.94 Yan et al. RM23748 CTCTGGGAACAAACTTGACAATGC CAATTTGCAGGCTATGCTACTGC 9 3.09 Chen et al. RM24718.CNR111 GACCAACGTGCATGTGACTT GCTTGCACTAGGGCTCCTT 9 20.9 Xie et al. RM171 AACGCGAGGACACGTACTTAC ACGAGATACGTACGCCTTTG 10 18.79 Chen et al. RM6745 TGTTCTCAACACAAAATTCTCT ATCGTAAGCAAAGTGCATAA 10 18.9 Tan et al. RM27073 CCACGTGTCAGTCATCCATCTAGG GGTCTGCTCGATTACCATCAAACTCC 11 23.47 Chen et al. RM27792 GAAGAAGAGAGACTAGGGAGAAGACGCTTGTACCAGCAATTCTCTGTCC 12 7.1 He et al. RM28502 CGAGCAGATCTGATGTCGTCTTCC CTTTGCTTTGCATGCCTCACG 12 23.41 Terao et al.

10.3864/j.issn.0578-1752.2017.04.017

2016-08-01；接受日期：2016-09-12

國家自然科學(xué)基金（地區(qū)科學(xué)基金項目）（31460412）、甘肅農(nóng)業(yè)大學(xué)盛彤笙科技創(chuàng)新基金（GSAU-STS-1432）、國家科技支撐計劃（2012BAD06B00）

聯(lián)系方式：姜紅，E-mail：1473240451@qq.com。通信作者畢陽，E-mail：biyang@gsau.edu.cn。通信作者王毅，E-mail：wangyi@gsau.edu.cn