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MYB轉(zhuǎn)錄因子在水稻抗逆基因工程中的應(yīng)用進展

2021-12-09 16:14段俊枝李瑩馮麗麗孫巖齊紅志齊學禮楊翠蘋王楠燕照玲陳海燕張會芳卓文飛平西栓
江蘇農(nóng)業(yè)科學 2021年21期
關(guān)鍵詞:抗逆性轉(zhuǎn)基因籽粒

段俊枝 李瑩 馮麗麗 孫巖 齊紅志 齊學禮 楊翠蘋 王楠 燕照玲 陳海燕 張會芳 卓文飛 平西栓

摘要:水稻(Oryza sativa)經(jīng)常遇到干旱、高鹽、低溫等非生物脅迫及病蟲害等生物脅迫,抑制其生長發(fā)育,甚至降低籽粒產(chǎn)量。MYB(myeloblastosis)在調(diào)控水稻響應(yīng)各種非生物脅迫(干旱、高鹽、低溫等)及生物脅迫(病蟲害等)反應(yīng)中具有重要作用。本文闡述了水稻MYB轉(zhuǎn)錄因子的結(jié)構(gòu)、分類及其在水稻抗旱、耐鹽、耐冷、耐熱、耐低磷、抗病原菌、抗蟲等抗逆基因工程中的應(yīng)用進展,為MYB 在水稻及其他作物抗逆育種中的應(yīng)用提供參考。

關(guān)鍵詞:水稻;MYB 轉(zhuǎn)錄因子;抗旱;耐鹽;耐冷;耐熱;耐低磷;抗病;抗蟲;基因工程

中圖分類號: S511.01? 文獻標志碼: A

文章編號:1002-1302(2021)21-0046-08

收稿日期:2021-03-26

基金項目:國家自然科學基金(編號:31701510)。

作者簡介:段俊枝(1981—),女,河北滄州人,博士,助理研究員,主要從事作物遺傳育種研究,E-mail:junzhi2004@163.com;共同第一作者:李 瑩(1984—),女,河南焦作人,碩士,主要從事小麥栽培育種及期刊編輯方面的工作,E-mail:liying1233@163.com。

通信作者:卓文飛,博士,副研究員,主要從事農(nóng)業(yè)信息及期刊編輯方面的工作,E-mail:kjcankao@126.com;平西栓,研究員,主要從事農(nóng)業(yè)技術(shù)推廣管理工作,E-mail:njzpxs6410@126.com。

水稻(Oryza sativa)是我國乃至世界上重要的糧食作物之一,世界上半數(shù)以上的人口以大米為主食[1]。水稻在生長發(fā)育過程中,經(jīng)常遭遇干旱、高鹽、低溫及病蟲害等逆境脅迫,抑制其生長發(fā)育,甚至降低籽粒產(chǎn)量。減少這種逆境脅迫對水稻造成的危害,不僅可以提高水稻生產(chǎn)力,而且還可以擴大水稻種植范圍,將水稻種植于目前尚不能種植的邊際土地上[2-3],進而提高水稻總產(chǎn)量,這對于保障國家糧食安全具有重要的現(xiàn)實意義。培育抗逆水稻品種是減少這些逆境脅迫對水稻造成危害的有效途徑。采用傳統(tǒng)的遺傳育種方法培育抗逆水稻品種雖然簡便可行,但進展緩慢。隨著生物技術(shù)的快速發(fā)展,挖掘優(yōu)異的抗逆基因,然后通過基因工程育種技術(shù)提高水稻的抗逆性,是培育水稻抗逆新品種最有效的途徑。

為了應(yīng)對逆境脅迫,植物在長期的進化過程中,形成了一系列感知、適應(yīng)逆境脅迫的有效機制。當植物響應(yīng)并適應(yīng)逆境脅迫時,植物體內(nèi)的一系列生理、生化過程會發(fā)生改變,且許多抗逆相關(guān)基因會被激活,進而引起抗逆蛋白的積累,以抵御逆境脅迫??鼓婊虻谋磉_大多是由轉(zhuǎn)錄因子調(diào)控的,目前已知的逆境脅迫應(yīng)答轉(zhuǎn)錄因子有MYB(myeloblastosis)、NAC[NAM(No apical meristem)、ATAF1(Arabidopsis transcription activation factor 1)、ATAF2、CUC2(cup-shaped cotyledon 2)]、bHLH(Basic helix-loop-helix)、AP2/ERF (APETALA2/ethylene responsive factor)、bZIP(basic region/leucine zipper motif)、WRKY等[4-8]。其中,MYB轉(zhuǎn)錄因子廣泛存在于動物、植物、真菌中,是植物中最大的轉(zhuǎn)錄因子家族之一。目前,已在水稻[9]、擬南芥(Arabidopsis thaliana)[9]、小麥(Triticum aestivum L.)[10]、香蕉(Musa acuminata)[11]中分別鑒定了155、197、393、285個MYB轉(zhuǎn)錄因子。 MYB轉(zhuǎn)錄因子具有多種生物學功能,不僅參與植物生長發(fā)育的調(diào)控[12-18],而且還參與植物對干旱、高鹽、低溫、病原菌侵染等逆境脅迫的抗逆反應(yīng)[19-26]。目前,已經(jīng)從植物中分離獲得了許多MYB 轉(zhuǎn)錄因子,且在水稻中進行了遺傳轉(zhuǎn)化,MYB轉(zhuǎn)錄因子在不同程度上分別提高了水稻對干旱、高鹽、低溫、高溫、低磷及蟲等的抗性,有的甚至提高了轉(zhuǎn)基因水稻的籽粒產(chǎn)量。本文闡述了水稻MYB轉(zhuǎn)錄因子的結(jié)構(gòu)、分類及其在水稻抗旱、耐鹽、耐冷、耐熱、耐低磷、抗病、抗蟲等抗逆基因工程中的應(yīng)用進展,為MYB轉(zhuǎn)錄因子在水稻及其他作物抗逆遺傳改良中的應(yīng)用提供參考。

1 水稻MYB轉(zhuǎn)錄因子家族成員結(jié)構(gòu)、分類

水稻MYB 轉(zhuǎn)錄因子的N端均具有高度保守的MYB結(jié)構(gòu)域,該結(jié)構(gòu)域通常包含1~5個不完全重復序列(R,約含52個氨基酸殘基)[9,27],可形成3個α-螺旋結(jié)構(gòu),第2和第3個α-螺旋與R中均勻分布的3個色氨酸殘基形成螺旋-轉(zhuǎn)角-螺旋結(jié)構(gòu)[28];第3個螺旋是“識別螺旋”,可直接與DNA接觸并嵌入其大溝中,在接觸過程中,2個R緊密結(jié)合于DNA大溝中,使2個“識別螺旋”協(xié)同作用,結(jié)合到特定的DNA序列上[29]。MYB轉(zhuǎn)錄因子的C端通常含有一個富含酸性氨基酸的轉(zhuǎn)錄激活結(jié)構(gòu)域,其作用具有一定的可塑性,調(diào)控蛋白活性。

水稻 MYB 轉(zhuǎn)錄因子家族包含 155 個 MYB 基因,根據(jù)R數(shù)目,可以分為4種類型:MYB-related、MYB-R2R3、MYB-R1R2R3、Atypical MYB,分別包含62、88、4、1個MYB基因,以MYB-R2R3類型最多,占56.77%,MYB-related類型次之,占40.00%[9]。其中,MYB-related類型通常但不一定只包含1個R[30];MYB-R2R3類型包含2個R,MYB-R1R2R3類型包含3個R;Atypical MYB類型包含5個R[9]。水稻 MYB 基因不均勻地分布在12條染色體上,1號染色體分布最多,6號染色體次之,11號染色體次最少[9]。

2 MYB轉(zhuǎn)錄因子在水稻抗非生物脅迫基因工程中的應(yīng)用進展

目前,已經(jīng)克隆獲得眾多MYB基因,其中一部分MYB基因在水稻中超表達,提高了轉(zhuǎn)基因植株對非生物脅迫(干旱、高鹽、低溫、高溫、低磷、低氮等)的耐受性,有的甚至提高了轉(zhuǎn)基因水稻的籽粒產(chǎn)量[31-50]。但是,這些MYB基因的抗逆范圍不同,有的可以提高轉(zhuǎn)基因水稻植株的單一抗性,如抗旱、耐鹽、耐冷、耐熱、耐低磷、耐低氮等[31-45];有的可以提高轉(zhuǎn)基因水稻植株的綜合抗性,例如抗旱并耐鹽、耐冷、耐熱等[46-50]。

2.1 單一抗性

2.1.1 抗旱、耐鹽

水稻OsMYBR1基因受干旱誘導表達,正常生長條件下,超表達OsMYBR1水稻植株的表型與野生型對照無明顯差異,但是轉(zhuǎn)基因水稻植株對ABA(abscisic acid)的敏感性降低。干旱脅迫條件下,超表達OsMYBR1顯著增加了轉(zhuǎn)基因水稻植株可溶性糖和脯氨酸含量,提高了轉(zhuǎn)基因水稻植株存活率,增幅為1.3~1.5倍;進一步分析發(fā)現(xiàn),干旱脅迫條件下,轉(zhuǎn)基因植株中一些脅迫相關(guān)基因[OsP5CS1(Δ1-pyrroline-5-carboxylate synthetase 1)、OsProt(proline transporter ptotein)、OsTIJI3(late-embryogenesis-abundant protein 3)、OsRAB16(responsive to ABA? 16)]的表達量明顯提高[31]。類似的,谷子(Setaria italica)SiMYB56基因也受干旱誘導表達,在水稻中超表達該基因?qū)D(zhuǎn)基因植株的生長沒有任何不良影響,且提高了轉(zhuǎn)基因植株營養(yǎng)生長期和生殖生長期的抗旱性。與野生型對照相比,干旱脅迫條件下,營養(yǎng)生長期,超表達SiMYB56基因水稻植株存活率顯著提高4~7倍,丙二醛(MDA)含量降低,木質(zhì)素含量增加;田間生殖生長期,超表達SiMYB56基因水稻植株籽粒產(chǎn)量顯著增加,這主要得益于單株穗數(shù)、穗長的增加。進一步分析發(fā)現(xiàn),干旱脅迫條件下,SiMYB56能激活木質(zhì)素合成基因PAL(phenylalanine ammonia lyase)、4CL5(4-coumarate-coa ligase 5)、CAD(cinnamyl alcohol dehydrogenase)、CCR10(cinnamoyl-CoA reductase 10)等的表達。另外,超表達SiMYB56基因?qū)е翧BA在轉(zhuǎn)基因水稻種子中積累,深入分析發(fā)現(xiàn),轉(zhuǎn)基因水稻植株中ABA合成基因[NCED5(nine-cis epoxycarotenoid dioxygenase 5)]、ABA信號傳導相關(guān)基因[ABIL2(abscisic acid-insensitive-like 2)、ABF1(ABA? responsive element binding factors 1)、ABF2、bZIP23(basic leucine zipper 23)]、ABA響應(yīng)基因[P5CS1(Δ1-pyrroline-5-carboxylate synthetase 1)、LEA7]的表達量提高[32]。說明SiMYB56通過調(diào)控木質(zhì)素合成和ABA信號通路來提高轉(zhuǎn)基因植株的抗旱性。此外,甘蔗ScMYBAS1有4種可變剪接體,它們在調(diào)控植物生長及抗逆性方面作用不同,其中,超表達ScMYBAS1-3基因促進了干旱脅迫條件下轉(zhuǎn)基因水稻植株的生長發(fā)育,提高了葉片含水量,最終提高了轉(zhuǎn)基因植株的抗旱性[33]。

水稻OsMYB91基因受高鹽、干旱誘導表達,超表達OsMYB91基因延緩了轉(zhuǎn)基因水稻的生長發(fā)育,提高了轉(zhuǎn)基因植株體內(nèi)ABA含量;高鹽脅迫條件下,超表達OsMYB91基因提高了轉(zhuǎn)基因水稻植株體內(nèi)脯氨酸含量,降低了H2O2、MDA含量,且提高了鹽脅迫相關(guān)基因[SLR1(slender rice 1)、LEA3、SOS1(salt overly sensitive 1)、RAB16A、NHX1(Na+/H+antiporter 1)、P5CS1]的表達量[34]。說明超表達OsMYB91基因水稻植株耐鹽性的提高主要得益于活性氧清除能力、滲透調(diào)節(jié)能力和鹽脅迫相關(guān)基因表達量的提高。

2.1.2 耐冷、耐熱

水稻OsMYB3R-2基因受冷脅迫誘導,超表達OsMYB3R-2基因提高了水稻植株的耐冷性,低溫(2 ℃)處理72 h后,野生型對照存活率低于20%,而轉(zhuǎn)基因水稻植株存活率均高于50%,且轉(zhuǎn)基因水稻植株脯氨酸含量提高。進一步分析發(fā)現(xiàn),OsMYB3R-2的靶標基因是B型細胞周期蛋白基因OsCycB1;1,低溫條件下,轉(zhuǎn)基因水稻植株中一些細胞周期蛋白基因(OsCycB1;1、OsCycB2;1、OsCycB2;2)的表達量也均較野生型對照提高,且轉(zhuǎn)基因水稻植株細胞分裂指數(shù)較野生型對照提高。另外,超表達OsCycB1;1基因水稻植株的耐冷性也提高[35]。說明超表達OsMYB3R-2基因水稻植株耐冷性的提高主要是通過調(diào)控細胞循環(huán)來實現(xiàn)的。另外,水稻OsMYBS3基因在水稻所有組織中普遍存在,也受冷脅迫誘導表達,在田間條件下,超表達OsMYBS3基因水稻植株的表型和產(chǎn)量均與野生型對照相似;4 ℃脅迫條件下,轉(zhuǎn)基因水稻植株的耐冷性較野生型對照提高。表達分析發(fā)現(xiàn),轉(zhuǎn)基因水稻植株中一些脅迫響應(yīng)基因[GAD(qlutamate decarboxylase)、WRKY77、MRP4(multidrug resistance protein 4 )、TPP1(trehalose-6-phosphate phosphatase 1)、TPP2]的表達量提高;出人意料的是,OsMYBS3抑制DREB1/CBF(dehydration-responsive element binding protein 1/C-repeat binding factor)依賴冷信號途徑,DREB1快速短暫地響應(yīng)冷脅迫,OsMYBS3緩慢地響應(yīng)冷脅迫,兩者分別屬于適應(yīng)短期、長期冷脅迫的不同信號途徑[36]。此外,在水稻中超表達OsMYB30基因,提高了轉(zhuǎn)基因植株的冷敏感性;相反的,敲除OsMYB30基因突變體的耐冷性提高。進一步分析發(fā)現(xiàn),轉(zhuǎn)基因植株中BMY(β-amylase )基因表達量降低,BMY活性、麥芽糖(對細胞膜有保護作用)含量均降低。OsMYB30可與BMY基因啟動子區(qū)域結(jié)合,調(diào)控BMY基因的表達進而調(diào)控淀粉的分解[37]。說明OsMYB30通過負調(diào)控BMY基因的表達來調(diào)控淀粉的分解和麥芽糖含量,進而調(diào)控耐冷性,以后有望通過基因敲除技術(shù)敲除OsMYB30基因來提高水稻及其他作物的耐冷性。

水稻OsMYB55基因受高溫脅迫誘導表達,超表達該基因的轉(zhuǎn)基因水稻植株耐熱性提高。在高溫(35 ℃)脅迫條件下,轉(zhuǎn)OsMYB55基因水稻植株株高、地上部及根系生物量均較野生型對照顯著提高,籽粒產(chǎn)量降低幅度也顯著降低。轉(zhuǎn)錄組分析發(fā)現(xiàn),轉(zhuǎn)OsMYB55基因水稻植株中一些氨基酸代謝相關(guān)基因的表達量提高,OsMYB55可以與這些基因的啟動子區(qū)域結(jié)合并激活其表達,例如OsGS1;2(Glutamine synthetase 1;2)、GAT1(glutamine amidotransferase 1)、GAD3(glutamate decarboxylase 3)基因等。進一步分析發(fā)現(xiàn),高溫脅迫條件下,轉(zhuǎn)基因植株中總氨基酸含量顯著增加[38]。說明OsMYB55通過激活氨基酸代謝相關(guān)基因的表達來提高氨基酸代謝,進而提高轉(zhuǎn)基因植株的耐熱性。相反的,一些水稻MYB基因OsPL(purple leaf)會降低水稻植株的耐熱性。研究發(fā)現(xiàn),水稻突變體pl在籽粒灌漿后期表現(xiàn)紫色葉片、葉鞘,葉片衰老,該突變體細胞異常,葉綠體扭曲,葉綠素含量低,花青素含量高;進一步分析發(fā)現(xiàn),灌漿期,該突變體中一些花青素合成基因[PAL、CHS(chalcone synthase)、ANS(anthocyanidin synthase)]表達量提高,光合相關(guān)基因表達量下降,SOD(superoxide dismutase)、CAT(catalase)活性顯著增強,總可溶性糖及ABA、JA(jasmonic acid)、IAA(indoleacetic acid)含量顯著增加,耐40 ℃高溫[39]??梢娍梢酝ㄟ^敲除OsPL基因的方法來提高水稻及其他作物的耐熱性。

2.1.3 耐低磷、低氮等

水稻OsMYB2P-1基因受磷饑餓誘導表達,在水稻中超表達該基因提高了轉(zhuǎn)基因植株的磷饑餓耐性;相反的,沉默該基因表達,增強了轉(zhuǎn)基因植株對磷缺乏的敏感性。在豐磷條件下,超表達OsMYB2P-1基因植株主根長較野生型對照短;而在缺磷條件下,超表達OsMYB2P-1基因植株主根和側(cè)根長均較野生型對照長,說明OsMYB2P-1可能與根系結(jié)構(gòu)調(diào)控有關(guān)。進一步分析發(fā)現(xiàn),超表達OsMYB2P-1基因提高了轉(zhuǎn)基因水稻植株中磷響應(yīng)基因[SQD(UDP-sulfoquinovose synthase)、IPS1(induced by Pi starvation 1)、PAP10(purple acid phosphatase 10)、miR399a、miR399j]的表達量。另外,缺磷條件下,超表達OsMYB2P-1基因水稻植株中高親和磷轉(zhuǎn)運蛋白基因[PT6(phosphate transporter 6)、PT8、PT10]的表達量提高;豐磷條件下,超表達OsMYB2P-1基因水稻植株中低親和磷轉(zhuǎn)運蛋白基因PT2的表達量提高[40],說明OsMYB2P-1可能作為磷依賴的調(diào)節(jié)因子控制磷轉(zhuǎn)運蛋白的表達。綜上,OsMYB2P-1通過調(diào)控根系結(jié)構(gòu)、提高磷響應(yīng)基因及磷轉(zhuǎn)運蛋白基因的表達量來提高轉(zhuǎn)基因植株的磷饑餓耐性。類似的,缺磷條件下,OsMYB4P基因表達量也提高。無論高磷還是缺磷條件下,超表達OsMYB4P基因水稻植株的長勢均較野生型對照好,主根和側(cè)根長均較野生型對照長,根和嫩葉中磷濃度均高于野生型對照,尤其是根。高磷條件下,超表達OsMYB4P基因水稻植株的側(cè)根密度大于野生型對照,側(cè)根長也長于野生型對照,且轉(zhuǎn)基因水稻幼苗根和嫩葉中磷濃度顯著高于野生型對照;低磷條件下,超表達OsMYB4P基因水稻幼苗嫩葉中磷濃度急劇下降,但是根中磷濃度仍保持在較高水平。表達分析發(fā)現(xiàn),超表達OsMYB4P基因提高了轉(zhuǎn)基因水稻植株嫩葉中磷轉(zhuǎn)運蛋白基因(PT1、PT2、PT4、PT7、PT8)的表達量,但在根中降低或者不變(PT8基因除外)[41]。說明OsMYB4P通過調(diào)控根系結(jié)構(gòu)、激活磷穩(wěn)態(tài)相關(guān)基因來提高磷的利用,進而提高轉(zhuǎn)基因植株的磷饑餓耐性。另外,在水稻中超表達OsMYB5P基因,同樣提高了轉(zhuǎn)基因植株的磷饑餓耐性;相反,敲除OsMYB5P基因,提高了轉(zhuǎn)基因植株對磷缺乏的敏感性。高磷或缺磷條件下,超表達OsMYB5P基因水稻植株主根長、側(cè)根長均長于野生型對照,側(cè)根密度大于野生型對照,苗高均高于野生型對照,說明OsMYB5P調(diào)控植物生長發(fā)育;超表達OsMYB5P基因水稻植株中磷含量較野生型對照提高,說明OsMYB5P調(diào)控磷吸收和缺磷脅迫適應(yīng)。進一步分析發(fā)現(xiàn),OsMYB5P直接與OsPT5基因啟動子區(qū)的MBS (MYB binding site)元件結(jié)合,提高OsPT5基因的表達水平;且超表達OsMYB5P基因提高了Pht1;3(phosphate transporter1;3)的表達量[42]。說明轉(zhuǎn)OsMYB5P基因水稻植株磷饑餓耐性的提高主要是通過調(diào)控磷轉(zhuǎn)運蛋白基因的表達量來實現(xiàn)的。相反的,一些水稻MYB基因MYB1會降低水稻植株的磷饑餓耐性。研究發(fā)現(xiàn),水稻MYB1基因突變后導致磷的吸收和積累增加,并伴隨磷轉(zhuǎn)運蛋白家族基因(PHT1;2、PHT1;8)和參與磷饑餓信號途徑基因[PHO2.1(phosphate transporter 2.1)]的表達量提高。MYB1基因影響初生根和側(cè)根的伸長,對初生根伸長的影響依賴于磷,對側(cè)根伸長的影響不依賴于磷。此外,磷饑餓條件下,GA(gibberellic acid)誘發(fā)的側(cè)根伸長在野生型植株中被嚴重抑制,在myb1突變體中這種抑制作用得到部分緩解,這主要歸因于myb1突變體中GA合成基因表達量的提高[43]。說明MYB1既參與磷饑餓信號傳導、負調(diào)控磷的吸收和積累,又參與GA合成,可以通過敲除MYB1基因的方法提高水稻及其他作物的磷吸收和積累,增強磷饑餓耐性。

低氮脅迫條件下,對谷子轉(zhuǎn)錄組進行分析,發(fā)現(xiàn)了25個類MYB轉(zhuǎn)錄因子?;蚬δ芊治霭l(fā)現(xiàn),SiMYB3 基因受低氮脅迫誘導表達,低氮脅迫條件下,超表達SiMYB3基因水稻植株幼苗總根長、側(cè)根數(shù)高于野生型對照;2年的田間試驗結(jié)果表明,低氮脅迫條件下,轉(zhuǎn)基因植株的生物量顯著高于野生型對照,且粒質(zhì)量、總氮含量、籽粒氮含量均顯著高于野生型對照。進一步分析發(fā)現(xiàn),轉(zhuǎn)基因水稻植株中生長素合成相關(guān)基因TAR2(tryptophan aminotransferase related 2)的表達量提高[44]。說明SiMYB3通過調(diào)控根生長素合成來調(diào)控根系發(fā)育進而提高轉(zhuǎn)基因植株的耐低氮能力。

水稻OsARM1(arsenite-responsive MYB 1)調(diào)控砷(As)相關(guān)轉(zhuǎn)運蛋白基因。敲除OsARM1基因提高了轉(zhuǎn)基因水稻植株對As(Ⅲ)的耐受性,超表達OsARM1基因調(diào)高了轉(zhuǎn)基因水稻植株對As(Ⅲ)的敏感性。低As(Ⅲ)條件下,敲除OsARM1基因植株中更多的As從根轉(zhuǎn)運到嫩葉;高As(Ⅲ)條件下,超表達OsARM1基因植株中更多的As積累在根中,暗示OsARM1在調(diào)控As從根到嫩葉的轉(zhuǎn)移中具有重要作用。進一步分析發(fā)現(xiàn),關(guān)鍵的As響應(yīng)轉(zhuǎn)運蛋白基因[ Lsi1(silicon influx transporter 1 )、Lsi2、Lsi6]在超表達OsARM1基因水稻植株中表達量降低,在OsARM1基因敲除水稻植株中表達量提高[45]。說明OsARM1基因在調(diào)控As吸收和根到嫩葉的轉(zhuǎn)移中具有重要作用。

2.2 綜合抗性

2.2.1 抗旱+耐鹽

水稻OsMYB6基因受干旱、高鹽誘導表達,干旱和高鹽脅迫條件下,超表達OsMYB6基因水稻植株的存活率較野生型對照顯著提高約5倍。進一步分析發(fā)現(xiàn),轉(zhuǎn)基因植株脯氨酸含量和CAT、POD(peroxidase)活性大幅增加,相對電解質(zhì)滲漏率降低,一些脅迫相關(guān)基因(OsP5CS、OsDREB1A、OsDREB2A、OsCATA、OsLEA3、SNAC1)的表達量提高[46]。說明超表達OsMYB91基因提高了轉(zhuǎn)基因植株的活性氧清除能力、滲透調(diào)節(jié)能力和脅迫相關(guān)基因的表達量,進而提高了轉(zhuǎn)基因植株的抗旱和耐鹽性。類似的,水稻OsMYB48-1基因受PEG(polyethylene glycol)、高鹽、ABA、低溫誘導表達,超表達OsMYB48-1基因增強了轉(zhuǎn)基因水稻植株對ABA的敏感性,提高了轉(zhuǎn)基因水稻植株體內(nèi)ABA含量及對干旱、高鹽的耐受性。其中,干旱脅迫條件下,超表達OsMYB48-1基因顯著提高了轉(zhuǎn)基因水稻植株的存活率和葉片脯氨酸含量,降低了葉片失水速率和MDA含量。表達分析發(fā)現(xiàn),超表達OsMYB48-1基因顯著提高了轉(zhuǎn)基因水稻植株中一些ABA合成基因 (OsNCED4、OsNCED5)、早期信號基因 [OsPP2C68(serine/threonine protein phosphatases 2C 68)]、后期響應(yīng)基因 (RAB21、OsLEA3、RAB16C、RAB16D)的表達量[47]。說明超表達OsMYB48-1基因可促進轉(zhuǎn)基因水稻植株中ABA的合成,進而提高轉(zhuǎn)基因植株的抗旱性和耐鹽性。另外,在水稻中超表達金魚草(Antirrhinum majus)MYB 基因AmROSEA1同樣提高了轉(zhuǎn)基因植株的抗旱性和耐鹽性。正常生長條件下,超表達AmRosea1基因延緩了轉(zhuǎn)基因水稻植株的生長發(fā)育。干旱脅迫條件下,超表達AmRosea1基因顯著提高轉(zhuǎn)基因水稻植株的存活率,存活率較野生型對照提高2.7~3.1倍;高鹽脅迫條件下,野生型對照全部死亡,而超表達AmRosea1基因水稻植株存活率為69%~75%。表達分析發(fā)現(xiàn),干旱、高鹽脅迫條件下,超表達AmRosea1基因水稻植株中大量脅迫相關(guān)基因的表達量提高,主要涉及脅迫信號轉(zhuǎn)導基因[RD22(responsive to dehydration 22)、OsMYB48-1]、激素信號轉(zhuǎn)導途徑基因[COI1(coronatine-insensitive protein 1)、EBF1(early B cell factor 1) ]、離子平衡基因(鉀轉(zhuǎn)運子4基因)、活性氧清除酶基因[CAT、POD、APX(ascorbic acid peroxidase)、GST(glutathione-S-transferase )][48]。

2.2.2 抗旱+耐冷、耐熱等

水稻OsMYB2基因受高鹽、低溫、干旱誘導表達,正常生長條件下,超表達OsMYB2基因并沒有改變轉(zhuǎn)基因水稻植株的表型,但增強了轉(zhuǎn)基因水稻植株對ABA的敏感性,提高了轉(zhuǎn)基因水稻植株對高鹽、低溫和干旱的耐受性。高鹽、低溫和干旱脅迫條件下,超表達OsMYB2基因水稻植株中可溶性糖、脯氨酸含量和CAT、SOD、POD活性提高,H2O2和MDA含量降低。進一步分析發(fā)現(xiàn),超表達OsMYB2基因提高了脯氨酸合成酶基因及一些脅迫相關(guān)基因(OsLEA3、OsRab16A、OsDREB2A)的表達量[49]。說明超表達OsMYB2基因水稻植株抗逆性的提高主要得益于活性氧清除能力及脅迫相關(guān)基因表達量的提高。

與其他MYB基因不同,MYBS2基因受干旱、高溫抑制,在水稻中超表達MYBS2基因,降低了轉(zhuǎn)基因植株的抗旱性和耐熱性。干旱脅迫條件下,無論是水培還是土培條件下超表達MYBS2基因均顯著降低了轉(zhuǎn)基因水稻植株的存活率,更值得注意的是,超表達MYBS2基因轉(zhuǎn)基因水稻植株籽粒產(chǎn)量顯著降低36%~45%。干旱脅迫條件下,無論是水培還是土培條件下沉默MYBS2基因水稻植株的存活率均顯著提高,且轉(zhuǎn)基因水稻植株的籽粒產(chǎn)量也顯著提高26%~54%。進一步分析發(fā)現(xiàn),MYBS2通過調(diào)控αAmy3(α-amylase 3)基因的表達來調(diào)控糖平衡,進而調(diào)控轉(zhuǎn)基因植株的生長發(fā)育及抗逆性[50]。說明可以通過敲除MYBS2基因的方法提高水稻及其他作物的抗旱性和耐熱性。

3 MYB轉(zhuǎn)錄因子在水稻抗生物脅迫基因工程中的應(yīng)用進展

目前,關(guān)于MYB基因?qū)ι锩{迫的響應(yīng)研究較少,尤其在水稻上的研究更少?,F(xiàn)有研究發(fā)現(xiàn),有些MYB基因在水稻中超表達可以提高轉(zhuǎn)基因植株對病原菌、稻飛虱等的耐受性[51-52]。

苯丙素類包括各種化合物,如木質(zhì)素單體和羥基肉桂酸(HCAAs),是植物保護自身免受非生物脅迫和病原體感染所必需的物質(zhì)。在水稻中超表達MYB30、MYB55、MYB110基因,轉(zhuǎn)基因水稻中肉桂酸/木質(zhì)素單體途徑基因(磷酸合成酶基因、脫氫醌合成酶基因、兒茶酚氧位甲基轉(zhuǎn)移酶基因、PAL1等)的表達量提高,阿魏酸(一種羥基肉桂酸)含量較野生型對照和轉(zhuǎn)空載體對照增加,對病原真菌和病原細菌的抗性增強[51]。

在水稻中超表達OsPAL6、OsPAL8基因均提高了轉(zhuǎn)基因植株對褐飛虱的抗性,轉(zhuǎn)基因植株中水楊酸和木質(zhì)素含量提高;相反的,抑制OsPAL6、OsPAL8基因表達降低了轉(zhuǎn)基因植株對褐飛虱的抗性。進一步分析發(fā)現(xiàn),OsMYB30直接調(diào)控OsPAL6、OsPAL8基因的表達,從而調(diào)控褐飛虱抗性[52]??梢奜sMYB30基因在作物抗褐飛虱育種中具有重要作用,可以通過超表達OsMYB30基因的方法提高水稻及其他作物對褐飛虱的抗性。

4 展望

水稻是全球重要的糧食作物之一,在農(nóng)業(yè)發(fā)展中占有舉足輕重的地位,穩(wěn)定并提高其產(chǎn)量是保證國家糧食安全的重要途徑。但水稻在生長發(fā)育過程中,不可避免地遭受到干旱、高鹽、低溫及病蟲害等逆境脅迫,抑制其生長發(fā)育,甚至降低籽粒產(chǎn)量。因此,培育抗逆水稻品種以提高其抗逆性對保障糧食安全具有重要的現(xiàn)實意義。植物的抗逆性狀是多基因控制的復雜數(shù)量性狀,且易受環(huán)境影響,采用傳統(tǒng)遺傳育種方法選育抗逆品種存在效率低、周期長等缺點;而分子育種具有定向創(chuàng)造遺傳變異、實現(xiàn)優(yōu)良基因高效重組和聚合等優(yōu)點。因此,利用分子生物學技術(shù)挖掘利用優(yōu)異的抗逆基因,并通過基因工程育種技術(shù)提高水稻的抗逆性,是培育水稻抗逆新品種最有效的途徑。

MYB轉(zhuǎn)錄因子在水稻生長發(fā)育及抵御非生物脅迫和生物脅迫過程中具有重要的調(diào)控作用。超表達MYB基因可以提高轉(zhuǎn)基因水稻植株的抗逆性,有的甚至可以提高轉(zhuǎn)基因植株在逆境條件下的籽粒產(chǎn)量,這為水稻抗逆育種提供了非常重要的基因資源。除了水稻,MYB基因在其他作物上也表現(xiàn)出很強的抗逆性[53-61],有望在水稻及其他作物抗逆改良育種中應(yīng)用,尤其是那些可以在生殖生長期提高轉(zhuǎn)基因植株抗逆性并提高籽粒產(chǎn)量的MYB基因。盡管,一些MYB基因可以提高生殖生長期轉(zhuǎn)基因水稻的抗旱性,并最終提高籽粒產(chǎn)量,但是大部分MYB基因只是提高了苗期水稻的抗逆性,生殖生長期是否抗逆尚未得知。因此,對這些MYB基因的轉(zhuǎn)基因植株應(yīng)該進行生殖生長期抗逆性鑒定,以確定其功能強弱,為以后的抗逆育種應(yīng)用提供依據(jù)。另外,抗逆性鑒定最好在田間進行,因為室內(nèi)模擬環(huán)境與田間自然環(huán)境終究不同,田間自然環(huán)境更復雜,也更貼近于生產(chǎn)。

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