枳PtMLP1啟動(dòng)子的克隆和表達(dá)分析

姚利曉，蘇娟，郭興茹，李鳳龍，何永睿，鄒修平，陳善春

枳啟動(dòng)子的克隆和表達(dá)分析

姚利曉，蘇娟，郭興茹，李鳳龍，何永睿，鄒修平，陳善春

西南大學(xué)柑桔研究所/國(guó)家柑桔工程技術(shù)中心/國(guó)家柑桔品種改良中心，重慶 400712

【目的】基因工程是柑橘品種改良的一種重要手段。本研究基于枳根消減文庫(kù)中主要乳膠蛋白基因片段，克隆根特異啟動(dòng)子序列，為研究外源基因在柑橘根組織的特異表達(dá)奠定基礎(chǔ)?！痉椒ā客纯寺〖皢?dòng)子序列。利用ExPASy、PSIPRED、SWISS-MODEL等在線軟件對(duì)編碼蛋白的理化特征、二級(jí)結(jié)構(gòu)和三級(jí)結(jié)構(gòu)進(jìn)行生物信息學(xué)分析，利用PlantCARE數(shù)據(jù)庫(kù)對(duì)啟動(dòng)子的順式作用元件進(jìn)行預(yù)測(cè)。實(shí)時(shí)熒光定量PCR法對(duì)在不同樹(shù)齡枳根和葉中的表達(dá)進(jìn)行分析。構(gòu)建啟動(dòng)子與標(biāo)記基因的融合載體，利用根癌農(nóng)桿菌轉(zhuǎn)化法轉(zhuǎn)化枳上胚軸，GUS染色觀察標(biāo)記基因的表達(dá)部位。【結(jié)果】枳含2個(gè)外顯子和1個(gè)內(nèi)含子，開(kāi)放閱讀框長(zhǎng)471 bp。PtMLP1蛋白由156個(gè)氨基酸組成，分子量17.63 kDa，等電點(diǎn)5.49，含Bet v I功能域。其二級(jí)結(jié)構(gòu)含3個(gè)-螺旋和7個(gè)-折疊，三級(jí)結(jié)構(gòu)包含一個(gè)保守疏水基結(jié)合位點(diǎn)和一個(gè)富含甘氨酸的回環(huán)結(jié)構(gòu)。5′端1 666 bp的上游調(diào)控序列不僅有TATA-box、CAAT-box等啟動(dòng)子結(jié)構(gòu)的核心元件，還具有多個(gè)根組織特異表達(dá)元件，以及TGACG-motif、P-box和ABRE等激素應(yīng)答相關(guān)的順式作用元件。3′端非翻譯區(qū)具有加尾信號(hào)AATAAA。該基因在1月齡苗、6月齡苗、20年生成年枳根中的表達(dá)量分別是葉中的46.34、74.82、110.25倍。構(gòu)建啟動(dòng)子的融合表達(dá)載體pBI121-ProPtMLP1::，獲得枳轉(zhuǎn)基因植株。啟動(dòng)子驅(qū)動(dòng)在轉(zhuǎn)基因枳幼苗根中特異表達(dá)，在3個(gè)轉(zhuǎn)基因枳株系的根中表達(dá)量分別為葉中表達(dá)量的124.78、11.53和7.76倍?！窘Y(jié)論】獲得柑橘主要乳膠蛋白及啟動(dòng)子序列，該啟動(dòng)子可驅(qū)動(dòng)標(biāo)記基因在柑橘根組織特異表達(dá)。

枳；主要乳膠蛋白；根特異性啟動(dòng)子；

0 引言

【研究意義】柑橘是世界第一大水果，也是我國(guó)南方地區(qū)農(nóng)民脫貧致富和鄉(xiāng)村振興的支柱產(chǎn)業(yè)。我國(guó)柑橘的產(chǎn)量和種植面積均居世界第一位。然而，柑橘面臨著嚴(yán)重的干旱、凍害等非生物脅迫和黃龍病、潰瘍病等生物脅迫。轉(zhuǎn)基因技術(shù)是改良柑橘品質(zhì)和增強(qiáng)抗性的重要手段[1-2]。啟動(dòng)子是決定外源基因轉(zhuǎn)錄效率的關(guān)鍵因素。在柑橘轉(zhuǎn)基因研究中，來(lái)自煙草花葉病毒的35S啟動(dòng)子（cauliflower mosaic virus，CaMV35S）是最常用的組成型啟動(dòng)子[3]。但組成型啟動(dòng)子驅(qū)動(dòng)外源基因在植物體內(nèi)持續(xù)、高效表達(dá)，不僅耗費(fèi)植物大量能量和養(yǎng)分，也可能會(huì)改變某些性狀，影響植株的正常生長(zhǎng)發(fā)育。因此，組織特異性啟動(dòng)子和誘導(dǎo)型啟動(dòng)子開(kāi)始受到研究者的關(guān)注，前者驅(qū)動(dòng)目的基因在特定的植物組織表達(dá)，后者可在特定的條件下誘導(dǎo)目的基因表達(dá)[4]。在植物轉(zhuǎn)基因研究中使用特異性啟動(dòng)子，既能夠減少轉(zhuǎn)基因植物能量和養(yǎng)分的消耗，又可以降低轉(zhuǎn)基因植物環(huán)境釋放的風(fēng)險(xiǎn)。【前人研究進(jìn)展】柑橘中研究相對(duì)較多的組織特異性啟動(dòng)子是韌皮部啟動(dòng)子，有柑橘來(lái)源的CsPP2.B1、CsVTE2[5]和CsSUS1p啟動(dòng)子[6]。也有外源韌皮部特異啟動(dòng)子用于柑橘轉(zhuǎn)基因的研究，如水稻東格魯桿狀病毒（RTBV）啟動(dòng)子、擬南芥蔗糖/質(zhì)子同向轉(zhuǎn)運(yùn)體基因（AtSUC2）啟動(dòng)子和豇豆富甘氨酸蛋白基因（GRP）啟動(dòng)子[7-8]。另外，來(lái)自馬鈴薯的KST1啟動(dòng)子在柑橘保衛(wèi)細(xì)胞特異表達(dá)[9]，花、果實(shí)、種子、胚和木質(zhì)部等組織和器官特異性啟動(dòng)子也有應(yīng)用于柑橘轉(zhuǎn)基因研究的報(bào)道[3]。誘導(dǎo)型啟動(dòng)子也用于柑橘轉(zhuǎn)基因研究，如低溫和光誘導(dǎo)的柑橘Ruby1啟動(dòng)子[10]和人工合成的可被柑橘潰瘍病菌效應(yīng)因子特異識(shí)別的啟動(dòng)子[11]?！颈狙芯壳腥朦c(diǎn)】根是植物的支撐器官，也是植物吸收水分和營(yíng)養(yǎng)元素及響應(yīng)外界脅迫的重要器官。已經(jīng)從擬南芥[12]、煙草[13-14]、大豆[15]、鷹嘴豆[16]、水稻[17]、蘋果[18]等植物中分離出根組織特異性啟動(dòng)子。目前，尚未發(fā)現(xiàn)柑橘根特異性啟動(dòng)子的報(bào)道。雖然異源植物根特異性啟動(dòng)子有可能用于柑橘轉(zhuǎn)基因的研究，但是，特異性啟動(dòng)子的異源表達(dá)存在組成型表達(dá)的風(fēng)險(xiǎn)，如草莓根特異性啟動(dòng)子FaRB7在煙草中異源轉(zhuǎn)化顯示為組成型表達(dá)特性[19]，脅迫誘導(dǎo)型啟動(dòng)子AtRD29A在柑橘中喪失了誘導(dǎo)表達(dá)功能[20]?！緮M解決的關(guān)鍵問(wèn)題】枳屬于冬季落葉性灌木或灌木狀小喬木，是柑橘產(chǎn)區(qū)常用的砧木。枳砧柑橘一般表現(xiàn)樹(shù)勢(shì)較矮化、抗寒性強(qiáng)、結(jié)果時(shí)間較早、果實(shí)品質(zhì)佳、抗腳腐病和衰退病等優(yōu)點(diǎn)，但易感裂皮病，對(duì)鹽堿性土壤敏感，易表現(xiàn)缺鐵性黃化癥狀。本研究在前期構(gòu)建枳根消減文庫(kù)和全長(zhǎng)文庫(kù)[21-22]基礎(chǔ)上，對(duì)枳主要乳膠蛋白基因和啟動(dòng)子進(jìn)行克隆和轉(zhuǎn)基因植物組織表達(dá)分析，為柑橘根部性狀的改良提供有利的基因資源，也為其他植物的根組織特異表達(dá)提供候選啟動(dòng)子。

1 材料與方法

試驗(yàn)于2021—2022年進(jìn)行。枳（）成年樹(shù)葉片和種子取自國(guó)家柑橘種質(zhì)資源圃（重慶）。枳實(shí)生苗和轉(zhuǎn)基因植株在國(guó)家柑桔品種改良中心網(wǎng)室中培養(yǎng)。試驗(yàn)所用引物見(jiàn)表1。

表1 試驗(yàn)用引物

1.1 總RNA提取方法

分別取棗陽(yáng)小葉枳根和葉各100 mg，在研缽中迅速加液氮研磨成粉末。轉(zhuǎn)入1.5 mL RNase-free離心管中，加入1 mL Trizol試劑，按照Trizol法提取樣品總RNA。

1.2 改良CTAB法提取DNA

稱取約1 g幼葉材料，置于研缽中液氮研磨至粉末狀，按照改良CTAB法提取葉片總DNA。溶于200 μL TE溶液，紫外分光光度法結(jié)合瓊脂糖凝膠電泳檢測(cè)DNA的濃度和純度，儲(chǔ)存于-20 ℃?zhèn)溆谩?/p>

1.3 PtMLP1啟動(dòng)子和內(nèi)含子序列克隆

根據(jù)5′端上游調(diào)控序列設(shè)計(jì)引物，以棗陽(yáng)小葉枳基因組DNA為模板，2PaR1和2PaS2引物對(duì)克隆啟動(dòng)子序列。反應(yīng)體系為10×Ex Taq Buffer 5 μL，MgCl2（25 mmol?L-1）4 μL，dNTP（10 mmol?L-1each）1 μL，上、下游引物（20 μmol?L-1）各1 μL，DNA模板1 μL，Ex Taq 0.5 μL，加ddH2O至50 μL。反應(yīng)條件為95 ℃ 1 min；94 ℃ 30 s，55—50 ℃（0.5 ℃/循環(huán)、30 s），72 ℃ 2 min，10個(gè)循環(huán)；94 ℃ 30 s，60 ℃ 30 s，72 ℃ 1—2 min，30個(gè)循環(huán)；72 ℃ 10 min。通過(guò)瓊脂糖凝膠電泳純化回收PCR產(chǎn)物，與pMD19 simple載體連接，將陽(yáng)性克隆送三博遠(yuǎn)志基因公司測(cè)序。

1.4 生物信息學(xué)分析

利用在線軟件ExPASy（http://web.expasy.org/ compute_pi）預(yù)測(cè)蛋白分子量、理論等電點(diǎn)等理化特性。PSIPRED 4.0（http://bioinf.cs.ucl.ac.uk/psipred/）預(yù)測(cè)蛋白的二級(jí)結(jié)構(gòu)，SWISS-MODEL（https://swissmodel.expasy.org/）預(yù)測(cè)蛋白的三級(jí)結(jié)構(gòu)。TMHMM和SignalP V5.0（https://services.healthtech.dtu.dk/）預(yù)測(cè)跨膜區(qū)域和信號(hào)肽區(qū)域。利用PlantCARE數(shù)據(jù)庫(kù)（http://bioinformatics.psb.ugent.be/webtools/plantcare/ html/）預(yù)測(cè)啟動(dòng)子序列的順式作用元件。

1.5 實(shí)時(shí)熒光定量PCR

以500 ng總RNA為模板，采用PrimeScript?RT Reagent Kit（Perfect Real Time）在37 ℃反應(yīng)15 min合成cDNA第一鏈，85 ℃作用5 min滅活逆轉(zhuǎn)錄酶。實(shí)時(shí)熒光定量PCR的反應(yīng)體系為：2×SYBR?Premix Ex Taq Buffer 12.5 μL，上、下游引物（10 μmol?L-1）各1 μL，cDNA模板1 μL，加ddH2O至25 μL。反應(yīng)條件為95 ℃ 90 s；95 ℃10 s，61 ℃20 s，72 ℃20 s，40個(gè)循環(huán)；72 ℃延伸1 min。以為內(nèi)參基因，采用2-ΔΔCT法計(jì)算目的基因的相對(duì)表達(dá)量。每樣品設(shè)3次生物學(xué)重復(fù)。

1.6 pBI121-ProPtMLP1::GUS表達(dá)載體構(gòu)建

PCR克隆啟動(dòng)子序列，構(gòu)建中間載體pMD19-ProPtMLP1。然后用限制性內(nèi)切酶d Ⅲ和Ⅰ對(duì)pMD19-ProPtMLP1和pBI121分別進(jìn)行酶切，瓊脂糖凝膠純化回收目的片段。將回收的pBI121載體片段和目的片段用T4連接酶連接，轉(zhuǎn)入大腸桿菌DH5α中，提取單克隆抽提質(zhì)粒進(jìn)行酶切檢測(cè)試驗(yàn)，結(jié)果顯示pBI121-ProPtMLP1::載體構(gòu)建成功。電擊法轉(zhuǎn)化到感受態(tài)根癌農(nóng)桿菌EHA105細(xì)胞。

1.7 枳的遺傳轉(zhuǎn)化

用2%次氯酸鈉對(duì)枳成熟種子消毒，將其播種于滅菌的MS固體培養(yǎng)基中。待上胚軸生長(zhǎng)至8—10 cm，光照培養(yǎng)5 d。將含有pBI121-ProPtMLP1::載體的農(nóng)桿菌EHA105侵染切割成1 cm小段的枳上胚軸，同時(shí)以含pBI121空載體的農(nóng)桿菌侵染枳作為對(duì)照組。當(dāng)枳不定芽長(zhǎng)到1—2 cm的時(shí)候，將其轉(zhuǎn)移到MS生根培養(yǎng)基（0.25 mg·L-1-萘乙酸、0.25 mg·L-1吲哚-3-丁酸、500 mg·L-1羧芐青霉素）生根培養(yǎng)4—6周，取葉片提取DNA，以GUSF/GUSR引物進(jìn)行候選轉(zhuǎn)基因植物的PCR檢測(cè)。

1.8 GUS組織化學(xué)染色

柑橘轉(zhuǎn)基因株系的根、葉按照Peng等[23]的方法進(jìn)行GUS組織化學(xué)染色。GUS染液成分如下：100 mmol?L-1NaH2PO4，100 mmol?L-1Na2HPO4，0.5 mmol?L-1K4[Fe(CN)6]，0.5 mmol?L-1K3[Fe(CN)6]，10 mmol?L-1EDTA-Na2，1 mmol?L-1X-Gluc，0.1% Sodium azide，0.1% TritonX-100。37 ℃下孵育過(guò)夜，用70%酒精進(jìn)行脫色，最后進(jìn)行顯微觀察拍照。

2 結(jié)果

2.1 PtMLP1序列及生物信息學(xué)分析

通過(guò)核苷酸序列比對(duì)，發(fā)現(xiàn)枳根消減cDNA文庫(kù)中contig 22和singleton 297[22]與枳根全長(zhǎng)文庫(kù)中的JK316196[21]為同一基因。三者經(jīng)序列拼接后得到含完整開(kāi)放閱讀框的基因，編碼主要乳膠蛋白（major latex protein），將其命名為。利用2PaS2和2PaR1引物克隆啟動(dòng)子序列，2IS和2IR引物克隆內(nèi)含子序列。分析結(jié)構(gòu)，發(fā)現(xiàn)含2個(gè)外顯子（186 bp和285 bp）、1個(gè)內(nèi)含子（104 bp）及5′端上游調(diào)控序列（1 666 bp）和具有poly(A)信號(hào)AATAAA的3′端非翻譯區(qū)序列（207 bp）。

該基因開(kāi)放閱讀框長(zhǎng)471 bp，以ATG為起始密碼子，以TAG為終止密碼子，共編碼156個(gè)氨基酸（圖1）。分析發(fā)現(xiàn)其編碼蛋白具有保守Bet v 1功能域（2—152氨基酸殘基），分子量為17.63 kDa，等電點(diǎn)為5.49，不含信號(hào)肽和跨膜區(qū)。預(yù)測(cè)PtMLP1蛋白二級(jí)結(jié)構(gòu)中有3個(gè)-螺旋和7個(gè)-折疊，三級(jí)結(jié)構(gòu)中包含一個(gè)保守疏水基結(jié)合位點(diǎn)和一個(gè)富含甘氨酸的回環(huán)結(jié)構(gòu)。

啟動(dòng)子序列中具有TATA-box、CAAT- box等啟動(dòng)子結(jié)構(gòu)的核心元件和多個(gè)根特異表達(dá)的順式作用元件，還具有MYBHv1結(jié)合位點(diǎn)CCAAT、分生組織表達(dá)元件CAT-box、MeJA反應(yīng)元件TGACG-motif，赤霉素反應(yīng)元件P-box，脫落酸反應(yīng)元件ABRE等應(yīng)答元件（圖1）。

2.2 PtMLP1組織表達(dá)分析

實(shí)時(shí)熒光定量PCR分析結(jié)果表明，1月齡幼苗根中的表達(dá)量是葉的46.34倍，6月齡苗根的表達(dá)量是葉的78.42倍，20年生成年樹(shù)根的表達(dá)量是葉的110.25倍（圖2-A）。

為進(jìn)一步確認(rèn)在根中的特異表達(dá)，下載其同源基因Cs2g_pb010910在甜橙不同組織中的轉(zhuǎn)錄組數(shù)據(jù)（http://citrus.hzau.edu.cn/）并進(jìn)行分析。發(fā)現(xiàn)該基因在根轉(zhuǎn)錄組FPKM平均值為9 930.67，遠(yuǎn)高于早期胚珠、晚期胚珠、種子、幼果果肉、成熟果果肉和葉等組織（圖2-B）。

2.3 ProPtMLP1::GUS在枳根中特異表達(dá)

同源克隆啟動(dòng)子序列，用其取代pBI121載體上d III和I酶切位點(diǎn)之間的CaMV35S啟動(dòng)子片段，成功構(gòu)建pBI121-ProPtMLP1::載體（圖3）。

對(duì)候選轉(zhuǎn)基因枳抽提DNA進(jìn)行標(biāo)記基因的PCR檢測(cè)，結(jié)果顯示獲得10株有ProPtMLP1啟動(dòng)子插入基因組的幼苗。組織化學(xué)染色結(jié)果表明轉(zhuǎn)基因枳的葉中無(wú)色，根顯現(xiàn)藍(lán)色，且根組織縱切觀察顯示維管束組織染色深于表皮。而對(duì)照組（CaMV35S啟動(dòng)子）枳幼苗的根和葉中均顯示藍(lán)色。對(duì)部分枳轉(zhuǎn)基因苗進(jìn)行實(shí)時(shí)熒光定量PCR分析，結(jié)果表明轉(zhuǎn)基因苗根中的表達(dá)量分別是葉中的124.78倍、11.53倍、7.77倍（圖4）。

3 討論

3.1 PtMLP1是柑橘中新發(fā)現(xiàn)的一種主要乳膠蛋白基因

主要乳膠蛋白（major latex protein，MLP）是植物特有的一種蛋白家族，其三級(jí)結(jié)構(gòu)可形成疏水空腔結(jié)構(gòu)和富含甘氨酸的回環(huán)結(jié)構(gòu)，與疏水化合物相結(jié)合[24]。MLP首次從罌粟的乳膠中鑒定出來(lái)[25]，廣泛存在于其他植物中，目前已經(jīng)從葡萄、蘋果、黃瓜、西葫蘆等園藝植物基因組中鑒定出多個(gè)家族成員[26-29]，在柑橘中尚未見(jiàn)關(guān)于主要乳膠蛋白基因的報(bào)道。本研究從枳中克隆出，其編碼蛋白不僅具有MLP蛋白二級(jí)結(jié)構(gòu)中存在的-螺旋和-折疊，而且三級(jí)結(jié)構(gòu)含有疏水基結(jié)合位點(diǎn)和富含甘氨酸的回環(huán)結(jié)構(gòu)，是一種主要乳膠蛋白的編碼基因。轉(zhuǎn)錄組數(shù)據(jù)和實(shí)時(shí)熒光定量PCR結(jié)果顯示在柑橘根中特異表達(dá)。這與擬南芥和、棉花、西葫蘆在根中優(yōu)勢(shì)表達(dá)[26,30-32]結(jié)果相一致。MLP基因家族成員在植物的其他組織中也存在優(yōu)勢(shì)或特異表達(dá)，如蘋果主要在花中表達(dá)[33]。關(guān)于MLP功能的解析尚處于初級(jí)階段，研究顯示MLP正向調(diào)控種子休眠、營(yíng)養(yǎng)生長(zhǎng)，抑制生殖生長(zhǎng)[31-32]，在植物中過(guò)表達(dá)可增強(qiáng)對(duì)凍害、干旱等非生物脅迫的抗性[33-34]，但具體調(diào)控機(jī)制未知，其在柑橘中的生物學(xué)功能值得關(guān)注。

黑色小寫字體表示啟動(dòng)子序列，棕色橫線為順式作用元件：①根特異基序，②TGACT基序，③ P盒子，④CCAAT盒子，⑤CAT盒子，⑥ABRE，⑦CAAT盒子，⑧TATA盒子。紅色大寫字體表示外顯子區(qū)域和編碼氨基酸；綠色小寫字體表示內(nèi)含子；藍(lán)色小寫字體表示3′非翻譯區(qū)，具有加尾信號(hào)AATAAA

The black lowercases mean promoter sequence, with the cis-acting elements marked with brown horizontal lines: ① root specific motif, ② TGACT-motif, ③ P-box, ④ CCAAT-box, ⑤ CAT-box, ⑥ ABRE, ⑦ CAAT-box, ⑧TATA-box. The red uppercases mean exons and their encoded amino acids. The green lowercases mean intron sequence. The blue lowercases mean a 3′-terminal untranslated region with the poly (A) signal (AATAAA)

圖1及啟動(dòng)子序列

Fig. 1 The DNA sequence of

A：PtMLP1在不同生長(zhǎng)期枳根和葉中的表達(dá)結(jié)果；B：PtMLP1同源基因Cs2g_pb010910在甜橙不同組織轉(zhuǎn)錄組中的FPKM值（數(shù)據(jù)來(lái)源于http://citrus.hzau.edu.cn/）A: The expression of PtMLP1 in roots and leaves of 1-month, 6-month, and 20-year-old Poncirus trifoliate; B: FPKM value of orthologous gene with PtMLP1 from RNA-seq data of sweet orange (Data from http://citrus.hzau.edu.cn/)

圖3 PtMLP1啟動(dòng)子克?。ˋ）和植物表達(dá)載體構(gòu)建示意圖（B）

圖4 部分ProPtMLP1::GUS轉(zhuǎn)基因枳中GUS的表達(dá)分析

3.2 PtMLP1啟動(dòng)子是一種根特異表達(dá)啟動(dòng)子

在柑橘轉(zhuǎn)基因研究中，常用煙草花葉病毒的35S啟動(dòng)子作為外源基因的組成型啟動(dòng)子，也利用誘導(dǎo)型啟動(dòng)子和韌皮部、木質(zhì)部、花、果實(shí)、種子和胚等組織和器官特異性啟動(dòng)子[3]。根是植物生長(zhǎng)、發(fā)育和抵抗不良環(huán)境的重要組織，關(guān)于柑橘根特異性表達(dá)基因已有研究成果，尚未發(fā)現(xiàn)關(guān)于柑橘根特異性啟動(dòng)子的報(bào)道或其他植物根特異性啟動(dòng)子應(yīng)用于柑橘轉(zhuǎn)基因研究的報(bào)道。

柑橘根中高表達(dá)或優(yōu)勢(shì)表達(dá)的基因常常來(lái)自功能基因家族的研究。海藻糖-6-磷酸合成酶（trehalose-6- phosphate synthase，TPS）在柑橘基因組中有8個(gè)成員，其中6個(gè)在根組織高表達(dá)，另外2個(gè)在莖中高表達(dá)[35]。6個(gè)多胺氧化酶基因在柑橘根中都存在優(yōu)勢(shì)表達(dá)[36]。和是8個(gè)H+-ATPase家族基因中在根中優(yōu)勢(shì)表達(dá)的成員[37]。筆者課題組在資陽(yáng)香橙中發(fā)現(xiàn)與鐵吸收和轉(zhuǎn)運(yùn)相關(guān)的鐵螯合還原酶和5在根中優(yōu)勢(shì)表達(dá)，并受到缺鐵脅迫的誘導(dǎo)[38]。另外，筆者課題組前期構(gòu)建了枳根的消減cDNA文庫(kù)，豐富了根組織中優(yōu)勢(shì)表達(dá)基因的資源[22]。MLP家族基因的啟動(dòng)子如擬南芥啟動(dòng)子可驅(qū)動(dòng)標(biāo)記基因在擬南芥根中特異表達(dá)[31]。本研究結(jié)果增加了MLP根特異性啟動(dòng)子的種類，也為柑橘轉(zhuǎn)基因功能研究和種質(zhì)創(chuàng)制提供了本源的根組織特異啟動(dòng)子。

4 結(jié)論

通過(guò)序列比對(duì)，發(fā)現(xiàn)枳根消減文庫(kù)中contig22和singleton 297為的片段。的啟動(dòng)子可在轉(zhuǎn)基因枳中驅(qū)動(dòng)標(biāo)記基因在根中特異表達(dá)。這種從根的特異表達(dá)基因發(fā)掘根特異啟動(dòng)子的研究思路可篩選和鑒定更多的柑橘組織特異性啟動(dòng)子，為柑橘砧木的改良提供更多的候選啟動(dòng)子種類，也為該啟動(dòng)子在柑橘和其他植物基因改良中的應(yīng)用奠定了理論基礎(chǔ)。

[1] 姚利曉, 何永睿, 鄒修平, 雷天剛, 許蘭珍, 彭愛(ài)紅, 陳善春. 柑橘基因工程育種研究策略及其進(jìn)展. 果樹(shù)學(xué)報(bào), 2013, 30(6): 1056-1064.

YAO L X, HE Y R, ZOU X P, LEI T G, XU L Z, PENG A H, CHEN S C. Advances and strategies in citrus genetic engineering and breeding. Journal of Fruit Science, 2013, 30(6): 1056-1064. (in Chinese)

[2] SOARES J M, TANWIR S E, GROSSER J W, DUTT M. Development of genetically modified citrus plants for the control of citrus canker and huanglongbing. Tropical Plant Pathology, 2020, 45(3): 237-250.

[3] CONTI G, XOCONOSTLE-CáZARES B, MARCELINO-PéREZ G, HOPP H E, REYES C A.genetic transformation: An overview of the current strategies and insights on the new emerging technologies. Frontiers in Plant Science, 2021, 12: 768197.

[4] ZHONG V, ARCHIBALD B N, BROPHY J A N. Transcriptional and post-transcriptional controls for tuning gene expression in plants. Current Opinion in Plant Biology, 2023, 71: 102315.

[5] DOS ANJOS BEZERRA Y C, MARQUES J P R, STIPP L C L, ATTíLIO L B, FREITAS-ASTúA J, DE ASSIS ALVES MOUR?O FILHO F. How to drive phloem gene expression? A case study with preferentially expressed citrus gene promoters. Revista Brasileira De Fruticultura, 2021, 43(4): e-005.

[6] SINGER S D, HILY J M, COX K D. The sucrose synthase-1 promoter fromdirects expression of the-glucuronidase reporter gene in phloem tissue and in response to wounding in transgenic plants. Planta, 2011, 234(3): 623-637.

[7] DUTT M, ANANTHAKRISHNAN G, JAROMIN M K, BRLANSKY R H, GROSSER J W. Evaluation of four phloem-specific promoters in vegetative tissues of transgenic citrus plants. Tree Physiology, 2012, 32(1): 83-93.

[8] 許蘭珍, 彭愛(ài)紅, 何永睿, 姚利曉, 雷天剛, 劉小豐, 姜國(guó)金, 鄒修平, 陳善春. 異源韌皮部特異啟動(dòng)子在轉(zhuǎn)基因枳中的表達(dá). 園藝學(xué)報(bào), 2014, 41(1): 1-8.

XU L Z, PENG A H, HE Y R, YAO L X, LEI T G, LIU X F, JIANG G J, ZOU X P, CHEN S C. Expression analysis of three phloem-specific promoters in transgenic. Acta Horticulturae Sinica, 2014, 41(1): 1-8. (in Chinese)

[9] KELLY G, LUGASSI N, BELAUSOV E, WOLF D, KHAMAISI B, BRANDSMA D, KOTTAPALLI J, FIDEL L, BEN-ZVI B, EGBARIA A, ACHEAMPONG A K, ZHENG C L, OR E, DISTELFELD A, DAVID-SCHWARTZ R, CARMI N, GRANOT D. TheKST1 partial promoter as a tool for guard cell expression in multiple plant species. Journal of Experimental Botany, 2017, 68(11): 2885-2897.

[10] HUANG D, YUAN Y, TANG Z Z, HUANG Y, KANG C Y, DENG X X, XU Q. Retrotransposon promoter of Ruby1 controls both light- and cold-induced accumulation of anthocyanins in blood orange. Plant, Cell & Environment, 2019, 42(11): 3092-3104.

[11] SHANTHARAJ D, R?MER P, FIGUEIREDO J F L, MINSAVAGE G V, KR?NAUER C, STALL R E, MOORE G A, FISHER L C, HU Y, HORVATH D M, LAHAYE T, JONES J B. An engineered promoter driving expression of a microbial avirulence gene confers recognition of TAL effectors and reduces growth of diversestrains in citrus. Molecular Plant Pathology, 2017, 18(7): 976-989.

[12] GONG J M, LEE D A, SCHROEDER J I. Long-distance root-to-shoot transport of phytochelatins and cadmium in. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100(17): 10118-10123.

[13] CAI T C, CHEN H, YAN L M, ZHANG C, DENG Y, WU S X, YANG Q, PAN R L, RAZA A, CHEN S H, ZHUANG W J. The root-specific NtR12 promoter-based expression of RIP increased the resistance against bacterial wilt disease in tobacco.Molecular Biology Reports, 2022, 49(12): 11503-11514.

[14] ZHANG C, PAN S F, CHEN H, CAI T C, ZHUANG C H, DENG Y, ZHUANG Y H, ZENG Y H, CHEN S H, ZHUANG W J. Characterization of NtREL1, a novel root-specific gene from tobacco, and upstream promoter activity analysis in homologous and heterologous hosts. Plant Cell Reports, 2016, 35(4): 757-769.

[15] CHEN L, JIANG B J, WU C X, SUN S, HOU W S, HAN T F. The characterization of, a root-specific gene from soybean, and the expression analysis of its promoter.Plant Cell, Tissue and Organ Culture, 2015, 121(2): 259-274.

[16] KHANDAL H, GUPTA S K, DWIVEDI V, MANDAL D, SHARMA N K, VISHWAKARMA N K, PAL L, CHOUDHARY M, FRANCIS A, MALAKAR P, SINGH N P, SHARMA K, SINHAROY S, SINGH N P, SHARMA R, CHATTOPADHYAY D. Root-specific expression of chickpea cytokinin oxidase/dehydrogenase 6 leads to enhanced root growth, drought tolerance and yield without compromising nodulation. Plant Biotechnology Journal, 2020, 18(11): 2225-2240.

[17] LI Y Y, LI C X, CHENG L Z, YU S S, SHEN C J, PAN Y. Over- expression ofunder a rice root specific promoter. Plant Physiology and Biochemistry, 2019, 136: 52-57.

[18] LV D M, ZHANG Y H. Isolation and functional analysis of appleandgene promoters in transgenic. Plant Cell, Tissue and Organ Culture, 2017, 129(1): 133-143.

[19] VAUGHAN S P, JAMES D J, LINDSEY K, MASSIAH A J. Characterization of FaRB7, a near root-specific gene from strawberry (× ananassa Duch.) and promoter activity analysis in homologous and heterologous hosts. Journal of Experimental Botany, 2006, 57(14): 3901-3910.

[20] ORBOVI? V, ALI RAVANFAR S, ACANDA Y, NARVAEZ J, MERRITT B A, LEVY A, LOVATT C J. Stress-induciblepromoter constitutively drivesandexpression and precocious flowering in transgenicspp. Transgenic Research, 2021, 30(5): 687-699.

[21] 姚利曉, 王金萍, 何永睿, 雷天剛, 許蘭珍, 彭愛(ài)紅, 鄒修平, 陳善春. 枳根cDNA全長(zhǎng)文庫(kù)的構(gòu)建與分析. 園藝學(xué)報(bào), 2015, 42(1): 149-156.

YAO L X, WANG J P, HE Y R, LEI T G, XU L Z, PENG A H, ZOU X P, CHEN S C. Construction and analysis of a root full-length cDNA library of. Acta Horticulturae Sinica, 2015, 42(1): 149-156. (in Chinese)

[22] 姚利曉, 何永睿, 許蘭珍, 雷天剛, 彭愛(ài)紅, 鄒修平, 陳善春. 應(yīng)用抑制性消減雜交技術(shù)從枳中篩選根特異表達(dá)基因. 園藝學(xué)報(bào), 2014, 41(12): 2481-2488.

YAO L X, HE Y R, XU L Z, LEI T G, PENG A H, ZOU X P, CHEN S C. Identification of root-specific genes with subtractive suppression hybridization from. Acta Horticulturae Sinica, 2014, 41(12): 2481-2488. (in Chinese)

[23] PENG A H, ZOU X P, XU L Z, HE Y R, LEI T G, YAO L X, LI Q, CHEN S C. Improved protocol for the transformation of adultOsbeck ‘Tarocco’ blood orange tissues.Cellular & Developmental Biology-Plant, 2019, 55(6): 659-667.

[24] FUJITA K, INUI H. Review: Biological functions of major latex-like proteins in plants. Plant Science, 2021, 306: 110856.

[25] NESSLER C L, KURZ W G W, PELCHER L E. Isolation and analysis of the major latex protein genes of opium poppy.Plant Molecular Biology, 1990, 15(6): 951-953.

[26] FUJITA K, CHITOSE N, CHUJO M, KOMURA S, SONODA C, YOSHIDA M, INUI H. Genome-wide identification and characterization of major latex-like protein genes responsible for crop contamination in. Molecular Biology Reports, 2022, 49(8): 7773-7782.

[27] ZHANG N B, LI R M, SHEN W, JIAO S Z, ZHANG J X, XU W R. Genome-wide evolutionary characterization and expression analyses of major latex protein (MLP) family genes in. Molecular Genetics and Genomics, 2018, 293(5): 1061-1075.

[28] YUAN G P, HE S S, BIAN S X, HAN X L, LIU K, CONG P H, ZHANG C X. Genome-wide identification and expression analysis of major latex protein () family genes in the apple (Borkh.) genome. Gene, 2020, 733: 144275.

[29] KANG Y Y, TONG J L, LIU W, JIANG Z L, PAN G Z, NING X P, YANG X, ZHONG M. Comprehensive analysis of major latex-like protein family genes in cucumber (L.) and their potential roles inblight resistance. International Journal of Molecular Sciences, 2023, 24(1): 784.

[30] YANG C L, LIANG S, WANG H Y, HAN L B, WANG F X, CHENG H Q, WU X M, QU Z L, WU J H, XIA G X. Cotton major latex protein 28 functions as a positive regulator of the ethylene responsive factor 6 in defense against. Molecular Plant, 2015, 8(3): 399-411.

[31] CHONG S N, RAVINDRAN P, KUMAR P P. Regulation of primary seed dormancy by major latex protein-like protein329 inis dependent on dna-binding one zinc finger6. Journal of Experimental Botany, 2022, 73(19): 6838-6852.

[32] GUO D, WONG W S, XU W Z, SUN F F, QING D J, LI N.-cinnamic acid-enhanced 1 gene plays a role in regulation ofbolting. Plant Molecular Biology, 2011, 75(4/5): 481-495.

[33] LIU H, DU B Y, MA X C, WANG Y, CHENG N N, ZHANG Y H. Overexpression of major latex protein 423 () enhances the chilling stress tolerance in. Plant Science, 2023, 329: 111604.

[34] WANG Y P, YANG L, CHEN X, YE T T, ZHONG B, LIU R J, WU Y, CHAN Z L. Major latex protein-like protein 43 (MLP43) functions as a positive regulator during abscisic acid responses and confers drought tolerance in. Journal of Experimental Botany, 2016, 67(1): 421-434.

[35] LIU K H, ZHOU Y. Genome-wide identification of the trehalose-6- phosphate synthase gene family in sweet orange () and expression analysis in response to phytohormones and abiotic stresses. PeerJ, 2022, 10: e13934.

[36] WANG W, LIU J H. Genome-wide identification and expression analysis of the polyamine oxidase gene family in sweet orange (). Gene, 2015, 555(2): 421-429.

[37] SHI C Y, SONG R Q, HU X M, LIU X, JIN L F, LIU Y Z.PH5-like H(+)-ATPase genes: Identification and transcript analysis to investigate their possible relationship with citrate accumulation in fruits. Frontiers in Plant Science, 2015, 6: 135.

[38] YAO L X, HE Y R, FAN H F, XU L Z, LEI T G, ZOU X P, PENG A H, LI Q, CHEN S C. Identification and expression analysis of multiple ferric chelate reductases in. Journal of the American Society for Horticultural Science, 2017, 142(6): 419-424.

Cloning and Expression Analysis ofPromoter in

YAO LiXiao, SU Juan, GUO XingRu, LI FengLong, HE YongRui, ZOU XiuPing, CHEN ShanChun

Citrus Research Institute, Southwest University/National Citrus Engineering Technology Research Center/National Center for Citrus Varieties Improvement, Chongqing 400712

【Objective】Genetic transformation plays a significant role in exploring gene function and improving traits in citrus. Tissue-specific promoters is a key to regulate the expression of transgenes in particular tissues. Here, expression characteristics of thepromoter, isolated from the root subtractive library of, was thoroughly examined, which could lay a foundation for the specific expression of exogenous genes in citrus root tissue. 【Method】The complete sequence ofgene was cloned by PCR using DNA as a template. The physiochemical attributes, secondary and tertiary structures of PtMLP1 protein were predicted by ExPASy, PSIPRED, and SWISS-MODEL tools. Cis-acting elements inpromoter were predicted by PlantCARE. The expression pattern oftrees of diverse ages was examined by employing real-time qPCR. Furthermore, to investigate the tissue-specific expression of thepromoter in citrus, a pBI121-ProPtMLP1::plasmid, in whichexpression was controlled by thepromoter, was constructed and then introduced intothrough-mediated hypocotyl transformation. 【Result】consisted of two exons and one intron, which possessed a 471 bp open reading frame encoding a protein with 156 amino acid residues. This protein had a molecular weight of 17.63 kilodaltons with an isoelectric point of 5.49 and contained a Bet v I functional domain in its primary structure. Moreover, the secondary structure of PtMLP1 contained three α-helices and seven β-folds, while its tertiary structure had a conserved hydrophobic binding site and a cyclic domain, which was rich in glycine. Thepromoter was 1 666 bp long. Multiple root-specific expression elements, phytohormone response elements (such as the TGACG motif, P-box, and ABRE), and the TATA box and CAAT box core elements were predicted in the promoter. Additionally, the 3-terminal untranslated region ofwas predicted to contain a poly (A) signal AATAAA. Notably, the expression ofwas significantly higher in the roots of 1-month, 6-month, and 20-year-old, with fold changes of 46.34, 74.82, and 110.25, respectively, compared with those in leaves. GUS expression analysis of pBI121-ProPtMLP1::transgenic plants showed thatpromoter exhibited specific and high expression in roots, and its expression levels were 7.76 to 124.78 times of that in the leaves. 【Conclusion】The sequences of thegene and its promoter were successfully obtained, and the promoter demonstrated the ability to drive specific expression ofgene in citrus roots.

; major latex protein; root-specific promoter;

10.3864/j.issn.0578-1752.2023.24.009

2023-05-31；

2023-08-04

國(guó)家重點(diǎn)研發(fā)計(jì)劃（2021YFD140080，2021YFD160080）、國(guó)家現(xiàn)代農(nóng)業(yè)（柑橘）產(chǎn)業(yè)技術(shù)體系（CARS-26）

姚利曉，E-mail：yaolixiao@cric.cn。通信作者陳善春，E-mail：chenshanchun@cric.cn

（責(zé)任編輯 趙伶俐）