CN103388005B - 一种水稻钙依赖性蛋白激酶基因及其应用 - Google Patents
一种水稻钙依赖性蛋白激酶基因及其应用 Download PDFInfo
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Abstract
本发明公开了一种水稻钙依赖性蛋白激酶基因及其应用。该水稻钙依赖性蛋白激酶基因编码序列表中的SEQ ID No:1所示的钙依赖性蛋白激酶21(OsCPK21),该蛋白在花粉发育中特异性高表达。用OsCDPK21的RNAi载体转化水稻,获得营养生长正常、花粉隐性致死的转化体,从而为水稻雄性不育系的创制提供了一种有效的方法。
Description
技术领域
本发明涉及水稻转基因新品种培育,特别涉及一种介导水稻花粉隐性致死的钙依赖性蛋白激酶的编码基因在水稻雄性不育系创制方面的应用。
背景技术
水稻是人类赖以生存的主要粮食作物之一。利用水稻的优质遗传素材,应用转基因技术结合杂交育种方法,成为培育高抗与高产水稻新品种的重要途径。然而,水稻雄性不育系创制有价值相关基因的匮乏,一定程度上制约了应用转基因新技术高效、精准培育水稻新品种。因此,深化研究水稻花粉发育分子机制,并获得拥有自主知识产权的调控水稻花粉发育关键功能基因,不仅可为水稻智能化分子育种提供理论依据,且可为利用转基因技术创制水稻不育系提供优质基因资源。
近年来,许多研究通过利用水稻雄性发育相关突变体筛选调控水稻花粉发育相关功能基因,并开展了系统的水稻雄性不育的分子机制研究。研究发现,水稻tdr突变体绒毡层的凋亡受到抑制,造成小孢子的发育停滞并最终无法形成花粉;OsTDR基因于绒毡层中特异表达,编码一个核定位的DNA结合蛋白,推测其通过激活下游基因的表达从而调控绒毡层的凋亡(Li et al.,(2006)The rice tapetum degeneration retardation gene is required for tapetumdegradation and anther development.Plant Cell 18,2999-3014.)。此外,水稻Wax-deficientanther1(Wda1)突变体的雄蕊为白色且比正常水稻的雄蕊小,其绒毡层功能受损,使花药表皮的蜡质和花粉粒外壁缺失,最终造成雄性不育(Jung et al.,(2006)Wax-deficient anther1 isinvolved in cuticle and wax production in rice anther walls and is required for pollen development.Plant Cell 18,3015-3032.)。利用图位克隆等技术方法,已从tdr突变体和Wda1突变体中成功地克隆获得了雄性不育的效应基因。同时,仍然有很多雄性发育相关突变体未克隆出相应的突变基因,例如Osnop(Jiang et al.,(2005)The Oryza sativa no pollen(Osnop)gene playsa role in male gametophyte development and most likely encodes a C2-GRAM domain-containingprotein.Plant Mol.Biol.57,835-853.)、rip1(Han et al.,(2006)Rice Immature Pollen 1(RIP1)isa regulator of late pollen development.Plant Cell Physiol.47,1457-1472.)、pss1(Li et al.,(2007)Fine mapping of pss1,a pollen semi-sterile gene in rice(Oryza sativa L.).Theor.Appl.Genet.114,939-946.)、ms91(Liu et al.,(2007)Genetic analysis and molecular mapping of a nuclear recessivemale sterility gene,ms91(t),in rice.Genome 50,796-801.)、xs1(Zuo et al.,(2008)Phenotypiccharacterization,genetic analysis,and molecular mapping of a new mutant gene for male sterilityin rice.Genome 51,303-308.)和add(Zhang et al.,(2008)Characterization and mapping ofa newmale sterility mutant of anther advanced dehiscence(t)in rice.J Genet Genomics 35,177-182.)。由此可见,应用水稻花粉发育相关的功能基因创制水稻雄性不育系,对于精准与高效培育水稻新品种具有重要的现实意义。
已有研究表明,植物钙依赖性蛋白激酶(Calcium-dependent protein kinase,CDPK)是主要的Ca2+信号传导蛋白,通过介导Ca2+的信号通路,参与调节植物的生殖发育(Harper etal.,(2004)Decoding Ca2+signals through plant protein kinases.Annu.Rev.Plant Biol.55,263-288.;Harper and Harmon,(2005)Plants,symbiosis and parasites:A calcium signallingconnection.Nat.Rev.Mol.Cell Biol.6,555-566.)。CDPK广泛分布于高等植物、藻类和一些原生动物中(Harmon et al.,(2001)The CDPK superfamily of protein kinases.New Phytol.151,175-183.;Ishino et al.,(2006)A calcium-dependent protein kinase regulates Plasmodium ookineteaccess to the midgut epithelial cell.Mol.Microbiol.59,1175-1184.)。CDPK在结构上的特点为:具有N端可变区,并同时含有可以结合Ca2+的拟钙调素结构域(calmodulin-like regulatorydomain,CaM-LD)和激酶结构域(kinase domain),二者由连接区(junction domain)相连(Harper et al.,(1991)A Calcium-Dependent Protein-Kinase with a Regulatory Domain Similarto Calmodulin.Science 252,951-954.;Harmon et al.,(2000)CDPKs-a kinase for every Ca2+signal?Trends Plant Sci.5,154-159.)。CDPK的激酶结构域由300多个氨基酸组成,具有典型的Ser/Thr蛋白激酶亚结构域;连接区由20~30个氨基酸组成,是蛋白激酶活性自抑制的区域;拟钙调素结构域是钙结合区,也是CDPK的Ca2+激活结构域(Harper and Harmon,(2005)Plants,symbiosis and parasites:A calcium signalling connection.Nat.Rev.Mol.Cell Biol.6,555-566.);N端可变区的同源性很低,与CDPK的亚细胞分布和底物特异性有关(Breviario et al.,(1995)Molecular cloning of two novel rice cDNA sequences encoding putativecalcium-dependent protein kinases.Plant Mol.Biol.27,953-967.;Hrabak et al.,(1996)Characterization of eight new members of the calmodulin-like domain protein kinase gene familyfrom Arabidopsis thaliana.Plant Mol.Biol.31,405-412.)。
目前,在水稻中发现了31个CDPK编码基因(Asano et al.,(2005)Genome-wideidentification of the rice calcium-dependent protein kinase and its closely related kinase genefamilies:Comprehensive analysis of the CDPKs gene family in rice.Plant Cell Physiol.46,356-366.;Ray et al.,(2007)Expression analysis of calcium-dependent protein kinase gene familyduring reproductive development and abiotic stress conditions in rice(Oryza sativa L.ssp indica).Mol.Genet.Genomics 278,493-505.)。应用基因芯片研究发现,水稻钙依赖性蛋白激酶(OsCDPK)9个编码基因于雄花中特异表达:OsCDPK2、OsCDPK11、OsCDPK14、OsCPK21、OsCDPK22、OsCDPK25、OsCDPK26、OsCDPK27和OsCDPK29(Ye et al.,(2009)Expressionprofile of calcium-dependent protein kinase(CDPKs)genes during the whole lifespan and underphytohormone treatment conditions in rice(Oryza sativa L.ssp.indica).Plant Mol.Biol.70,311-325.)。
发明内容
本发明的目的在于筛选水稻花粉发育相关的功能基因,以用于创制水稻雄性不育系。
本发明研究发现,水稻的一个钙依赖性蛋白激酶基因OsCPK21于雄花中表达量相对于其他OsCDPK较高,推测OsCPK21可能在水稻雄花发育中发挥非常关键的调控作用。本发明通过RNAi技术在水稻中干扰OsCPK21的表达,获得了花粉败育的转基因水稻材料。由此推测,利用转基因技术,干扰、沉默或敲除OsCPK21基因,都可使创制水稻雄性不育系成为可能。鉴于该基因的应用价值及其利用潜力的巨大应用前景,有必要通过专利加以保护。
本发明所涉及的可介导花粉隐性致死的钙依赖性蛋白激酶基因,名称为OsCPK21,来源于水稻(Oryza sativa),编码序列表中的SEQ ID No:1所示的钙依赖性蛋白激酶21(OsCPK21)。该蛋白具有典型的钙依赖性蛋白激酶结构特征,拥有钙依赖性的激酶活性,且在花粉发育中特异性高表达。
序列表中的SEQ ID No:1序列由565个氨基酸残基组成,具有N端脂酰化位点(第1位至第8位氨基酸残基)、激酶结构域(第77位至第358位氨基酸残基)、自抑制区(第364位至第395位氨基酸残基)和4个EF-手型结构域(第415位至第529位氨基酸残基)。
本发明的钙依赖性蛋白激酶基因OsCPK21可以是所述钙依赖性蛋白激酶基因的cDNA序列,也可以是基因组DNA序列,或者是与这些序列具有90%以上同源性且编码相同功能蛋白的DNA序列。例如序列表中SEQ ID NO:2所示的cDNA序列编码SEQ ID No:1中1至565位氨基酸序列。
本发明通过体外磷酸化实验证明,OsCPK21具有典型的钙依赖性蛋白激酶活性(图1)。利用原位杂交技术,对OsCPK21在水稻生殖器官发育过程中生理功能研究结果表明,水稻花粉和花粉囊中表达的OsCPK21,可能直接参与了花粉发育的调控(图2A-D)。通过实时定量RT-PCR分析确定OsCPK21主要在开花当天的花粉中特异表达(图2E-G)。
基于上述OsCPK21基因的生化特性研究,本发明通过RNAi技术成功地抑制了OsCPK21基因在水稻转化植株中的表达,所获得的转基因水稻的花粉发育不正常,并失去育性。可见,利用转基因技术,干扰、沉默或敲除OsCPK21基因,可获得花粉败育的转基因水稻,这使得创制水稻雄性不育系成为可能。
在本发明的一个具体实例中,选取序列表SEQ ID NO:2的第1-533bp序列作为RNAi的引导序列,将该核酸片段以正反两个方向插入到植物表达载体pTCK-303中构建成为OsCPK21的RNAi双元表达载体pTCK-21i。将此RNAi质粒经由农杆菌转入水稻中,发现转基因水稻的OsCPK21表达均被有效抑制(图3)。同时,针对OsCPK21-RNAi转基因水稻植株的表型分析表明,相比对照野生型植株,OsCPK21-RNAi转化子的分蘖数、株高、叶长、穗长和颖果数均未有明显改变,显示其营养生长正常(表1)。然而,转化子的雄花发育状态检测发现,虽然颖壳和雌花发育都正常,但是雄蕊白色干瘪,其中未见任何花粉(图4)。
综上,将OsCPK21的RNAi双元表达载体经农杆菌介导转入水稻中,T-DNA插入并整合到水稻染色体上(如图5所示),可以获得营养生长正常,但雄性不育(花粉隐性致死)的转化体。该技术可为水稻智能化育种,遗传改造定向创制水稻雄性不育系,提供一种有效的方法。
附图说明
图1显示了OsCPK21具有典型的钙依赖性蛋白激酶活性,其中:A图所示为体外表达重组蛋白OsCPK21-Trx和其标签蛋白Trx的SDS-PAGE电泳检测结果;B图为体外磷酸化实验结果,左侧图为Pro-Q磷酸化蛋白染色试剂盒染色结果,中空箭头所示为OsCPK21-Trx自我磷酸化条带,黑色箭头所示为底物Histone III磷酸化条带;右侧图为相应SDS-PAGE胶的考马斯亮蓝染色结果。
图2显示OsCPK21于开花时在花粉中特异表达,其中:A-D图为OsCPK21的组织原位杂交结果,A、B和C、D分别为雄花的横切及雌蕊的纵切,A和C图为正义探针杂交结果,B和D为反义探针杂交结果,po和aw分别表示花粉和花粉囊;E和F为OsCPK21于雄花不同组织中的实时定量RT-PCR结果,其中E图以UBQ5为内参,F图以eEF-1α为内参;G图为OsCPK21于水稻生殖发育期中的实时定量RT-PCR结果,其中P1-P6示水稻花穗的形态建成时期,0DAH-20DAH示水稻颖果的不同发育时期,DAH为抽穗后天数。
图3是OsCPK21-RNAi双元表达载体pTCK-21i构建示意图和转基因水稻中OsCPK21的表达检测结果图,其中:上图为OsCPK21-RNAi双元表达载体(pTCK-21i)的结构图,OsCPK21的RNAi引导序列以正反两个方向插入pTCK-303载体,图中,UBi1 promoter region示玉米泛素基因启动子,Intron示内含子,Nos示终止子;下图为OsCPK21-RNAi转基因植株和对照转化植株中OsCPK21的表达研究,图中,R1-R9是RNAi转化子,control是对照转化植株,UBQ5为内参。
图4是OsCPK21-RNAi转基因植株的表型分析,其中:A和B为颖果成熟时期对照植株和RNAi转化子株型的比较,C和D为对照植株和RNAi转化子花穗形态的比较,E和F、G和H及I和J分别为对照植株和RNAi转化子的颖花、去除内外颖的颖花和雄蕊的比较,标尺为1mm。
图5是OsCPK21-RNAi转基因植株基因组DNA的PCR检测结果,其中R1-R9是RNAi转化子,control是对照转化植株。
具体实施方式
下述实验方法,如无特殊说明,均为常规方法。下述实验方法所用的试剂,如无特殊说明,均为自常规生化试剂公司购买得到。
植物材料:水稻粳稻品种中花15号(Oryza sativa L.ssp.japonica cv.Zhonghua 15)。
1、实验材料的获得和RNA的提取
(1)水稻不同器官和不同发育期颖果取材方法
水稻不同器官的取材:成叶、叶鞘、茎、花、颖果、胚乳和颖壳取自抽穗后4天的水稻植株,幼叶和根取自2周的水稻幼苗,取材后液氮冷冻并保存于-80℃低温冰箱。
水稻不同发育期颖果的取材:取抽穗后0、1、2、4、6、8、10、12、15和20天的稻穗,于冰上快速分离出初级颖果,取材后液氮冷冻并保存于-80℃低温冰箱。
(2)玻璃制品、塑料制品和电泳槽的去RNA酶处理
RNA相关实验过程中所用的玻璃制品在使用前于180℃烘烤8h。塑料制品,包括各种类型的枪头和离心管,用0.1%DEPC水溶液浸泡过夜,高压灭菌后置于80℃干燥箱中干燥。用于RNA电泳的电泳槽经清洗后,用无水乙醇中浸泡30min,然后用30%H2O2中浸泡30min,最后用灭菌的DEPC处理水冲洗5次。
(3)水稻不同器官和不同发育期颖果总RNA的提取
取0.5g材料,在液氮中研磨成粉末并转移到装有3mL预冷的RNA提取缓冲液的离心管中,充分混匀后加入3mL缓冲液饱和酚和1mL氯仿,混匀。4℃、13,000rpm离心30min。上清转移至另一离心管中,加入两倍体积的无水乙醇,-20℃放置2h。4℃13,000rpm离心10min,干燥沉淀。DEPC处理水溶解沉淀并转移至1.5mL离心管中,加入等体积8M LiCl,冰上放置1h后13,000rpm离心15min。沉淀溶于0.4mL DEPC处理水中。酚/氯仿抽提,乙醇沉淀后4℃13,000rpm离心15min。75%乙醇清洗RNA沉淀,室温干燥。加适量DEPC处理水溶解RNA。
(4)RNA质量检测
在GBC Cintra 10e紫外分光光度计上测RNA样品在260nm和280nm的光吸收值,通过吸收值计算RNA样品的浓度和判断RNA样品的纯度。RNA光吸收值和浓度换算公式:1OD260=40μg/mL。纯度判断方法:纯的RNA,其OD260/OD280为2.0,若污染了蛋白质或酚,OD260/OD280比值明显低于此值。然后通过1%琼脂糖凝胶电泳检测RNA的完整性。
(5)RNA样品中少量DNA的去除
通过无RNase的DNase消化RNA样品中残留的DNA,反应体系包括:1×RQ1 RNase-freeDNase Buffer、RNase inhibitor 20unit、RQ1 RNase-free DNase 1μL、RNA样品50μg,用DEPC-H2O补足体系至50μL。上述体系于37℃温育30min。DNA酶解反应结束后,用酚/氯仿抽提,乙醇沉淀回收RNA样品。
2、OsCPK21全长cDNA的克隆
提取抽穗0天颖果总RNA,逆转录为cDNA,并以此cDNA为模板PCR扩增OsCPK21基因全长序列。具体流程如下:
SS II逆转录总RNA:配制反应体系I:2μg总RNA、1μL Oligo dT15和6μLDEPC处理水,于65℃温育5min后迅速置于冰上。配制反应体系II:4μL 5×first-strand buffer、1μL RNase inhibitor、2μL 0.1M DTT、1μL 10mM dNTP Mixture和1μL SS II Reversetranscriptase。混合反应体系I和II,50℃逆转录反应60min,70℃处理15min灭活SS II反转录酶,置于冰上2min,加入1μL RNase H于37℃温育20min消化与cDNA链结合的mRNA。反应结束后70℃处理15min灭活RNase H,分装并保存于-20℃。
PCR扩增OsCPK21编码基因:使用高保真DNA聚合酶pfx扩增OsCPK21编码基因。反应体系如下:10×pfx Amplication Buffer 2.0μL、10×pfx Enhancer Buffer 2.0μL、50mMMgSO4 0.4μL、2.5mM dNTPs 2.4μL、上游引物OsCPK21-U 0.3μM、下游引物OsCPK21-L0.3μM、pfx DNA聚合酶0.2μL、cDNA 1.0μL,用dd H2O补充反应体系至20.0μL。PCR反应条件:95℃预变性5min;95℃变性30sec,54℃退火30sec,68℃延伸2min,30个循环;最后68℃延伸7min。
PCR所用引物序列如下:
上游引物OsCPK21-U:5’-ATGGGGGGCTGCTACTC-3’(SEQ ID No:3);
下游引物OsCPK21-L:5’-TCAGGGAGTATGAGTATCCTTGCAGAGC-3’(SEQ ID No:4)。
PCR产物的加A尾和T载体克隆:按Tiangen胶回收试剂盒使用说明切胶回收PCR产物。配置加A尾反应体系,于70℃温育30min进行加A尾反应。加A尾反应体系包括:1×Taq PCR buffer、胶回收产物7μL、Taq DNA聚合酶1μL和2mM dATP 1μL,总体积10μL。取上述加A产物按照Tiangen pGM-T载体试剂盒说明书进行T载体克隆,得到质粒pGMT-OsCPK21,经测序验证序列无误。
3、OsCPK21体外磷酸化功能验证
pET32a-OsCPK21原核表达载体的构建:以pGMT-OsCPK21为模板,通过引物OsCPK21-pET32a-U和OsCPK21-pET32a-L扩增获得OsCPK21的ORF,克隆至T载体并测序验证序列无误。其中,引物序列如下:
OsCPK21-pET32a-U:5’-TTGGATCCATGGGGGGCTGCTACTC-3’(SEQ ID No:5);
OsCPK21-pET32a-L:5’-TTCTCGAGTCAGGGAGTATGAGTATCCTTGCAGAGC-3’(SEQ ID No:6)。
通过Bam HI和Xho I将OsCPK21的ORF亚克隆至pET32a载体,转化菌株DH5α。从DH5α转化菌中提取pET32a-OsCPK21载体并转化原核表达菌株BL21(DE3)pLysS。
Trx-OsCPK21融合蛋白的原核表达:将转化有质粒pET32a-OsCPK21的菌株BL21(DE3)pLysS于LB-Amp/CM平板上划线,37℃培养过夜。挑单克隆接菌到10mLLB-Amp/CM液体培养基中,37℃250rpm震荡培养过夜。次日,将10mL过夜培养物接菌到1L LB-Amp/CM液体培养基中,37℃250rpm震荡培养至OD600介于0.5-0.7之间(大约需要120min)。加入IPTG至终浓度为1mM,21℃150rpm诱导表达8h。冰上放置5min,4000g、4℃离心10min,弃上清并将细胞沉淀保存于-20℃。
Trx-OsCPK21融合蛋白的提取及电泳检测:重悬细菌沉淀于30mL Lysis Buffer中。加入溶菌酶至终浓度为1mg/mL,DNase至终浓度为0.3unit/mL,RNase A至终浓度为12.5μg/mL,冰上放置30min。超声破碎菌体(裂解液始终置于冰上),功率强度36%,超声2sec、停8sec,共超声破碎20sec。4℃10000g离心20min,将上清液(可溶蛋白)转移至另一离心管中,冰上放置。用适量Solution Buffer重悬沉淀,获得包涵体蛋白。取20μL 可溶和包涵体蛋白样品,分别加入5μL 5×SDS-PAGE Sample Buffer,沸水中煮样4min,13000rpm离心2min,上样SDS-PAGE电泳检测,分离胶浓度为12%。
可溶性蛋白镍柱亲和纯化及透析:提取的可溶蛋白加入含2cm高Ni-NTA树脂的亲和层析柱中,收集流出液,用50mL Washing Buffer洗柱,待液体流尽后用4mL Elution BufferI和Elution Buffer II洗脱目的蛋,收集洗脱液,用50kDa超滤管快速脱盐,12%SDS-PAGE电泳检测。含有目的蛋白的组分在25mM Tris中4℃透析过夜。透析袋预处理方法:将透析袋浸泡在处理液I中,沸水煮沸10min,蒸馏水洗3次;将透析袋浸泡于处理液II中,沸水中煮10min,蒸馏水洗3次。透析结束后收集重组蛋白并进行分子筛纯化。
分子筛纯化方法:分子筛实验前一天先后用25mM Tris溶液和Mili-Q水平衡预装柱,次日上样2ml待纯化蛋白样品,之后用25mM Tris溶液洗柱,检测280nm吸收峰并收集各个组分。SDS-PAGE电泳检测各收集管,将目的蛋白组分合并,经超滤浓缩后液氮速冻并保存于-80℃。整个实验过程中蛋白始终处于4℃。Trx标签蛋白的表达与纯化方法与Trx-OsCPK21相同。
OsCPK21的体外磷酸化实验:在1.5mL EP管中配置反应体系,体系中各组分用量如下:Trx-CDPK21或Trx标签蛋白5μg、底物histone III-S 10μM、1×kinase assay buffer、ATP100μΜ、Ca2+1mM或EGTA2mM、W7 200μM,反应体积50μL。将反应体系置于30℃水浴中反应30min,每管加入12.5μL 5×SDS-PAGE Sample Buffer终止反应,沸水中煮样4min。13000rpm离心2min,上样,进行SDS-PAGE电泳,分离胶浓度为12%。使用Pro-QDiamond Phosphoprotein Gel Stain kit进行磷酸化蛋白染色,并用多功能激光分子成像系统(pharos FX)采集图像。体外磷酸化实验检测结果如图1所示,显示了OsCPK21具有典型的钙依赖性蛋白激酶活性。
4、实时定量RT-PCR(Q-RT-PCR)
(1)水稻生殖器官发育中OsCDPKs的实时定量RT-PCR
取材:在水稻的孕穗期,根据叶鞘中正在发育的花穗的长度将此时期化分为6个阶段(Itoh et al.,2005 Rice plant development:from zygote to spikelet.Plant Cell Physiology 46,23-47),即P1(0~3cm)、P2(3~5cm)、P3(5~10cm)、P4(10~15cm)、P5(15~20cm)、P6(20~30cm)。在水稻开花后,根据抽穗后颖果发育的天数(DAH)将颖果发育化分为8个阶段,即0 DAH、1 DAH、2 DAH、4 DAH、8 DAH、12 DAH、15 DAH、20 DAH,其中0 DAH为抽穗当天。所有材料取材后立即置于液氮中冷冻,并于-80℃保存。
实时定量RT-PCR:使用Trizol Plant试剂盒(Invitrogen)提取总RNA,去DNA后逆转录为cDNA,将cDNA稀释5倍并保存于-20℃待用。在8联定量PCR管中加入10μL 2×ABIpower SYBR green PCR master mix,上、下游引物(10μM)和cDNA各1μL,加dd H2O补足反应体系至20μL,混匀并短暂离心。将上述反应体系置于ABI 7500实时荧光定量PCR仪中,按标准流程进行PCR反应。循环条件为:50℃2min,94℃预变性10min;95℃变性15sec,60℃退火及延伸1min,40个循环;最后添加一个溶解曲线测定循环。反应结束后应用软件ABI 7500 Software v2.0分析实验结果。待测基因的扩增引物:
OsCPK21-F:5’-CTACTCCGCCTACGCCTCC-3’(SEQ ID No:7);
OsCPK21-R:5’-AGCTCCTTCCCCAGCACGTA-3’(SEQ ID No:8)。
以OsActin11(AK100267)的表达为内参。结果见图2G,可以看出抽穗时初级颖果中的OsCPK21相对于抽穗前的幼穗及抽穗后不同发育状态的颖果特异高表达。
(2)水稻花粉发育中OsCDPKs的实时定量PCR
取材:水稻开花当天,取将要开颖的花,在解剖镜下将颖壳、雌蕊和雄蕊分离;取花药自然开裂散落的花粉和花粉囊。使用Trizol Plant试剂盒提取总RNA,去DNA后逆转录为cDNA。
实时定量RT-PCR:按上述方法进行Q-RT-PCR反应,以eEF-1α(AK061464)和UBQ5(AK061988)的表达为内参。结果见图2E和F,表明抽穗时初级颖果中特异高表达的OsCPK21主要取决于在成熟花粉和花粉囊中的mRNA的积累水平。
5、OsCPK21 mRNA组织原位杂交
原位杂交探针制备:为使探针具有较好的通透性和特异性,本实验在OsCPK21的3’UTR区域选择360bp左右的片段为探针合成模板。设计带酶切位点的上、下游引物,其中上游引物序列:5’-TCTCGAGCCGCATCAGCTACCAGGAGT-3’(SEQ ID No:9);下游引物序列:5’-GTCTAGACCCATTTACCGAAAGAGTAG-3’(SEQ ID No:10)(酶切后产生5’突出末端),进行PCR扩增,将产物克隆至pBS-T载体并测序。
体外转录:提取测序正确的质粒,通过单酶切线性化载体作为体外转录的模板,为获得正义探针应用HindIII单酶切载体,为获得反义探针应用EcoRI单酶切载体,通过QIAGEN胶回收试剂盒回收获得线性化质粒,并以此为模板利用载体本身的T7、T3RNA转录位点进行体外转录获得反义和正义RNA探针。体外转录体系包括:4μL 5×transcription buffer、1μg线性化质粒、2μL T7或T3 RNA polymerase、2μL RNase inhibitor,用DEPC-H2O补足至20μL。以上反应体系于37℃反应2h。然后,进行质粒模板的消化和RNA探针的回收。在体外转录体系中加入2μL RNase free DNaseI,37℃反应15min以消化DNA模板,加入0.8μL 500mM EDTA终止反应,加入2.5μL 4M LiCl和75μl无水乙醇,-20℃沉淀过夜。次日,13000rpm离心,弃上清,70%乙醇洗涤两次,干燥。100μL DEPC-H2O溶解沉淀,0.5×TBE电泳检测RNA探针质量,分装并于-80℃保存备用。
原位杂交探针的定量:将标准RNA样品及待测RNA探针进行梯度稀释,点于尼龙膜上,置于120℃烘箱中交联30min,马来酸缓冲液(MaB)中浸泡2min,blocking solution中室温封闭30min,抗体(anti-DIG-AP,1:5000稀释于blocking solution中)室温孵育30min,washing buffer室温洗15min,重复二次,TNM50洗膜3min,BCIP/NBP底物显色,通过与标准样品的颜色对比,对待测探针进行定量。
水稻花的固定和切片:取水稻雄花与雌花,放入FAA固定液中室温抽气2h,之后换新鲜固定液于室温固定12h以上(过夜)。次日对材料按如下梯度脱水,30%、50%、70%、85%、90%、100%、100%、100%乙醇,每步0.5~1h。脱水后对材料进行透明,流程如下:25%二甲苯-75%乙醇,50%二甲苯-50%乙醇,75%二甲苯-25%乙醇,90%二甲苯-10%氯仿,90%二甲苯-10%氯仿,90%二甲苯-10%氯仿,每步0.5~1h。透明后将材料浸入溶好的石蜡,60℃保温3天,期间更换6次纯蜡;之后进行石蜡包埋,包埋后迅速冷却材料,4℃短期保存。切片前一天下午将蜡块切至露出材料,将切口放在DEPC处理水制成的冰上过夜,次日切片,切片厚度为10μm。
OsCPK21 mRNA组织原位杂交:将DEPC处理水加在多聚赖氨酸载片上,蜡带置于水上并将其展平,展片后吸干多余的水。42℃烤片24h。之后按如下流程进行脱蜡复水:100%二甲苯室温20min,100%二甲苯室温20min,66%二甲苯-33%乙醇,33%二甲苯-66%乙醇,100%乙醇、100%乙醇、90%乙醇、70%乙醇、50%乙醇、30%乙醇、10%乙醇、H2O、H2O,室温每步2min。脱水后进行蛋白酶消化:于37℃预热PK buffer后加入蛋白酶(PK)(母液浓度为10mg/mL)至终浓度为2.5μg/mL,37℃处理15min,DEPC处理水室温洗片3次,每次2min。然后进行乙酰化:于100mM pH 8.0的三乙醇胺中10min,加入乙酸酐至终浓度0.25%,室温放置10min,2×SSC室温洗片两次,每次5min。乙酰化后进行脱水:10%乙醇、30%乙醇、50%乙醇、70%乙醇、90%乙醇、100%乙醇、100%乙醇,室温每步2min。脱水后42℃1h左右烘干载玻片,将杂交液均匀涂于载玻片上,100μL/片并加盖Parafilm膜,将载玻片置于含有0.3M NaCl-50%甲酰胺的湿室中42℃杂交过夜。杂交液配方:77.2μL杂交液A与22.8μL杂交液B混合;杂交液B:1μL RNA探针、17.8μL DEPC-H2O、1.5μLtRNA、2.5μL PolyA混合,80℃变性5min,立即置于冰上。次日洗片:40mL 4×SSC室温5-10min,重复三次。洗片后进行RNase A处理:37℃预热RNase buffer,加入RNase A至终浓度为25μg/mL,放入载玻片,37℃保温30min,之后RNase buffer 37℃洗15min,重复两次。RNase A消化后进行低/高严谨洗片:2×SSC(650mL)室温低严谨洗片30min,0.5×SSC 60℃高严谨洗片30min,洗片时以小转子低速搅动。最后进行封闭、抗体孵育和显色反应:1×PBS室温洗片5min,0.5%的封闭液(Blocking Reagent溶于1×PBS,现配)中室温封闭60min;1×PBS室温洗片5min,于湿室中抗体(1μL anti-DIG-AP+50μL 10mg/mLBSA+450μL 1×PBS)室温孵育120min,湿室中垫有1×PBS饱和的滤纸;1×PBST(650ml)室温洗片10min,重复洗片2次,1×TNM50室温5min,2%BCIP/NBT(用TNM50配制)室温黑暗中显色过夜。次日,于载玻片上滴加甘油,镜检并拍照。结果见图2A-D,图2B和2D为反义探针杂交结果,2A和2C为正义探针杂交结果(阴性对照),在图2B中可以看到水稻花粉和花粉囊中表达的OsCPK21,表明OsCPK21可能直接参与了花粉发育的调控。
6、OsCPK21基因沉默及过表达的转基因水稻的获得
(1)基因沉默triger序列的克隆及测序
在OsCPK21的ORF框中选取特异性较高的533bp DNA片段(即翻译起始位点起1-533bp的序列),以OsCPK21的ORF序列为模板,使用NEB Phusion DNA Polymerase试剂盒,分别用TCK-21RNAi-U(含有BamHI酶切位点)和TCK-21RNAi-L(含有KpnI酶切位点)、TCK-21RNAi-R-U(含有SacI酶切位点)和TCK-21RNAi-R-L(含有SpeI酶切位点)两对引物进行PCR,得到用作RNAi的DNA片段,连接T载体并测序。将测序正确的基因沉默triger序列分别正向和反向连接至pTCK303载体的intron序列两边,形成RNAi载体pTCK-21i。其中各引物的序列如下:
TCK-21RNAi-U:5’-TTGGATCCATGGGGGGCTGCTACTCC-3’(SEQ ID No:11);
TCK-21RNAi-L:5’-TTGGTACCATGACGAGGTGGACGGAG-3’(SEQ ID No:12);
TCK-21RNAi-R-U:5’-TTGAGCTCATGGGGGGCTGCTACTCC-3’(SEQ ID No:13);
TCK-21RNAi-R-L:5’-TTTCTAGAATGACGAGGTGGACGGAG-3’(SEQ ID No:14)。
如图3所示,533bp的OsCPK21特异性核酸片段以正反两个方向插入到植物表达载体pTCK-303中构建成为OsCPK21的RNAi双元表达载体pTCK-21i,两个特异片段由一水稻内含子隔开,由玉米泛素启动子(ZmUBi promoter)驱动表达。
之后,将pTCK-21i质粒转入农杆菌EHA105,并经由农杆菌将此质粒转入水稻品种中花11号中。图5显示了pTCK-21i转基因植株基因组DNA的PCR检测结果,其中R1-R9是RNAi转化子,control是对照转化植株。进而,通过RT-PCR方法对RNAi的效率进行了检测。结果显示,在R1至R9几个转化子中OsCPK21的表达均被有效抑制(图3)。
7、转基因水稻的表观表型观察统计及细胞学变化
(1)转基因水稻的表型观察及统计
待水稻成熟后(抽穗两周后),分别统计空载载体转化植株及21-RNAi水稻植株的分蘖数,测量每一个株系中最高分蘖的株高,箭叶长,穗长,颖花数(每一种转基因品种的统计样本数大于30株),同时进行取材保存,并对明显的表型形状用Nikon S 10进行数码拍照。待水稻结实后统计空载载体转化水稻植株的千粒重。最终数据用SPSS软件进行统计学分析,同时用实体镜观察水稻花器官。
表1给出了OsCPK21-RNAi转化子和对照植株的表型比较数据,表中数据均来自至少30个样本,分蘖数于植株开花时统计,株高、叶长、穗长、每穗颖果数和百粒重均在颖果成熟时统计。结果表明,相比对照野生型植株,转化子的分蘖数、株高、叶长、穗长和颖果数均未有明显改变,显示OsCPK21-RNAi转化子营养生长正常。
表1 转OsCPK21-RNAi的转基因植株和对照植株的生长发育数据比较
空载对照植株和RNAi转化子颖果成熟时期的株型分别如图4的A和B所示,花穗形态分别如图4的C和D所示,颖花分别如图4的E和F所示,去除内外颖的颖花分别如图4的G和H所示,雄蕊分别如图4的I和J所示。RNAi转化子的雄花发育状态检测发现,虽然颖壳和雌花发育都正常,但是雄蕊白色干瘪,其中未见任何花粉(图4)。
Claims (8)
1.水稻钙依赖性蛋白激酶基因OsCPK21在水稻育种中的应用,通过转基因技术干扰、沉默或敲除水稻的OsCPK21基因,从而创制水稻雄性不育系,所述基因编码序列表中SEQ ID No:1所示氨基酸序列的蛋白质。
2.如权利要求1所述的应用,其特征在于,所述水稻钙依赖性蛋白激酶基因的序列如序列表中SEQ ID No:2所示。
3.如权利要求1所述的应用,其特征在于,构建OsCPK21基因的RNAi载体并转化水稻,获得OsCPK21的表达受干扰的雄性不育转基因水稻。
4.如权利要求3所述的应用,其特征在于,所述OsCPK21基因的RNAi载体通过下述方法构建而成:以OsCPK21基因的特异性核酸片段作为引导序列,将其以正反两个方向插入到植物表达载体中。
5.如权利要求4所述的应用,其特征在于,所述OsCPK21基因的特异性核酸片段是序列表中SEQ ID No:2的第1-533位核苷酸序列。
6.如权利要求4所述的应用,其特征在于,所述OsCPK21基因的RNAi载体中,两个插入方向相反的OsCPK21基因特异性核酸片段由一个水稻内含子隔开,并由玉米泛素启动子驱动表达。
7.如权利要求4所述的应用,其特征在于,所述植物表达载体是pTCK-303。
8.如权利要求3所述的应用,其特征在于,所述OsCPK21基因的RNAi载体是双元表达载体,经农杆菌介导转入水稻中。
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CN110042089B (zh) * | 2019-03-18 | 2021-04-23 | 华南农业大学 | 芥蓝2-含氧依赖性双加氧酶基因Ba2ODD1及其应用 |
CN116064645B (zh) * | 2022-10-17 | 2024-12-06 | 惠州学院 | 一种降低水稻结实率的OsCDPK14基因及其编码得到的蛋白和应用 |
CN118515737A (zh) * | 2024-07-23 | 2024-08-20 | 浙江大学海南研究院 | 一种基于水稻生长素受体基因的蛋白质原核表达系统创建方法 |
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