青花菜C3H型鋅指蛋白基因BoCCCh1的克隆與表達(dá)

蔣明, 劉青娥, 章燕如, 祝琦, 龔秀, 俞可可, 周秀倩

(1.臺(tái)州學(xué)院生命科學(xué)學(xué)院生態(tài)學(xué)省重點(diǎn)學(xué)科，浙江 椒江318000;2.麗水學(xué)院生態(tài)學(xué)院，浙江 麗水 323000)

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青花菜C3H型鋅指蛋白基因BoCCCh1的克隆與表達(dá)

蔣明1*, 劉青娥2, 章燕如1, 祝琦1, 龔秀1, 俞可可1, 周秀倩1

(1.臺(tái)州學(xué)院生命科學(xué)學(xué)院生態(tài)學(xué)省重點(diǎn)學(xué)科，浙江 椒江318000;2.麗水學(xué)院生態(tài)學(xué)院，浙江 麗水 323000)

摘要以青花菜為材料，在克隆C3H型鋅指蛋白基因BoCCCH2的基礎(chǔ)上，研究該基因在不同器官及霜霉菌和灰葡萄孢菌侵染葉片中的表達(dá)模式。測序結(jié)果表明，BoCCCH2沒有內(nèi)含子，編碼區(qū)全長為1 740 bp，編碼579個(gè)氨基酸，推導(dǎo)的蛋白質(zhì)具2個(gè)ANK結(jié)構(gòu)域和2種CCCH鋅指結(jié)構(gòu)，鋅指結(jié)構(gòu)的類型分別為C—X8—C—X5—C—X3—H和C—X5—C—X4—C—X3—H。反轉(zhuǎn)錄聚合酶鏈反應(yīng)表明：BoCCCH2在根、葉、花莖、嫩角果、花蕾和花中均有表達(dá)，其中在根中的表達(dá)量最高；經(jīng)霜霉菌和灰葡萄孢菌侵染后，該基因表達(dá)量均有不同程度的增加，其中在霜霉菌侵染下，表達(dá)量在24 h后開始增加，72 h時(shí)下降，而在灰葡萄孢菌侵染下，6 h時(shí)的表達(dá)量最大，12 h時(shí)開始緩慢下降。聚類結(jié)果表明，BoCCCh1與其他十字花科植物的同源序列聚為一類，支持率達(dá)100%，而與豆科、大戟科和薔薇科等植物的序列處于不同分支。對(duì)BoCCCH2基因的克隆和表達(dá)分析為該基因功能研究奠定了基礎(chǔ)。

關(guān)鍵詞青花菜； C3H型鋅指； 霜霉菌； 灰葡萄孢菌； 克??； 表達(dá)

Cloning and expression of a C3H-type zinc finger protein gene BoCCCh1 from Brassica oleracea var. italica.JournalofZhejiangUniversity(Agric. &LifeSci.), 2016,42(2):143-149

JIANG Ming1*, LIU Qing’e2, ZHANG Yanru1, ZHU Qi1, GONG Xiu1, YU Keke1, ZHOU Xiuqian1

(1.EcologyKeyDisciplineofZhejiangProvince,CollegeofLifeSciences,TaizhouUniversity,Jiaojiang318000,Zhejiang,China; 2.CollegeofEcology,LishuiUniversity,Lishui323000,Zhejiang,China)

SummaryBrassicaoleraceavar.italicais an important vegetable crop worldwide, and in China, Taizhou City of Zhejiang Province is one of the major broccoli production areas. Downy mildew and grey mold rot are two common fungal diseases caused byHyaloperonosporaparasiticaandBotrytiscinerea, respectively. In recent years, broccoli production in Taizhou was frequently affected by these two fungal diseases, resulting in yield and quality loss. Broccoli germplasm resources resistance to disease is scarce; therefore, molecular breeding is regarded as an effective solution to solve the problem. This is critically important to isolate genes associated with disease resistance, which will act as potential target genes for broccoli breeding.

Zinc finger proteins are kinds of important transcription factors in eukaryotic organisms, which involve in various biological activities, such as replication, transcription, translation, repair, metabolism and signaling. According to the number and order of cysteine and histidine residues, zinc finger proteins were classified into several different types, such as C2h1, C2C2, C2C2C2, C2HC and C3H. For example, C3H-type ones contain one to six typical motifs with three cysteine residues and one histidine residue. However, their functions are little known, and no gene has been reported in broccoli.

In this study, a C3H-type zinc finger protein geneBoCCCH2 was isolated from broccoli, and later the expression patterns in different organs as well as leaves infected byH.parasiticaandB.cinereawere studied.

Results indicated thatBoCCCH2 contained no intron, and the full length of coding sequence was 1 740 bp encoding 579 amino acids. The deduced protein sequence contained two ANK domains and two CCCH zinc finger structures, respectively, and the CCCH zinc finger types were C—X8—C—X5—C—X3—H and C—X5—C—X4—C—X3—H. Reverse transcription-polymerase chain reaction results showed that theBoCCCH2 was expressed in roots, leaves, stalks, young siliques, flower buds and flowers, with highest level in roots. Expression levels increased when challenged by bothH.parasiticaandB.cinerea. When infected byH.parasitica, expression levels increased after 24 h, and decreased after 72 h, while infected byB.cinerea, the highest level was detected after 6 h, and slowed down in 12 h. Homologous sequences were downloaded from NCBI (National Center for Biotechnology Information) website, includingCitrussinensis,Gossypiumraimondii,Populustrichocarpa,Ricinuscommunis,Prunuspersica,P.mume,Malusdomestica,Fragariavesca,Phaseolusvulgaris,Glycinesoja,B.rapa,Camelinasativa,Capsellarubella,ArabidopsisthalianaandEutremasalsugineum. Phylogenetic analysis results revealed that BoCCCh1 was grouped with homogeneous sequences from other Cruciferae plants with bootstrap confidence of 100%, and sequences from Leguminosae, Euphorbiaceae and Rosaceae were found on different clades.

In conclusion, these results indicate that theBoCCCH2 might play an important role in defense responses challenged by eitherH.parasiticaorB.cinerea. Cloning and expression analysis ofBoCCCH2 provide evidence for further studies on gene function.

Key wordsBrassicaoleraceavar.italica; C3H-type zinc finger;Hyaloperonosporaparasitica;Botrytiscinerea; cloning; expression

青花菜(Brassicaoleraceavar.italica)為十字花科(Cruciferae)甘藍(lán)類蔬菜，是甘藍(lán)(B.oleracea)的一個(gè)變種，迄今已有2 500多年的栽培歷史[1]。青花菜以花蕾群和花莖為食用部位，可生食也可蒸煮，色澤碧綠，營養(yǎng)豐富，風(fēng)味獨(dú)特，深受人們的喜愛。浙江是我國青花菜主產(chǎn)省，在臺(tái)州、寧波和溫州等沿海地區(qū)均有大面積栽植，是當(dāng)?shù)夭宿r(nóng)的重要收入來源。霜霉病和灰霉病是青花菜在生長過程中常見的2種病害，分別由寄生霜霉菌(Hyaloperonosporaparasitica)和灰葡萄孢菌(Botrytiscinerea)引起，這2種病害在苗期和成株期均可發(fā)生，危害葉片和花球[2]。我國的青花菜種質(zhì)資源十分匱乏，常規(guī)育種受到很大的限制，已成為青花菜良種培育的重要制約因素[3]。分子育種是種質(zhì)創(chuàng)新的重要手段之一，具有成本低、目的性強(qiáng)和效率高等優(yōu)點(diǎn)，近年來在青花菜育種中已有一些報(bào)道[4-6]。合適的靶標(biāo)基因是基因工程育種的基礎(chǔ)，轉(zhuǎn)錄因子通過結(jié)合特定的啟動(dòng)子，控制一系列下游基因的活動(dòng)；導(dǎo)入一個(gè)抗病轉(zhuǎn)錄因子基因就相當(dāng)于轉(zhuǎn)入多個(gè)抗病相關(guān)基因，從而提高綜合抗病能力[7]。

鋅指蛋白是真核生物中一類重要的轉(zhuǎn)錄因子，參與細(xì)胞的諸多生命活動(dòng)，包括復(fù)制、轉(zhuǎn)錄、翻譯、修復(fù)、物質(zhì)代謝、信號(hào)轉(zhuǎn)導(dǎo)和RNA編輯等[8-9]。鋅指結(jié)構(gòu)由一段小分子肽鏈組成，可與Zn2+結(jié)合形成指形結(jié)構(gòu)，該結(jié)構(gòu)能與核酸、蛋白質(zhì)及一些小分子結(jié)合，從而發(fā)揮特定的功能[10]。根據(jù)鋅指結(jié)構(gòu)中半胱氨酸(C)和組氨酸(H)的數(shù)量及排列位置，可分為C2h1、CCCC、C2HC、C2C2、C2C2C2C2和C3H等類型[11-12]。C3H型鋅指蛋白通常含1～6個(gè)鋅指結(jié)構(gòu)，每個(gè)鋅指結(jié)構(gòu)由3個(gè)半胱氨酸和1個(gè)組氨酸組成，它們?cè)诜N子萌發(fā)、抗病免疫反應(yīng)、鹽脅迫響應(yīng)、葉片衰老和干旱脅迫反應(yīng)等方面起著重要作用[13-16]。目前，有關(guān)青花菜C3H型鋅指蛋白基因方面的研究尚未見報(bào)道；因此，本研究在克隆BoCCCH2的基礎(chǔ)上，利用反轉(zhuǎn)錄聚合酶鏈反應(yīng)(reverse transcription-polymerase chain reaction，RT-PCR)方法研究該鋅指基因在不同器官及在霜霉菌和灰葡萄孢菌侵染下葉片中的表達(dá)模式，旨在為開展該基因的功能鑒定奠定基礎(chǔ)。

1材料與方法

1.1實(shí)驗(yàn)材料

青花菜Bo0112在室內(nèi)栽植，其生育期為75 d，花球堅(jiān)實(shí)，花蕾深綠色，側(cè)枝少，具有較強(qiáng)的霜霉病和灰霉病抗性。于花期采集根、葉、花莖、花蕾、開放的花和嫩角果，用于DNA和RNA提取。霜霉菌病葉采自浙江省臨海市上盤鎮(zhèn)青花菜基地，用無菌ddH2O小心沖洗葉片上的白色霉層，將孢子溶液稀釋后備用，待青花菜長至2葉1心時(shí)用噴霧法接種[17]。灰葡萄孢菌也采自上盤鎮(zhèn)青花菜基地，利用分離法獲得菌株，在無菌室中把菌株接種到馬鈴薯葡萄糖瓊脂(potato-dextrose agar，PDA)培養(yǎng)基上，25 ℃培養(yǎng)6 d，在菌落邊緣用打孔器打出直徑為5 mm的瓊脂塊，反貼于葉片正面，對(duì)照為不接菌的PDA培養(yǎng)基。分別采集接種霜霉菌和灰葡萄孢菌0、6、12、24、36和72 h時(shí)的葉片，置于-80 ℃?zhèn)溆谩?/p>

從NCBI數(shù)據(jù)庫下載BoCCCh1的同源序列，分別來自橙(Citrussinensis，登錄號(hào)KDO66907.1)、雷蒙德氏棉(Gossypiumraimondii，KJB08973.1)、毛果楊(Populustrichocarpa，EEE84607.2)、蓖麻(Ricinuscommunis，EEF36773.1)、桃(Prunuspersica，EMJ09531.1)、梅(Prunusmume，XP_008220916.1)、蘋果(Malusdomestica，XP_008384793.1)、野草莓(Fragariavesca，XP_004294290.1)、菜豆(Phaseolusvulgaris，ESW14199.1)、野生大豆(Glycinesoja，KHN40076.1)、不結(jié)球白菜(Brassicarapa，XP_009120380.1)、亞麻薺(Camelinasativa，XP_010483463.1)、薺菜(Capsellarubella，EOA13094.1)、擬南芥(Arabidopsisthaliana，AED97077.1)和山萮菜(Eutremasalsugineum，ESQ42513.1)。

1.2DNA、RNA提取和cDNA合成

基因組DNA的提取采用十二烷基磺酸鈉法；RNA提取采用TRIzol法；cDNA合成試劑盒購自TaKaRa公司，第1鏈和第2鏈的合成根據(jù)說明書進(jìn)行。

1.3基因克隆

采用的上、下游引物分別為BoC3HUP：5′-ATGGGAGATGACGAGCTGT-3′和BoC3HDN：5′-TCAAGCAACGGTTTGTTCT-3′。反應(yīng)總體積為25 μL，含1×PCR緩沖液，0.8 UTaqDNA聚合酶(北京鼎國昌盛生物技術(shù)有限責(zé)任公司)，0.3 μmol/L dNTPs(上海生工生物工程股份有限公司)，各0.2 μmol/L上、下游引物，40 ng葉片基因組DNA或cDNA模板，最后加無菌ddH2O至25 μL。PCR程序：94 ℃預(yù)變性5 min；94 ℃變性30 s，53.3 ℃退火55 s，72 ℃延伸115 s，31個(gè)循環(huán)；72 ℃延伸10 min。

PCR產(chǎn)物經(jīng)1.2%瓊脂糖凝膠電泳，割取含目的條帶的膠塊，利用DNA凝膠回收試劑盒(碧云天生物技術(shù)研究所)回收，實(shí)驗(yàn)操作根據(jù)說明書進(jìn)行。取2 μL PCR回收產(chǎn)物克隆到pGEM-T Easy載體(Promega公司,美國)，于4 ℃冰箱中連接過夜，用熱激法將連接產(chǎn)物轉(zhuǎn)入DH5α大腸桿菌感受態(tài)細(xì)胞(北京鼎國昌盛生物技術(shù)有限責(zé)任公司)中，各取3個(gè)陽性克隆用于測序。

1.4基因表達(dá)分析

根據(jù)測序結(jié)果設(shè)計(jì)RT-PCR引物。上、下游引物分別為BoC3HRTUP：5′-TATGATCGCTGCCT TGTTT-3′和BoC3HRTDN：5′-AGTTCATTGCT GCGTTCTAT-3′，以各器官及經(jīng)霜霉菌和灰葡萄孢菌處理的葉片cDNA為模板進(jìn)行PCR擴(kuò)增，反應(yīng)體系及所采用的試劑同1.3節(jié)。PCR程序：94 ℃預(yù)變性5 min；94 ℃變性30 s，56.5 ℃退火55 s，72 ℃延伸80 s，33個(gè)循環(huán)；72 ℃延伸10 min。以肌動(dòng)蛋白基因?yàn)閮?nèi)標(biāo)，上、下游引物分別為5′-TCTCGATGGAAGAGCTGGTT-3′和5′-GATCC TTACCGAGGGAGGTT-3′。PCR程序：94 ℃預(yù)變性5 min；94 ℃變性30 s，55.6 ℃退火45 s，72 ℃延伸60 s，32個(gè)循環(huán)；72 ℃延伸10 min。PCR結(jié)束后，產(chǎn)物在1%瓊脂糖凝膠上電泳、拍照和記錄。

1.5生物信息學(xué)分析

BoCCCH2的編碼蛋白用Primer premier 5.0軟件翻譯；BoCCCh1及其同源序列比對(duì)采用ClustalX 1.81軟件；系統(tǒng)進(jìn)化樹的構(gòu)建采用Mega 3.1軟件，建樹方法為鄰接法，自舉檢驗(yàn)次數(shù)為1 000。

2結(jié)果與分析

2.1BoCCCh1的克隆與序列分析

分別以葉片基因DNA和cDNA為模板，用BoC3HUP/BoC3HDN引物進(jìn)行PCR擴(kuò)增，經(jīng)割膠、回收、連接、轉(zhuǎn)化和測序，獲得相應(yīng)的基因序列。測序結(jié)果(圖1)表明：BoCCCH2的基因組DNA和cDNA長度均為1 740 bp，該基因沒有內(nèi)含子，分別以ATG和TGA作為起始及終止密碼子；BoCCCH2編碼579個(gè)氨基酸，推導(dǎo)的蛋白質(zhì)具2個(gè)ANK結(jié)構(gòu)域，分別位于57～87和92～124處，另有2個(gè)不同類型的CCCH鋅指結(jié)構(gòu)，分別為C—X8—C—X5—C—X3—H和C—X5—C—X4—C—X3—H，位于249～267和284～299處。

下劃線處為ANK結(jié)構(gòu)域；陰影部分為2個(gè)CCCH結(jié)構(gòu)域。Underlined sequences indicate the ANK domains; sequences highlighted in shades demonstrate two CCCH domains.圖1　青花菜BoCCCh1編碼區(qū)及推導(dǎo)的氨基酸序列Fig.1　Complete coding sequence and deduced amino acid sequence of BoCCCh1 from Brassica oleracea var. italica

2.2BoCCCh1的表達(dá)分析

為研究BoCCCH2在不同器官及經(jīng)霜霉菌和灰葡萄孢菌侵染葉片中的表達(dá)模式，以肌動(dòng)蛋白基因?yàn)閮?nèi)標(biāo)，用BoC3HRTUP/BoC3HRTDN引物對(duì)進(jìn)行RT-PCR分析。結(jié)果(圖2)表明：BoCCCH2在根、葉、花莖、嫩角果、花蕾和花中均有表達(dá)；在根中的表達(dá)量最大，條帶最亮，在葉片、花莖和花中的表達(dá)量較小，條帶亮度較弱。

A:BoCCCh1基因表達(dá)；B:肌動(dòng)蛋白基因?qū)φ铡?:根;2:葉;3:花莖;4:嫩角果;5:花蕾;6:花。A: Expression of BoCCCh1 gene; B: Actin gene (CK). 1: Roots; 2: Leaves; 3: Stalks; 4: Young siliques; 5: Flower buds; 6: Flowers.圖2　BoCCCh1基因在不同器官中的表達(dá)Fig.2　Expression patterns of BoCCCh1 in different organs

RT-PCR結(jié)果表明：對(duì)照葉片中BoCCCH2的表達(dá)量沒有明顯變化(圖3E)；在霜霉菌侵染下，6 h和12 h時(shí)與0 h沒有區(qū)別，24 h后表達(dá)量增加，但增幅較小(圖3A)；葉片經(jīng)灰葡萄孢菌侵染后，6～36 h的表達(dá)量增加，其中6 h時(shí)的表達(dá)量最大，之后逐漸減少，72 h時(shí)的表達(dá)量與0 h相仿(圖3C)。

2.3系統(tǒng)發(fā)育分析

利用ClustalX 1.81軟件對(duì)BoCCCh1及其同源序列比對(duì)結(jié)果表明：桃(EMJ09531.1)和梅(XP_008220916.1)的相似性最大，達(dá)97%；其次為不結(jié)球白菜(XP_009120380.1)和BoCCCh1，相似性達(dá)94%；而菜豆(ESW14199.1)和不結(jié)球白菜、菜豆和BoCCCh1及野生大豆(KHN40076.1)和不結(jié)球白菜的序列相似性最低，僅為43%；BoCCCh1與不結(jié)球白菜的相似性最高，與亞麻薺(XP_010483463.1)、薺菜(EOA13094.1)、擬南芥(AED97077.1)及山萮菜(ESQ42513.1)等十字花科植物的相似性分別為77%、78%、78%和79%，而與其他植物的相似性較低，為43%～47%。

從圖4可以看出：BoCCCh1及15條同源序列均有2個(gè)CCCH鋅指結(jié)構(gòu)，它們是蛋白序列中最為

保守的區(qū)域；第1個(gè)CCCH鋅指結(jié)構(gòu)在+296、+298、+299和+302位存在差異，第2個(gè)鋅指結(jié)構(gòu)較第1個(gè)保守，有3處出現(xiàn)差異，分別位于+328、+329和+331；BoCCCh1、不結(jié)球白菜和山萮菜的2個(gè)鋅指結(jié)構(gòu)序列完全一致，類似現(xiàn)象也出現(xiàn)在擬南芥、薺菜和亞麻薺、菜豆和大豆、蘋果和草莓及桃和梅的組合中；另外，雷蒙德氏棉與十字花科植物第2個(gè)鋅指結(jié)構(gòu)序列完全一致。

利用Mega 3.1軟件構(gòu)建系統(tǒng)發(fā)育樹。結(jié)果(圖5)表明：16條蛋白序列可分為4類，BoCCCh1先與不結(jié)球白菜聚為一組，再與山萮菜、擬南芥、亞麻薺和薺菜等十字花科植物組成Ⅳ類，支持率達(dá)100%；豆科的菜豆和野生大豆聚為一類(Ⅲ)，支持率為100%；薔薇科的野草莓、蘋果、梅和桃聚為一類(Ⅱ)，支持率也是100%；橙、雷蒙德氏棉、毛果楊和蓖麻處于同一分支，歸為Ⅰ類，但支持率低，僅51%。

3討論

圖4　BoCCCh1及其同源序列鋅指模體的比較Fig.4　Comparisons of zinc finger motifs among BoCCCh1 and its homologous sequences

圖5　用鄰接法構(gòu)建的BoCCCh1及其同源序列的系統(tǒng)進(jìn)化樹Fig.5　Phylogenetic tree of BoCCCh1 and its homologous sequences constructed using neighbor-joining method

鋅指蛋白廣泛存在于真核生物中，在轉(zhuǎn)錄調(diào)控中起著重要作用。有關(guān)C2h1型鋅指蛋白的研究最多，而對(duì)C3H型的報(bào)道相對(duì)較少[8,18]，與青花菜鋅指蛋白相關(guān)的研究很少。GAO等[19]在研究種子和幼苗硫代葡萄糖苷代謝時(shí)，利用RNA-Seq鑒定出1 633個(gè)轉(zhuǎn)錄因子基因，其中C3H與C2h1鋅指蛋白基因的數(shù)量分別為69和65。C3H型鋅指結(jié)構(gòu)由3個(gè)半胱氨酸和1個(gè)組氨酸構(gòu)成，參與植物生長發(fā)育、物質(zhì)代謝和逆境響應(yīng)[13,20-21]。根據(jù)半胱氨酸和組氨酸之間的氨基酸殘基數(shù)，C3H型鋅指結(jié)構(gòu)共有序列的最初定義是C—X6-14—C—X4-5—C—X3—H[22]，在對(duì)擬南芥和水稻的C3H進(jìn)行全基因組鑒定時(shí)發(fā)現(xiàn)了一些新類型，重新定義為C—X4-15—C—X4-6—C—X3—H[23]。鋅指蛋白通常含1～6個(gè)鋅指結(jié)構(gòu)，但也有例外，如玉米的ZmC3p含7個(gè)C3H，類型為C—X17—C—X6—C—X3—H，除此之外，還有C—X8—C—X5—C—X3—H、C—X7—C—X5—C—X3—H、C—X7—C—X4—C—X3—H、C—X5—C—X4—C—X3—H、C—X7—C—X6—C—X3—H和C—X8—C—X4—C—X3—H[24]。在本研究中BoCCCh1具2個(gè)鋅指結(jié)構(gòu)，分別為C—X8—C—X5—C—X3—H和C—X5—C—X4—C—X3—H，它們均為常見類型，擬南芥C3H14和C3H15各具2個(gè)鋅指結(jié)構(gòu)，都是C—X8—C—X5—C—X3—H類型[23]；在毛果楊的211個(gè)C3H鋅指結(jié)構(gòu)中，C—X8—C—X5—C—X3—H有96個(gè)，C—X7—C—X5—C—X3—H有76個(gè)[25]，而在擬南芥的148個(gè)C3H鋅指結(jié)構(gòu)中，C—X8—C—X5—C—X3—H和C—X7—C—X5—C—X3—H類型的C3H分別為78和43個(gè)。

錨蛋白重復(fù)序列(ankyrin repeats，ANK)結(jié)構(gòu)域在蛋白質(zhì)-蛋白質(zhì)互作中起著重要作用，它們參與轉(zhuǎn)錄起始、細(xì)胞周期調(diào)控、細(xì)胞骨架、離子運(yùn)輸和信號(hào)轉(zhuǎn)導(dǎo)[26]。玉米C3H鋅指蛋白ZmC3H4、ZmC3H10、ZmC3H43和ZmC3H63均帶有2個(gè)ANK結(jié)構(gòu)域[24]；在毛果楊的91個(gè)C3H鋅指蛋白中，PtC3p2、PtC3p3、PtC3p4、PtC3p5、PtC3p6、PtC3p7、PtC3p8、PtC3p9、PtC3H81、PtC3H82、PtC3H90和PtC3H91等12個(gè)蛋白具ANK[25]；而在擬南芥中，有5個(gè)C3H型鋅指蛋白具ANK，它們是AtC3p0、AtC3H56、AtC3H66、AtC3H47和AtC3h19[23]，其中AtC3h19又名ZFAR1，它發(fā)生突變后，對(duì)灰葡萄孢菌的敏感性增加[27]。在本研究中，推導(dǎo)的BoCCCh1具2個(gè)ANK結(jié)構(gòu)域，分別位于57～87和92～124處，其功能尚需進(jìn)一步研究。

C3H型鋅指蛋白基因參與植物生長發(fā)育，如毛果楊的34個(gè)CCCH在根中大量表達(dá)，24個(gè)在新葉中高表達(dá)，43個(gè)在雌花序或雄花序中表達(dá)[25]；與毛果楊不同，擬南芥中的大部分CCCH在根、葉、花序和種子中均有表達(dá)[23]；擬南芥的C3H14和C3H15在花莖、花和角果中表達(dá)量最高，它們與細(xì)胞次生壁加厚相關(guān)[23]。在本研究中，BoCCCH2在根中的表達(dá)量最高，而在花莖、葉、嫩角果、花蕾和花中的表達(dá)量相對(duì)較低。C3H型鋅指蛋白參與逆境脅迫響應(yīng)，陸地棉(Gossypiumhirsutum)GhTZF1的表達(dá)受聚乙二醇、鹽、茉莉酸甲酯和過氧化氫的誘導(dǎo)，該基因的過量表達(dá)可增加擬南芥的耐旱性和延緩因干旱引起的衰老[16]。本研究青花菜BoCCCH2的表達(dá)受霜霉菌和灰葡萄孢菌的誘導(dǎo)，但表達(dá)量和表達(dá)模式存在一定差異，暗示該基因與2種病菌的抗病反應(yīng)相關(guān)。

對(duì)青花菜BoCCCH2基因的克隆與表達(dá)分析，為該基因在抗病反應(yīng)中的功能鑒定奠定了基礎(chǔ)。下一步我們將開展載體構(gòu)建和轉(zhuǎn)基因研究，以明確BoCCCH2在霜霉病和灰霉病抗性反應(yīng)中的功能。

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中圖分類號(hào)Q 78

文獻(xiàn)標(biāo)志碼A

收稿日期(Received)：2015-04-16；接受日期(Accepted)：2015-06-17；網(wǎng)絡(luò)出版日期(Published online)：2016-03-19

*通信作者(

Corresponding author)：蔣明(http://orcid.org/0000-0001-9556-2249)，E-mail:jiangming1973@139.com

基金項(xiàng)目：浙江省本科院校中青年學(xué)科帶頭人學(xué)術(shù)攀登項(xiàng)目(pd2013420)；浙江省自然科學(xué)基金(LY13C150003)；浙江省重點(diǎn)學(xué)科(浙江省臺(tái)州學(xué)院生態(tài)學(xué))開放課題(EKD2013-03)；浙江省公益技術(shù)應(yīng)用研究計(jì)劃項(xiàng)目(2016C32091).

URL:http://www.cnki.net/kcms/detail/33.1247.S.20160319.2344.002.html