水稻矮秆基因克隆研究进展

陈文娟,刘亚男,孙亚利,李万昌 ,李景原

(河南师范大学 生命科学学院,河南 新乡 453007)

水稻矮秆基因克隆研究进展

陈文娟,刘亚男,孙亚利,李万昌*,李景原

(河南师范大学 生命科学学院,河南 新乡 453007)

矮秆是水稻育种中最重要的农艺性状之一,对增强水稻抗倒伏性、提高水稻产量有重要作用。综述了水稻矮秆基因的分类,并从参与油菜素内酯、赤霉素、独角金内酯、生长素等生物合成或信号传导途径方面阐述了水稻矮秆基因的克隆情况,为矮秆突变基因在水稻育种中的应用提供理论依据。

水稻； 矮秆基因； 克隆

水稻(OryzasativaL．)是世界上最重要的粮食作物之一,其株型改良对提高产量至关重要[1]。矮秆是水稻育种中最重要的农艺性状之一,它可以增强水稻抗倒伏性并提高产量[2]。目前,已经克隆了多个矮秆基因,其中半矮秆基因sd1(semi-dwarf 1)是唯一用于水稻育种的,被称为“绿色革命”基因,它的成功运用,使水稻单产提高了20%～30%[3]。但为了避免依赖单一半矮秆基因,需要继续加大对水稻矮秆基因的研究,找到更多有利的矮秆基因资源,用于水稻育种,进一步提高水稻产量。研究表明,水稻株高的降低是由于植物内源激素如赤霉素(GA)、油菜素内酯(BR)、独角金内酯(SLs)的缺乏或者他们的信号传导途径的改变造成的[4]。目前，尚未见从这几方面具体阐述矮秆基因克隆情况的报道。为此,综述了水稻矮秆基因的分类,并从参与BR、GA、SLs、生长素(IAA)等生物合成或信号传导途径方面阐述了水稻矮秆基因的克隆情况,为矮秆突变基因在水稻育种中的应用提供理论依据。

1 水稻矮秆基因的分类

水稻矮秆基因有多种分类方法。根据株高可分为半矮秆、矮秆和极矮秆3种类型[5]。矮秆常指成熟时植株高度等于或低于原正常植株高度一半的矮秆突变系；半矮秆则是指株高介于矮秆和正常植株高度之间的突变系；极矮秆是指极其矮化的矮秆突变系。在具体划分时,卢永根等[6]把半矮秆株高定在75～102 cm,而马良勇等[7]把株高小于50 cm的定为矮秆,根据原株高或亲本的株高不同,将50～70 cm的定为矮秆或半矮秆,超过70 cm时一般归为半矮秆。根据矮秆基因对GA的反应,将矮秆基因分为在发芽期时α-淀粉酶显著增加而芽伸长较少的N型(normal type)；内源GA较少但对外源GA敏感的T型(Tan-ginbozu type)；对GA不敏感的D 型(Daikoku type)；内源GA正常,对外源GA敏感的E 型(Ebisu type)[8]。根据矮秆基因的表型,将众多矮源分为多蘖矮生、小粒矮生、畸形矮生和半矮生等4类[9]。正常的水稻植株,成花诱导的完成伴随着末梢4或5个节间的伸长,而底部其他节间基本不伸长。水稻植株的矮化是节间长度缩短或节间数减少的结果,也可能是两者共同作用的结果。根据水稻植株矮化与节间的关系,以节间长度占株高的比例为指数,将矮秆分为dn、dm、d6、nl和sh 等5种基本类型,而将节间比例正常的品种标定为N型[10]。 dn型的特征是节间比例与N型相同,即各节间按相同比例同时缩短；dm型的特征是倒2节间特别短；d6型的特征是只有穗下节间伸长,其他节间不伸长；nl型的特征是有颈叶,倒1节间短而第4节间长,偶尔第6节间伸长；sh型的特征是倒1节几乎不伸长,穗子包藏在剑叶叶鞘中。其中,除dn型外,所有矮秆类型都存在某一节间显著缩短的特征,不同的矮秆基因作用于水稻植株的不同伸长节间,其矮化作用只发生在某一特定生长时期,导致某些节间显著缩短。

2 水稻矮秆基因的克隆

株高是由复杂的遗传途径决定的,矮秆来自BR、GA、SLs、IAA等的生物合成或信号传导途径的缺陷突变。目前,研究者利用图位克隆等方法得到多个矮秆相关基因,其参与不同的生物合成途径或信号传导途径。

2.1 参与BR生物合成或信号传导途径的矮秆基因

BR是重要的植物激素,在植物生长发育中起重要作用,能促进植物茎秆伸长和细胞分裂。BR缺陷矮秆突变体一般分为2种,一种是信号传导缺陷突变,另一种是自身合成缺陷突变。无论是哪一种都会导致矮秆表型。

BR自身合成缺陷突变的矮秆基因有d2(dwarf 2)[11]、d11[12]、CPB1(clustered primary branch 1)[13]、SDG725(set domain group 25)[14]、brd1(BR-deficient dwarf 1)[15]。其中,前3个基因都属于细胞色素P450(CYP450)家族,编码BR生物合成过程中的一种关键酶。d2编码CYP90D2蛋白,它的隐性突变导致BR生物合成受阻,致使水稻植株矮化[11]。d11编码CYP724B1蛋白,其突变体产生移码突变,不能合成此蛋白质[12]。CPB1是d11的一个新等位基因,编码CYP724B1蛋白,其突变体中His360亮氨酸取代了保守的CPB1/D11区域,此区域控制突变体的穗结构和种子大小[13]。水稻SDG725基因编码H3K36甲基转移酶,其下调引起表型缺陷,包括矮秆、节间缩短、叶片直立、小粒[14]。brd1是BR合成缺陷突变体,在突变体中导入野生型OsDWARF基因可以使突变体恢复正常表型[15]。OsDWARF编码水稻中的BR合成酶C-6氧化酶,此酶的缺失会抑制叶和茎中细胞的有序排列和极性生长分裂,使植株矮化[15]。

另外,还有BR合成补充途径缺失突变的矮秆基因brd2(BR-deficient dwarf 2)[16]和OsDWARF4[17]。brd2编码DIM(DIMINUTO)/DWF1蛋白,催化BR生物合成早期的24-亚甲基胆固醇(24-MC)到菜油甾醇(CR)的反应,与拟南芥DIM1/DWF1基因有同源序列,brd2中一个外显子的单碱基G缺失导致移码突变,BR合成受阻,造成成熟植株严重矮化[16]。OsDWARF4基因编码CYP90B2蛋白,催化BR生物合成后期步骤的C-22 的羟基化,该基因突变后,致使BR合成量减少,植株略矮、叶片直立,产量增加[17]。

BR信号传导缺陷突变的矮秆基因有d1352[18]、TUD1(Taihu dwarf)[19]、编码LRR(leucine-rich repeat)激酶的基因XIAO[20]、OsBLE3(brassinolide-enhanced 3)[21]、SG1(short grain 1)和SGL1(short grain like 1)[22]、DLT(dwarf and low tillering)[23]、OsBZR1(brassinosteroid resistant 1)[24]、d61和OsBRI1(brassinosteroid insensitive 1)[25]、OsDof12(DNA binding with one finger)[26]、OsMDP1(MADS-domain-containing protein 1)[27]、OsBRL1(brassinosteroid insensitive like 1)和OsBRL3[28]、BU1(brassinosteroid upregulated 1)[29]。基因d1352在U-box E3泛素连接酶区插入一个24 bp的片段 ,故在U-box保守结构域插入了8个氨基酸,使其失去识别BR的功能[18]。TUD1基因编码U-Box E3泛素连接酶,它和异源三聚体G蛋白α亚基一起共同调节BR介导的水稻生长；TUD1是d1的上位基因,二者协同互作对BR信号途径进行调节,二者的双突变体呈现植株第2节间变短、叶直立、谷粒变短的表型[19]。基因XIAO对BR 信号传递和细胞分裂起调控作用,其T-DNA插入突变体的表型为植株矮化、叶直立且结实率下降[20]。OsBLE3基因编码芸苔素内酯(BL)上调蛋白,是一个BR增强型基因,可通过对BL和IAA的双重调节参与细胞伸长,导致植株生长缓慢、矮化[21]。SG1和SGL1分别编码SG1蛋白和类SG1蛋白,SG1降低了对BR的应答,SG1和SGL1过表达会产生短粒和矮化表型[22]。基因DLT编码一个新的GRAS[GAI(GA insensitive)、RGA(repressor of gal-3)、SCR(SCARECROW)]家族蛋白,参与BR合成基因表达的反馈抑制, 其突变体dlt表现为矮化、少分蘖[23]。OsBZR1 基因编码拟南芥BZR1同源蛋白,经过RNAi技术沉默该基因后,BR信号传递受阻,产生矮化、叶直立表型[24]。d61和OsBRI1是等位基因,单碱基的突变就导致了BR受体,即OsBRI1激酶结构域中的1834F被替换,从而使OsBRI1活性大大降低,而OsBRI1活性对BR调节水稻植株的正常生长和发育是必需的[25]。OsDof12编码Dof蛋白,OsDof12过表达时,BR信号的2个信号正调节因子OsBR1 和OsZR1显著下调,表明OsDof12在水稻中是一个负调节因子,通过抑制BR信号传导使植株形态发生改变[26]。另外一个编码BR信号负调节因子的基因是OsMDP1,它编码MADS-box转录因子。OsMDP1缺陷导致主根系变短、胚芽鞘伸长、叶节倾角变大,还可导致编码木葡聚糖转葡糖苷酶的基因OsXTR1表达增强[27]。OsBRL1和OsBRL3在根中高度表达,在嫩枝中表达量较少,其编码蛋白是BR信号途径中的正调控因子；同时,OsBRL1 和OsBRL3也会部分参与根中BR的感知过程,二者的转基因反义植株表现出不同程度的矮化现象[28]。BU1编码螺旋-环-螺旋蛋白质BU1,参与BR信号传导并控制叶的弯曲度；BU1蛋白也是BR反应的正调节因子,通过OsBRI1和 RGA1参与BR反应的2个通路,BU1过表达会使株高变矮、种子变大、叶的弯曲度增大、育性降低[29]。

2.2 参与GA生物合成或信号传导途径的矮秆基因

GA在高等植物生长和发育的多个阶段是至关重要的。它促进茎的伸长、开花、种子萌发、种子和果实的生长。GA缺陷矮秆突变体一般也分为2种,一种是信号传导缺陷突变,另一种是自身合成缺陷突变。

GA信号传导缺陷突变的矮秆基因有d89[30]、dwt1(dwarf tiller 1)[31]、d1[32]、gid1(GA-insensitive dwarf 1)[33]、gid2[34]、OsGAI(GA-insensitive)[35]、EUI1(elongated uppermost internode 1)[36]。d89编码异源三聚体G蛋白的α亚基,D89中1个碱基的替换(A—G)使苏氨酸突变为丙氨酸,致使α螺旋变短,不能与GSP结合,故导致了G蛋白的失活,GA信号传导受阻,最终表现为植株矮化[30]。dwt1编码WUSCHEL相关同源框(WOX)转录因子,与拟南芥WOX8和WOX9同源,dwt1与GA的信号传导有关,在突变体中细胞分裂和细胞伸长受抑,主枝的穗长发育正常,矮化分蘖的穗长变短[31]。d1编码GTP 结合蛋白的α 亚基,在突变体HO541中该位点上有833 bp的缺失,造成G蛋白失活,抑制了GA的信号传导,节间的细胞分裂减少,导致植株矮化[32]。GID1编码GA 受体,其类似于激素敏感的脂肪酶,是核定位蛋白质,突变体gid1中GA信号传递受阻,导致植株矮化[33]。GID2蛋白是GA信号传导中的正调节因子,调节抑制因子SLR1(slender rice 1)的降解,突变体gid2中SLR1不能正常降解,从而抑制GA信号向下游的传导,致使植株严重矮化、不育等[34]。OsGAI与EUI1编码蛋白则是负调节因子,OsGAI即SLR1,编码水稻GA负调控因子DELLA蛋白,属于GRAS基因超家族,OsGAI的过表达可阻碍GA的信号传导,致使植株矮化[35,37]。基因EUI1编码细胞色素P450单加氧酶,其通过改变水稻中GA的反应进而负调节节间的伸长,过表达该基因可导致植株极度矮化[36]。

GA自身合成缺陷突变的矮秆基因有sd1[5]、ph1(plant height 1)[38]、pad(plant architecture determinant)[39]、sgd1(t)(small-grain dwarf mutant 1)[40]、OsDOG(dwarf rice with overexpression of gibberellin-induced gene)[41]、d35[42]、OsKO2(ent-kaurene oxidase 2)[43]、dit1(dwarf increased tillering 1)[44]、d18[45]、OsGA2ox6(Oryzasativagibberellin 2-oxidase 6)[46]、OsWOX3A(WUSCHEL-related homeobox 3A)[47]、BC12(brittle culm 12)/GDD1(gibberellin-deficient dwarf 1)[48]。半矮秆基因sd1,编码由389个氨基酸组成的GA20氧化酶(GA20ox),GA20ox是GA合成途径中的关键酶,sd1突变将导致植株不同程度的矮化[5]。基因ph1与sd1紧密连锁,编码几丁质诱导GA应答蛋白(CIGR),属于GRAS家族,ph1可调控株高,且对株高有很大的遗传效应[38]。pad基因编码由SNP导致的单个氨基酸改变的细胞质膜蛋白OsMCA1,该基因可能参与GA代谢与信号传导的调控,且能降低GA的生物活性,产生矮化、小叶的株型[39]。sgd1(t)与BC12/GDD1是等位基因,编码kinesin-4亚家族驱动蛋白,该基因第4个外显子上发生由G到T的碱基突变,使第186位保守氨基酸由甘氨酸突变为缬氨酸,影响该蛋白质的功能,sgd1(t)对外源GA敏感,呈现植株矮化、穗及各节间显著缩短的表型[40]。OsDOG基因编码GA诱导的A20/AN1型锌指蛋白,其调节GA稳态,并负调节水稻植物细胞伸长[41]。d35与OsKO2位于同一基因位点,d35编码贝壳杉烯氧化酶,该酶催化GA生物合成早期步骤,D35缺失植株严重矮化[42]；OsKO2还影响糊粉层细胞程序化死亡进程,并促进种子萌发[43]。基因dit1编码类胡萝卜素裂解酶7(CCD7),是htd1(high tillering dwarf 1)的等位基因,dit1的第6个外显子中CC置换AA,使终止密码子取代丝氨酸,形成截短的蛋白质,丧失功能,dit1为外源GA敏感型基因,可产生矮化多分蘖表型[44]。基因D18编码GA 3β羟化酶,其突变体GA活性下降,产生矮秆表型[45]。基因OsGA2ox6编码GA2氧化酶(GA2ox),GA2ox通过2β-羟基化作用使有活性的GA失活,过量表达OsGA2ox6后植株表现为半矮秆性状[46]。OsWOX3A是GA响应基因,其编码的OsWOX3A参与GA生物合成通路的负反馈调节,保持GA的稳态。OsWOX3A直接与编码贝壳杉烯酸的基因KAO(ent-kuarenoic acid oxidase)的启动子相互作用,过表达OsWOX3A的植株中GA合成量下降,导致植株极度矮化[47]。基因BC12/GDD1编码驱动蛋白类似蛋白BC12,与GA生物合成基因启动子结合,抑制内源性GA的生成,使细胞伸长受阻,使植株产生矮化表型[48]。

2.3 参与SLs生物合成或信号传导途径的矮秆基因

SLs生物合成或信号传导缺陷突变的矮秆基因有d27[49]、D10[50]、D3[51]、D53[52]、HTD2/D88/D14[53]、HTD1/D17[54]、OsTB1(teosinte branched 1)[55]、FIE1(fertilization-independent endosperm 1)[56]。d27参与MAX/RMS/D通路,编码位于叶绿体的含铁蛋白质,是BL生物合成的一个新成员,d27突变体呈现多分蘖、矮秆的表型[49]。D10编码类胡萝卜素裂解双加氧酶,参与SLs/SLs衍生物的生物合成,隐性突变导致SLs合成受阻,致使植株矮化[50]。D3编码拟南芥MAX2(more axillary branches 2)/ORE9(oresara 9) 同源的D3蛋白,对SL信号是必需的,D3组装成一个SCFD3[SKP(S-phase kinase-associated protein)、cullin、F-box protein dwarf 3]复合体,并与D14联合抑制水稻分枝,d3突变体呈现株高变矮、分蘖增多的表型[51]。D53编码SCFD3泛素复合物中的一种底物,是SLs信号途径的抑制子,SLs可诱导其降解,D53负调节SLs信号传导,其过表达可使植株矮化[52]。基因HTD2、HTD1/D17和OsTB1都对水稻分蘖数进行负调节。HTD2也称D88或D14,编码酯酶/脂肪酶,抑制水稻分枝发生,D14可能是SLs信号途径的一个组分,在其下游起作用,其突变体呈现多蘖、矮秆表型[53]。HTD1/D17编码拟南芥MAX3的同源蛋白胡萝卜素裂解双加氧酶OsCCD7,参与SLs的生物合成,突变体htd1中SLs合成受阻,产生多蘖矮秆表型[54]。OsTB1编码转录因子,此转录因子携带与DNA启动子结合的螺旋-环-螺旋结构,命名为TCP结构域,参与SLs信号传递；OsTB1过表达,导致侧枝明显减少,说明OsTB1负调节侧枝生长[55]。此外,DNA甲基化和去甲基化,也能阻断遗传信息的传递过程,从而影响植株正常生长发育,例如：FIE1基因甲基化后使植株呈现矮秆、多蘖的Epi-df(epi-allele)的突变表型[56]。

2.4 参与IAA生物合成或信号传导途径的矮秆基因

IAA在许多植物的侧根起始、维管束分化、细胞伸长、细胞分裂、胚的形成、芽伸长和根尖形成等重要发育过程中起关键作用。IAA自身合成或信号传导缺陷突变的矮秆基因有NAL1(narrow leaf 1)[57]、OsIAA1[58]、tdd1(tryptophan deficient dwarf 1)[59]。nal1-2(narrow leaf 1-2)是NAL1的无义等位基因突变,因整个启动子和NAL1的第一外显子404 bp的片段被删除,而使得NAL1无法编码特定的胰蛋白酶样丝氨酸和半胱氨酸蛋白酶,阻碍IAA的信号传导,产生矮秆、窄叶表型[57]。OsIAA1编码Aux/IAA蛋白,OsIAA1在IAA和BR的信号通路的相互作用中及植株的形态建成中起重要作用,OsIAA1过表达会造成植株矮化、株型松散[58]。TDD1编码邻氨基苯甲酸合酶β亚基同源蛋白,参与IAA合成,催化色氨酸生物合成途径的第一步，并在色氨酸依赖性IAA生物合成的上游起作用,突变体tdd1中由于色氨酸和IAA的不足,导致植株出现矮化、窄叶、花异常等表型[59]。

2.5 与其他生物合成或信号传导途径有关的矮秆基因

虽然控制水稻矮秆相关基因的大多是激素相关基因,但也有一些控制其他因素导致水稻矮秆的基因,如Psd1(photoperiod sensitive dwarf 1)[60]、OsCESA7(cellulose synthase A subunit 7)[61]、RLS2(rapid leaf senescence 2)[62]、d6/OSH15(homeobox 15)[63]。在长日照条件下,显性矮秆突变体Psd1出现严重矮化表型,但短日照时接近正常生长。突变位点在编码特定的脂质转移蛋白的基因中,突变后蛋白质C末端缩短,不能发挥正常功能,使细胞分裂和伸长受损,导致矮秆[60]。基因OsCESA7编码纤维素酶A的第七亚基,错义突变改变该酶锌指结构域中高度保守的C40为Y,造成茎中厚壁细胞细胞壁增厚有缺陷且纤维素含量减少,使植株呈现脆秆和矮化等表型[61]。RLS2基因编码OsEXO70A1,rls2突变触发EXO70同源基因转录波动,并影响到与大量元素的吸收和运输有关的基因,造成维管束不规则和矿质营养同化的紊乱,产生矮秆等性状[62]。基因OSH15 编码含有同源异型结构域的蛋白质,属于KNOX(knotted-like homeobox) 家族Ⅰ类。其与Oskn3是同一基因,参与调节SAM(shoot apicalm eristem) 的形成,控制水稻节间的发育,OSH15缺失会造成植株矮化[63]。d6突变体植株矮化,表现为第2、3、4 节间变短,而稻穗和第1节间没有变短,导入OSH15基因可以使突变体恢复正常表型[63]。

3 展望

水稻矮秆基因的研究已经有了很大进展,发现了很多有利突变体,但是现在能真正应用到生产中并发挥重要作用的只有水稻半矮秆基因sd1。因此，为了避免依赖单一半矮秆基因,需要找到更多的可用基因,培育理想株型,以应对人口的增加和更加多变的环境条件。首先,应该注重野生稻的发展和利用。亲本材料遗传背景狭窄，便得不到理想的产量、质量和抗性材料,而野生稻提供了丰富的具有优良性状的遗传资源,如病虫害强抗性和胁迫耐受性等。其次,利用人工诱变,如空间诱变、射线诱变等，选育出具有优良性状的矮秆品系。同时，考虑到环境因素如光、温等对水稻生长的影响。最后,要克隆出具有优良性状的基因,搞清楚其调控网络。确保新品种在产量、谷粒质量、抗逆性等方面具有优良农艺性状,有助于进一步保护国家粮食安全。

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Research Advances in Cloning of Dwarf Genes in Rice

CHEN Wenjuan,LIU Ya’nan,SUN Yali,LI Wanchang*,LI Jingyuan

(College of Life Sciences,Henan Normal University,Xinxiang 453007,China)

Dwarf is one of the most important agricultural traits in rice(OryzasativaL.) breeding,which plays an important role in enhancing the lodging resistance and production of rice.Here,classification and cloning of rice dwarf genes were reviewed from the aspects of the biosynthesis and signal transduction pathways of brassinosteroid,gibberellins,strigolactones and auxins,which could provide a theoretical basis for the application of dwarf mutant genes in rice breeding.

rice(OryzasativaL．); dwarf gene; clone

2016-09-04

国家自然科学基金项目(U1304317)

陈文娟(1992-)，女，河南商丘人，在读硕士研究生，研究方向：作物遗传育种。E-mail：1273298205@qq.com

*通讯作者：李万昌(1974-),男,河南汝州人，教授，博士，主要从事水稻遗传育种研究。E-mail：li_wan_chang@163.com

S511

A

1004-3268(2017)03-0001-07