陳杉彬 孫思凡 聶 楠 杜 冰 何紹貞 劉慶昌 翟 紅

甘薯基因的克隆及耐鹽性、抗旱性鑒定

陳杉彬 孫思凡 聶 楠 杜 冰 何紹貞 劉慶昌 翟 紅*

中國(guó)農(nóng)業(yè)大學(xué)/ 農(nóng)業(yè)農(nóng)村部甘薯生物學(xué)與生物技術(shù)重點(diǎn)實(shí)驗(yàn)室/ 教育部作物雜種優(yōu)勢(shì)研究與利用重點(diǎn)實(shí)驗(yàn)室/ 北京市作物遺傳改良重點(diǎn)實(shí)驗(yàn)室, 北京 100193

(CCR4-associated factor 1)基因在植物發(fā)育、抗病等方面發(fā)揮著重要的作用。本研究根據(jù)前期獲得的差異表達(dá)EST序列, 克隆得到甘薯基因?；虻拈_放閱讀框(open reading frame, ORF)長(zhǎng)度為846 bp, 編碼281個(gè)氨基酸, 分子量為32.13 kD, 等電點(diǎn)為4.83。氨基酸序列比對(duì)和系統(tǒng)進(jìn)化樹分析表明, IbCAF1與甘薯近緣野生種(2)同源蛋白ItlCAF1有較高的同源性, 序列一致性為96.8%?；蚴艿絅aCl、PEG、ABA和H2O2的誘導(dǎo)表達(dá)。利用根癌農(nóng)桿菌介導(dǎo)法將基因轉(zhuǎn)入煙草, 過表達(dá)基因顯著提高了轉(zhuǎn)基因煙草植株的耐鹽性和抗旱性。在200 mmol L-1NaCl和10% PEG-6000的脅迫下,基因的過表達(dá)顯著上調(diào)了轉(zhuǎn)基因煙草植株中活性氧清除系統(tǒng)和脯氨酸合成相關(guān)基因的表達(dá), 增加了SOD活性、POD活性、脯氨酸含量, 降低了H2O2含量和丙二醛含量。表明基因能夠提高轉(zhuǎn)基因煙草植株的耐鹽性和抗旱性。本研究為后續(xù)甘薯基因耐鹽抗旱基因工程研究奠定了基礎(chǔ)。

甘薯;; 轉(zhuǎn)基因煙草; 耐鹽; 抗旱

土壤鹽害和干旱嚴(yán)重影響了作物的產(chǎn)量, 成為限制農(nóng)業(yè)生產(chǎn)的主要因素之一。據(jù)統(tǒng)計(jì), 世界上存在著8億公頃鹽漬化土地, 約20%灌溉農(nóng)業(yè)用地受到鹽堿化的影響[1]。我國(guó)耕地中鹽漬化面積達(dá)到920.9萬(wàn)公頃, 占全國(guó)耕地面積6.62%, 其中只有少部分被改良利用, 絕大部分仍未脫鹽及不斷遭受鹽漬危害[2]。全球氣候變化和人口增長(zhǎng)引發(fā)的全球水資源短缺問題威脅著農(nóng)業(yè)的可持續(xù)發(fā)展[3]。因此, 提高作物的耐鹽性和抗旱性迫在眉睫。

Ccr4-Not復(fù)合物是一種多亞基的蛋白質(zhì)復(fù)合物, 在真核生物中高度保守, 主要參與轉(zhuǎn)錄調(diào)控、mRNA降解、組蛋白修飾等重要生理過程[4-5]。CAF1 (CCR4-associated factor 1)是Ccr4-Not復(fù)合物中關(guān)鍵的亞基, 對(duì)復(fù)合物的形成及功能的行使起著重要作用[6]。CAF1屬于DEDD家族, 是參與mRNA降解的主要脫腺苷酸酶之一, 在植物生長(zhǎng)、脅迫響應(yīng)和抵御微生物病原菌等方面發(fā)揮著重要作用[7-8]。Sarowar等[9]將辣椒基因轉(zhuǎn)入番茄發(fā)現(xiàn),基因的過量表達(dá)不但顯著促進(jìn)番茄的生長(zhǎng), 還增強(qiáng)了對(duì)番茄晚疫病病菌()的抗性。Liang等[10]研究發(fā)現(xiàn),基因的過量表達(dá), 上調(diào)了和基因的表達(dá)量, 從而增強(qiáng)了擬南芥轉(zhuǎn)基因植株對(duì)丁香假單胞菌(DC3000)的抗性。基因的過表達(dá)增強(qiáng)了擬南芥轉(zhuǎn)基因植株對(duì)壞死性真菌病原菌()的敏感性[11]。Shimo等[12]研究發(fā)現(xiàn), 甜橙基因與甜橙潰瘍病的抗性有關(guān)。有關(guān)基因提高植物的耐鹽性報(bào)道較少。Walley等[13]研究發(fā)現(xiàn), 在200 mmol L-1NaCl脅迫下, 擬南芥突變體種子的發(fā)芽率顯著高于野生型對(duì)照。目前, 有關(guān)基因提高植物的抗旱性研究還未見報(bào)道。

甘薯是世界上第七大重要糧食作物, 同時(shí)也是飼料、工業(yè)原料、生物質(zhì)能源作物[14]。然而, 其產(chǎn)量也受到鹽和干旱脅迫的嚴(yán)重制約。因此, 培育耐鹽抗旱品種是甘薯育種的重要目標(biāo)之一?；蚬こ虨槎ㄏ蚋牧几适砟望}抗旱性提供了可行的方法[15-29]。本研究從甘薯中克隆得到基因, 其過量表達(dá)顯著提高了轉(zhuǎn)基因煙草植株的耐鹽性和抗旱性。

1 材料與方法

1.1 植物材料

以甘薯品種魯薯3號(hào)為材料克隆基因。利用煙草品種Wisconsin 38 (W38)分析基因的功能。

1.2 IbCAF1基因的克隆與序列分析

根據(jù)前期逆境脅迫獲得的基因EST序列, 在甘薯近緣野生種(2=2=30)數(shù)據(jù)庫(kù)(http://sweetpotato-garden.kazusa.or.jp/blast.html)中進(jìn)行BLAST分析, 獲得的ORF序列。參照楊元軍等[30]的方法提取甘薯總RNA。用TaKaRa公司的PrimeSript RT Kit合成cDNA。使用Primer Premier 5軟件, 根據(jù)基因ORF序列設(shè)計(jì)擴(kuò)增引物(表1), 以cDNA為模板, 進(jìn)行PCR擴(kuò)增。PCR擴(kuò)增體系為25 μL, 包含模板1.0 μL、10×Reaction buffer 2.5 μL、DNA Polymerase (5 U μL-1) 0.2 μL、dNTP (10 mmol L-1) 0.5 μL、-F和-R (10 μmol L-1) 各0.5 μL、ddH2O 19.8 μL。PCR擴(kuò)增程序?yàn)?4℃ 5 min; 95℃ 30 s, 57℃ 30 s, 72℃ 1 min, 35個(gè)循環(huán); 72℃ 5 min; 4℃保溫。

利用ProtParam (https://web.expasy.org/protparam/) 分析蛋白質(zhì)的理化性質(zhì), 利用SOPMA (https://npsa- prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html) 在線預(yù)測(cè)蛋白質(zhì)二級(jí)結(jié)構(gòu), 利用CDS (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)預(yù)測(cè)保守結(jié)構(gòu)域, 利用NCBI數(shù)據(jù)庫(kù)進(jìn)行Blastp分析(http://blast.ncbi.nlm.nih.gov/Blast.cgi), 尋找該基因的同源序列, 并利用MEGA7.0 (http://www. megasoftware.net/)構(gòu)建同源進(jìn)化樹。

1.3 IbCAF1基因的表達(dá)分析

選取長(zhǎng)勢(shì)良好的甘薯試管苗, 在MS固體培養(yǎng)基中繼代培養(yǎng)30 d, 在1/2 Hoagland溶液中培養(yǎng)3 d, 然后將試管苗分別浸入含有200 mmol L-1NaCl、20% PEG-6000、100 μmol L-1ABA和10 mmol L-1H2O2的1/2 Hoagland溶液中處理0、0.5、1、3、6、12、24、48 h, 處理后的試管苗存于?80℃冰箱待用。以甘薯肌動(dòng)蛋白基因()作為內(nèi)參基因, 用Primer Premier 5軟件根據(jù)基因的ORF非保守區(qū)間設(shè)計(jì)實(shí)時(shí)定量PCR引物(表1)。使用SYBR(Tli RNaseH Plus)熒光定量試劑盒(寶生物工程(大連)有限公司, RR420)進(jìn)行qRT-PCR分析。

1.4 煙草遺傳轉(zhuǎn)化

將構(gòu)建的pCAMBIA1300-質(zhì)粒通過凍融法轉(zhuǎn)入根癌農(nóng)桿菌EHA105中。采用葉盤法對(duì)煙草品種W38進(jìn)行轉(zhuǎn)化[31]。將侵染后的煙草葉盤接種于含有15 mg L-1潮霉素、400 mg L-1頭孢氨芐、1.0 mg L-16-BA和0.1 mg L-1NAA的MS培養(yǎng)基上, 每天13 h、54mmol m-2s-1光照, (27±1)℃培養(yǎng)30 d, 然后將再生芽培養(yǎng)于含有15 mg L-1潮霉素、400 mg L-1頭孢氨芐、1.0 mg L-16-BA和0.1 mg L-1NAA的1/2 MS培養(yǎng)基上, 直至長(zhǎng)成完整植株。對(duì)再生植株進(jìn)行PCR檢測(cè), 所用引物序列見表1。

表1 本研究所用引物

1.5 耐鹽性、抗旱性鑒定

參照J(rèn)iang等[32]和喻娜等[33]的方法(略有改動(dòng)), 對(duì)轉(zhuǎn)基因煙草植株進(jìn)行耐鹽和抗旱性離體鑒定。將轉(zhuǎn)基因植株和野生型對(duì)照植株分別培養(yǎng)于含有200 mmol L-1NaCl和10% PEG-6000的MS培養(yǎng)基上, 培養(yǎng)條件為(27±1)℃, 每天13 h、54mmol m-2s-1光照, 脅迫培養(yǎng)4周后, 觀察植株生長(zhǎng)狀態(tài), 并測(cè)定相關(guān)生理生化指標(biāo)。參考Zhai等[26]的方法測(cè)定脯氨酸、MDA、H2O2含量、SOD和POD活性。

1.6 抗逆相關(guān)基因的表達(dá)分析

對(duì)0、200 mmol L-1NaCl或10% PEG-6000處理4周的轉(zhuǎn)基因煙草植株的抗逆相關(guān)基因的表達(dá)量進(jìn)行分析。用Primer Premier 5軟件設(shè)計(jì)基因的特異擴(kuò)增引物。抗逆相關(guān)基因包括活性氧清除相關(guān)基因、、, 以及脯氨酸合成相關(guān)基因。參照Huo等[34]設(shè)計(jì)內(nèi)標(biāo)基因煙草肌動(dòng)蛋白基因()的檢測(cè)引物。引物序列見表1。

1.7 統(tǒng)計(jì)分析

每個(gè)試驗(yàn)設(shè)置3次重復(fù), 通過<0.01或<0.05的檢驗(yàn)分析差異顯著性。

2 結(jié)果與分析

2.1 IbCAF1基因的克隆與序列分析

從甘薯品種魯薯3號(hào)中克隆了基因, 該基因的ORF長(zhǎng)度為846 bp, 編碼281個(gè)氨基酸, 分子量為32.13 kD, 理論等電點(diǎn)pI為4.83, 是酸性蛋白, 不穩(wěn)定系數(shù)為36.36, 親水性平均系數(shù)為-0.167, 說明該蛋白是一個(gè)穩(wěn)定的親水蛋白?；蚓幋a蛋白是由45.55%的α螺旋、36.30%的不規(guī)則卷曲、13.52%的延伸鏈和4.63%的β折疊組成。IbCAF1屬于CAF1超家族成員(圖1-A)。基因與甘薯近緣野生種(2=2=30)親緣關(guān)系最近, 序列一致性為96.8% (圖1-B, C)。

2.2 IbCAF1基因的表達(dá)分析

基因在魯薯3號(hào)莖中的表達(dá)水平顯著高于在葉和根中(圖2-A)。其在離體培養(yǎng)的魯薯3號(hào)植株中的表達(dá)受到NaCl、PEG-6000、ABA和H2O2的強(qiáng)烈誘導(dǎo), 在200 mmol L-1NaCl處理24 h時(shí)達(dá)到高峰(1.4倍), 在20% PEG-6000處理12 h時(shí)達(dá)到高峰(15.4倍), 在100 μmol L-1ABA處理12 h時(shí)達(dá)到高峰(4.4倍), 在10 mmol L-1H2O2處理0.5 h時(shí)達(dá)到高峰(1.7倍)(圖2-B)。

圖1 IbCAF1基因序列分析

A: IbCAF1蛋白序列分析; B: 甘薯CAF1蛋白和其他植物中的CAF1蛋白序列比對(duì); C: 甘薯CAF1蛋白和其他植物中的CAF1蛋白的同源進(jìn)化樹分析。ItlCAF1: 三裂葉野牽牛(XP_031110715.1); InCAF1: 牽牛(XP_019199562.1); NtCAF1: 煙草(XP_016511744.1); SpCAF1: 番茄近緣野生種(XP_015079674.1); CaCAF1: 辣椒(NP_001312000.1); SlCAF1: 番茄(XP_004241342.1); StCAF1: 馬鈴薯(XP_006361099.1)。

A: sequence analysis of IbCAF1 protein. B: multiple sequence alignment of IbCAF1 and CAF1 proteins from other plants. C: phylogenetic analysis of IbCAF1 and CAF1 proteins from other plants. ItlCAF1:(XP_031110715.1); InCAF1:(XP_019199562.1), NtCAF1:(XP_016511744.1); SpCAF1:(XP_015079674.1), CaCAF1:(NP_001312000.1), SlCAF1:(XP_004241342.1), StCAF1:(XP_006361099.1).

2.3 轉(zhuǎn)基因煙草植株的獲得

利用Wang等[31]的方法獲得12株轉(zhuǎn)基因煙草植株, 即L1, L2, …, L12。qRT-PCR結(jié)果顯示, 轉(zhuǎn)基因植株中的表達(dá)量均顯著高于WT (圖3)。選取表達(dá)量較高的L1、L5和L9株系進(jìn)行后續(xù)的分析。

2.4 過表達(dá)IbCAF1基因能夠提高煙草的耐鹽性和抗旱性

將3個(gè)轉(zhuǎn)基因株系(L1、L5和L9)和野生型植株分別在200 mmol L-1NaCl和10% PEG-6000的MS培養(yǎng)基上和無脅迫條件下培養(yǎng)4周。轉(zhuǎn)基因植株和野生型植株在無脅迫條件下生長(zhǎng)無明顯差異。在鹽脅迫和干旱脅迫下, 轉(zhuǎn)基因植株生長(zhǎng)良好(圖4-A), 葉片中積累了較少的H2O2(圖4-B, D)和O2-(圖4-C), SOD活性、POD活性和脯氨酸含量均顯著高于WT, MDA含量在鹽和干旱脅迫下顯著低于WT (圖4-E~H)。

2.5 轉(zhuǎn)基因植株抗性相關(guān)基因的表達(dá)分析

在200 mmol L-1NaCl和10% PEG-6000的脅迫下, 過表達(dá)株系與WT相比, ROS清除相關(guān)基因、和上調(diào)表達(dá), 脯氨酸合成相關(guān)基因上調(diào)表達(dá)(圖5)。

圖2 IbCAF1基因在魯薯3號(hào)中的表達(dá)分析

A:基因在魯薯3號(hào)不同組織中的表達(dá); B: 200 mmol L–1NaCl、20% PEG-6000、100mmol L–1ABA和10 mmol L–1H2O2分別處理不同時(shí)間后, 魯薯3號(hào)中基因的表達(dá)分析。*與**分別表示在0.05和0.01水平下差異顯著。

A: expression analysis ofgene in different tissues of Lushu 3; B: expression analysis of thegene in Lushu 3 after different times (h) in response to 200 mmol L–1NaCl, 20% PEG-6000, 100 μmol L–1ABA and 10 mmol L–1H2O2, respectively.* and ** indicate significantly different at the 0.05 and 0.01 probability levels, respectively.

圖3 轉(zhuǎn)基因煙草植株的IbCAF1基因的qRT-PCR分析

**表示在0.01水平下差異顯著。**: significantly different at the 0.01 probability level.

3 討論

CAF1是參與mRNA降解的主要脫腺苷酸酶之一, 在調(diào)控基因表達(dá)和影響生物學(xué)性狀方面起著重要作用[7]。目前, CAF1在酵母和動(dòng)物中被廣泛研究,在植物中的作用還不清楚。只有少數(shù)植物, 如擬南芥[10-11,13]、辣椒[9]、甜橙[12]等有基因功能的研究報(bào)道, 表明該基因在植物的生長(zhǎng)發(fā)育及生物和非生物脅迫的應(yīng)答過程中發(fā)揮重要作用。到目前為止,基因在植物中的耐鹽作用還不清楚, 抗旱性研究還未見報(bào)道。本研究發(fā)現(xiàn)基因能夠被NaCl、PEG、ABA和H2O2誘導(dǎo)上調(diào)表達(dá)(圖2-B), 并且基因的過量表達(dá)增強(qiáng)了轉(zhuǎn)基因煙草植株的耐鹽抗旱性(圖4)。

圖4 IbCAF1增強(qiáng)了轉(zhuǎn)基因煙草植株的耐鹽性和抗旱性

A: 轉(zhuǎn)基因煙草植株和WT煙草植株在無脅迫或添加200 mmol L–1NaCl或10% PEG-6000的1/2 MS培養(yǎng)基上培養(yǎng)4周; B~H: 在無脅迫、200 mmol L–1NaCl或10% PEG-6000的1/2 MS培養(yǎng)基上培養(yǎng)4周的轉(zhuǎn)基因煙草和WT煙草葉片的DAB染色(B)、NBT染色(C)、H2O2含量(D)、SOD活性(E)、POD活性(F)、脯氨酸含量(G)、丙二醛含量(H)。*與**分別表示在0.05和0.01水平下差異顯著。

A: responses of-transgenic and WT tobacco plants cultured for 4 weeks on half-MS medium supplemented without stress or with 200 mmol L–1NaCl or 10% PEG-6000; B–H: DAB staining (B), NBT staining (C), H2O2content (D), SOD activity (E), POD activity (F), proline content (G), and MDA content (H) in the leaves oftransgenic and WT tobacco plants cultured for 4w on half-MS medium supplemented with no stress, 200 mmol L–1NaCl or 10% PEG-6000. * and ** indicate significantly different at the 0.05 and 0.01 probability levels, respectively.

在鹽或干旱脅迫下, 植物體內(nèi)常常會(huì)產(chǎn)生大量的活性氧(ROS), 如超氧陰離子(O2?)和過氧化氫(H2O2)等。ROS的大量累積會(huì)造成細(xì)胞氧化損傷, 對(duì)植物有很大的毒害作用。超氧化物歧化酶(SOD)、抗壞血酸過氧化物酶(APX)和過氧化物酶(POD)等ROS清除系統(tǒng), 可以解毒ROS, 以減少植物細(xì)胞中的氧化損傷而增強(qiáng)抗逆性[19,35]。在鹽或干旱脅迫下, 脯氨酸水平的升高增強(qiáng)了植物的耐鹽性和抗旱性[28,35]。脯氨酸可以調(diào)節(jié)植物細(xì)胞質(zhì)pH防止其酸化, 保護(hù)膜完整性, 同時(shí)還具有清除活性氧的功能[36]。丙二醛(MDA)的含量變高時(shí)會(huì)致使細(xì)胞發(fā)生膜損傷, 植物的耐鹽抗旱性也會(huì)因此減弱[37-38]。在本研究中, 鹽或干旱脅迫條件下, 在過量表達(dá)的煙草植株中, 活性氧清除相關(guān)基因、和和脯氨酸合成相關(guān)基因被顯著上調(diào)表達(dá), 增加了SOD活性, POD活性, 脯氨酸含量, 降低了H2O2含量和MDA含量, 從而增強(qiáng)了轉(zhuǎn)基因煙草植株的耐鹽性和抗旱性(圖4和圖5)。

4 結(jié)論

基因的過表達(dá)通過上調(diào)活性氧清除和脯氨酸合成相關(guān)基因的表達(dá), 增加SOD活性、POD活性、脯氨酸含量, 降低H2O2含量和丙二醛含量, 從而增強(qiáng)轉(zhuǎn)基因煙草植株的耐鹽性和抗旱性?；?qū)⒃谔岣吒适淼戎参锬望}抗旱性方面具有一定的應(yīng)用潛力。

圖5 轉(zhuǎn)基因植株及WT植株的抗逆相關(guān)基因的表達(dá)分析

*與**分別表示在0.05和0.01水平下差異顯著。

* and ** indicate significantly different at the 0.05 and 0.01 probability levels, respectively.

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Cloning ofand identification on tolerance to salt and drought stress in sweetpotato

CHEN Shan-Bin, SUN Si-Fan, NIE Nan, DU Bing, HE Shao-Zhen, LIU Qing-Chang, ZHAI Hong*

Key Laboratory of Sweetpotato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs / Laboratory of Crop Heterosis and Utilization, Ministry of Education / Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing 100193, China

(CCR4-associated factor 1)gene plays an important role in plant development and disease resistance. In this study, thegene of sweetpotato was cloned according to the EST sequence. The ORF ofwas 846 bp, encoding 281 amino acids, with a molecular weight of 32.13 kD and an isoelectric point of 4.83. The results of amino acid sequence alignment and phylogenetic tree analysis showed that IbCAF1 had higher homology with ItlCAF1, a homologous protein of(2), and the homology was 96.8%.gene was induced and expressed by NaCl, PEG, ABA, and H2O2. Thegene was transferred into tobacco bymediated transformation. The overexpression ofgene significantly improved the salt and drought tolerance of transgenic tobacco plants. After 200 mmol L-1NaCl and 10% PEG-6000 treatments, the transgenic tobacco plants showed significant upregulation of the genes involved in ROS scavenging system and proline biosynthesis related genes, significant increase of SOD activity, POD activity and proline content and significant decrease of H2O2and malondialdehyde contents. These results demonstrate that thegene could improve salt and drought tolerance in transgenic tobacco. This study will lay a foundation on salt and drought tolerance gene engineering ofgene in sweetpotato for the following research.

sweetpotato;; transgenic tobacco; salt tolerance; drought tolerance

本研究由國(guó)家自然科學(xué)基金項(xiàng)目(31872878), 國(guó)家重點(diǎn)研發(fā)計(jì)劃項(xiàng)目(2018YFD1000700, 2018YFD1000704)和國(guó)家現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系建設(shè)專項(xiàng)(CARS-10)資助。

This study was supported by the National Natural Science Foundation of China (31872878), the National Key Research and Development Program of China (2018YFD1000700, 2018YFD1000704), and the China Agriculture Research System (CARS-10).

翟紅, E-mail: zhaihong@cau.edu.cn

E-mail: 757015572@qq.com

2020-02-27;

2020-06-02;

2020-07-02.

URL: https://kns.cnki.net/kcms/detail/11.1809.S.20200702.1029.002.html

10.3724/SP.J.1006.2020.04045