CN116083443A - 一种芍药PlMYB103基因及其蛋白的应用和鉴定方法 - Google Patents
一种芍药PlMYB103基因及其蛋白的应用和鉴定方法 Download PDFInfo
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Abstract
本发明公开了一种芍药PlMYB103基因、芍药PlMYB103基因编码的蛋白。本发明还公开了含有芍药PlMYB103基因的表达盒、重组载体、重组菌或细胞。本发明还公开了芍药PlMYB103基因、芍药PlMYB103蛋白、表达盒、重组载体、重组菌或细胞在增强植物茎秆强度中的应用。本发明还公开了一种获得具有高茎秆强度植物的方法。本发明还公开了鉴定由所述方法获得的具有高茎秆强度植物的方法。本发明通过将构建的PlMYB103基因过量表达载体转化到烟草中进行表达,植株茎秆强度增强,茎秆木质部径宽增大、木质部细胞壁厚度增加、木质素沉积水平提高,创制了高茎秆强度的烟草新种质。
Description
技术领域
本发明涉及一种增强植物茎秆强度的方法,尤其涉及一种芍药PlMYB103基因及其蛋白的应用和鉴定方法,属于芍药生物技术领域。
背景技术
转录因子在植物生长发育中起着重要的作用。MYB类转录因子家族是植物中成员数量最多、分布最广的转录因子家族,广泛参与植物的各种生理活动及对外界环境的响应过程(Roy S.Function of MYB domain transcription factors in abiotic stress andepigenetic control of stress response in plant genome.Plant Signaling andBehavior,2016,11:e1117723)。MYB类转录因子分子结构中包含1~4个不完全重复序列(R),每个重复序列由50~53个保守的氨基酸残基及中间的间隔序列组成,每个重复序列形成3个螺旋。根据R序列的数量和位置,MYB类转录因子家族可以分为4类:1R(R1/2,R3-MYB)、2R(R2R3-MYB)、3R(R1R2R3-MYB)及4R(R1R2R2R1/2-MYB)(Dubos C,et al.MYBtranscription factors in Arabidopsis.Trends in PlantScience,2010,15:573-581)。其中,2R蛋白成员是植物中MYB家族中数量最多的一类,它根据蛋白质羧基端DNA结合区保守氨基酸的基序又可以分为28个亚类。这类MYB成员广泛参与植物初生及次生代谢、细胞分化、激素信号传导、生长发育调控、生物及非生物逆境响应等生命过程(Wu Y,etal.Evolution and functional diversification of R2R3-MYB transcription factorsin plants.Horticulture Research,2022,9:uhac058)。
芍药(Paeonialactiflora Pall.)为芍药科芍药属多年生宿根草本花卉,是我国的传统名花。由于缺乏机械支撑,部分芍药品种花茎弯曲,这严重降低了其切花质量和商业价值。前期研究表明,HCT、COMT等结构基因均能增强芍药花茎强度(Zhao DQ,etal.Silicon enhances stem strength by promoting lignin accumulation inherbaceous peony(Paeonialactiflora Pall.).International Journal of BiologicalMacromolecules,2021,190:769-779;Zhao DQ,et al.Melatonin enhances stemstrength by increasing lignin content and secondary cell wall thickness inherbaceous peony.Journal of Experimental Botany,2022,73:5974-5991)。但迄今为止,有关芍药花茎强度的转录调控层面研究鲜有报道。
发明内容
发明目的:本发明所要解决的技术问题是提供了一种芍药PlMYB103基因,其核苷酸序列如SEQ ID NO.1所示。
本发明还要解决的技术问题是提供了所述的芍药PlMYB103基因编码的蛋白,其氨基酸序列如SEQ ID NO.2所示。
本发明还要解决的技术问题是提供了表达盒、重组载体、重组菌或细胞。
本发明还要解决的技术问题是提供了芍药PlMYB103基因、芍药PlMYB103蛋白、表达盒、重组载体、重组菌或细胞在增强植物茎秆强度中的应用。
本发明还要解决的技术问题是提供了一种获得具有高茎秆强度植物的方法。
本发明还要解决的技术问题是提供了鉴定由所述方法获得的具有高茎秆强度植物的方法。
技术方案:为解决上述技术问题,本发明提供如下技术方案:
本发明提供一种芍药PlMYB103基因,所述芍药PlMYB103基因的核苷酸序列如SEQID NO.1所示。
本发明还提供所述芍药PlMYB103基因编码的蛋白,所述蛋白的氨基酸序列如SEQID NO.2所示。
本发明还提供表达盒、重组载体、重组菌或细胞,其含有所述的芍药PlMYB103基因。
其中,所述重组载体是将芍药PlMYB103基因导入载体质粒。
其中,所述重组菌是将所述重组载体导入宿主菌,筛选鉴定后保留阳性菌株即得。
本发明还提供所述芍药PlMYB103基因、所述芍药PlMYB103蛋白、所述表达盒、重组载体、重组菌或细胞在增强植物茎秆强度中的应用。
本发明还提供一种获得具有高茎秆强度植物的方法,包括以下步骤:
1)使植物包含芍药PlMYB103基因;或
2)使植物表达芍药PlMYB103基因编码的蛋白。
其中,所述方法还包括转基因、杂交、回交或无性繁殖。
本发明还提供鉴定由所述方法获得的具有高茎秆强度植物的方法,包括以下步骤:
(1)测定所述植物是否包含芍药PlMYB103基因;或
(2)测定所述植物是否表达芍药PlMYB103基因编码的蛋白。
其中,所述应用、所述方法或所述的鉴定方法中所述植物包括烟草。
有益效果:与现有技术相比,本发明具有如下显著优点:
本发明通过将构建的PlMYB103基因过量表达载体转化到烟草中进行表达,植株茎秆强度增强,茎秆木质部径宽增大、木质部细胞壁厚度增加、木质素沉积水平提高,创制了高茎秆强度的烟草新种质。
附图说明
图1为芍药PlMYB103与来自拟南芥R2R3-MYBs的同源进化分析;
图2A为芍药PlMYB103基因在不同组织部位的表达模式分析图;图2B为芍药PlMYB103基因在茎微管束不同发育时期的表达模式分析图(图2B中S1:初生细胞壁合成,S2:初生与次生细胞壁合成过渡期,S3:次生细胞壁生成,S4:次生细胞壁发育成熟,不同小写字母表示差异显著(P<0.05));
图3为芍药PlMYB103的亚细胞定位观察;
图4为芍药PlMYB103的转录激活活性分析;
图5为野生型和转PlMYB103基因烟草植株的PCR鉴定(图中WT:野生型烟草K326;OE-PlMYB103-L1:转基因植株1;OE-PlMYB103-L2:转基因植株2);
图6为野生型和转PlMYB103基因烟草植株的qRT-PCR鉴定(图中WT:野生型烟草K326;OE-PlMYB103-L1:转基因植株1;OE-PlMYB103-L2:转基因植株2);
图7为野生型和转PlMYB103基因烟草植株的表型比较(图中WT:野生型烟草K326;OE-PlMYB103-L1:转基因植株1;OE-PlMYB103-L2:转基因植株2);
图8为野生型和转PlMYB103基因烟草植株的茎秆形态指标比较,**表示极显著差异(P<0.01)(图中WT:野生型烟草K326;OE-PlMYB103-L1:转基因植株1;OE-PlMYB103-L2:转基因植株2);
图9为野生型和转PlMYB103基因烟草植株的茎秆横截面微观结构及木质素沉积水平比较(图中WT-1:野生型烟草K326植株1;WT-2:野生型烟草K326植株2;OE-PlMYB103-L1:转基因植株1;OE-PlMYB103-L2:转基因植株2;Ixf,束间木质部纤维;Xv,木质部导管);
图10为野生型和转PlMYB103基因烟草植株的茎秆木质部细胞壁厚度比较。**表示极显著差异(P<0.01)(图中WT:野生型烟草K326;OE-PlMYB103-L1:转基因植株1;OE-PlMYB103-L2:转基因植株2)。
具体实施方式
下面结合附图对本发明的技术方案作进一步说明。
实施例1芍药PlMYB103基因的克隆与序列分析
芍药PlMYB103基因的克隆:采用RACE技术进行基因的克隆,以扬州大学国家芍药种质资源库中芍药主栽品种‘红艳争辉’盛花期花下12cm的茎秆为植物材料,采后使用液氮速冻,并保存在-80℃下。
样品使用MiniBEST Plant RNA Extraction Kit(TaKaRa Biotechnology Co.,Ltd.,简称TaKaRa)试剂盒进行RNA提取,并使用核酸蛋白仪检测RNA浓度和OD值,利用1.5%的琼脂糖凝胶电泳检测总RNA的完整性。
芍药PlMYB103基因的3'端序列利用3'-RACE技术获取。采用3'-Full RACE Kit(TaKaRa)试剂盒,先进行3'cDNA的合成,以获得的3'cDNA产物作为3'-Outer-PCR扩增的模板,用于3'-Outer-PCR引物为(5'-TCAGCATTATTCCCAGTTC-3'),随后以获得的3'-Outer-PCR产物作为3'-Inner-PCR扩增的模板,用于3'-Inner-PCR扩增引物为(5'-TAGTTCACTTGATGGGATTG-3')。3'-Inner-PCR扩增产物经过琼脂糖凝胶电泳分析,采用DNAGel Extraction Kit(Tsingke Biotechnology Co.,Ltd.,简称Tsingke)凝胶回收试剂盒回收目的片段。使用5min TA/Blunt-Zero Cloning Kit试剂盒(Vazyme BiotechnologyCo.,Ltd.,简称Vazyme)将回收获得的纯化目的片段产物连接到TOPO载体上。随后将连接产物转化TreliefTM5α感受态细胞(Tsingke),而后在LB平板上(含有Kan 50mg/L)37℃过夜培养,挑取阳性单克隆扩大培养,进行测序验证,直至获得芍药PlMYB103基因的3'端序列。
芍药PlMYB103基因的5'端序列利用5'-RACE技术获取。采用5'-Full RACE Kit(TaKaRa)试剂盒,先进行5'cDNA的合成,以获得的5'cDNA产物作为5'-Clontech PCR扩增的模板,用于5'-Clontech PCR扩增引物为(5'-CTATTTGAGCCCATCTGTTCCCCAAAAC-3')。5'-Clontech PCR扩增产物经过琼脂糖凝胶电泳分析,采用DNA Gel Extraction Kit(Tsingke)凝胶回收试剂盒回收目的片段。使用5min TA/Blunt-Zero Cloning Kit试剂盒(Vazyme)将回收获得的纯化目的片段产物连接到TOPO载体上。随后将连接产物转化TreliefTM5α感受态细胞(Tsingke),而后在LB平板上(含有Kan 50mg/L)37℃过夜培养,挑取阳性单克隆扩大培养,进行测序验证,直至获得芍药PlMYB103基因的5'端序列。
芍药PlMYB103基因的完整开放阅读框序列利用PCR扩增获取。采用Full LengthKit(TaKaRa)试剂盒,先进行Full-length cDNA的合成,以获得的Full-length cDNA产物作为Full-lengthPCR扩增的模板,用于Full-lengthPCR扩增引物为(Forward Primer:5'-GGGCAAGCAGTGGTATCA-3'、Reverse Primer:5'-ACTGCATTTGCTCACACT-3')。Full-lengthPCR扩增产物经过琼脂糖凝胶电泳分析,采用DNA Gel Extraction Kit(Tsingke)凝胶回收试剂盒回收目的片段。使用5min TA/Blunt-Zero Cloning Kit试剂盒(Vazyme)将回收获得的纯化目的片段产物连接到TOPO载体上。随后将连接产物转化TreliefTM5α感受态细胞(Tsingke),而后在LB平板上(含有Kan 50mg/L)37℃过夜培养,挑取阳性单克隆扩大培养,进行测序验证,直至获得芍药PlMYB103基因的完整开放阅读框序列。
芍药PlMYB103基因的序列分析:使用DNAMAN(v9)软件分析芍药PlMYB103基因开放阅读框长度及氨基酸数目。序列分析显示,芍药PlMYB103基因长1,147bp,具有由924个碱基组成的完整开放阅读框,由113个碱基组成的5'端非编码区,由110个碱基组成的3'端非编码区,共编码307个氨基酸。所述芍药PlMYB103基因的核苷酸序列如SEQ ID NO.1所示,氨基酸序列如SEQ ID NO.2所示。
使用MEGA(v7.0.26)软件邻接法(NJ)构建芍药PlMYB103推导的氨基酸序列与来自拟南芥的R2R3-MYBs蛋白的系统进化树。同源进化分析显示,芍药PlMYB103与拟南芥AtMYB103具有最高的同源性(图1)。
实施例2芍药PlMYB103基因的表达模式分析
芍药PlMYB103基因的表达模式分析:以芍药主栽品种‘红艳争辉’盛花期的根、叶、花、茎、茎的表皮皮层区、维管束区和髓区为植物材料,用于芍药PlMYB103基因的空间表达模式分析;以芍药主栽品种‘红艳争辉’四个茎秆发育阶段的茎的维管束区为植物材料,用于芍药PlMYB103基因的时间表达模式分析。样品采集后使用液氮速冻,并保存在-80℃下。
样品使用MiniBEST Plant RNA Extraction Kit(TaKaRa)试剂盒进行RNA的提取,并使用核酸蛋白仪检测RNA浓度和OD值,利用1.5%的琼脂糖凝胶电泳检测总RNA的完整性;采用HiScript III RT SuperMix for qPCR(+gDNA wiper)(Vazyme)试剂盒进行cDNA的合成。以获得的cDNA产物用SYBR qPCR SuperMix Plus(Novoprotein)试剂盒进行实时荧光定量PCR检测。在此基础之上,以芍药PlActin(JN105299)基因为内参(ForwardPrimer:5'-GTTGCCCTTGATTACGAG-3'、Reverse Primer:5'-CAGCTTCCATTCCGATTA-3'),同时设计PlMYB103基因的特异性引物(Forward Primer:5'-ATGGGTCATCACTCTTGC-3'、ReversePrimer:5'-CTTTCTCAGGGACTTCAC-3')进行实时荧光定量PCR检测。反应体系:5.0μLSYBR qPCR SuperMix(Novoprotein E096-01A),0.4μL Forward Primer、0.4μL Reverse Primer,0.8μL cDNA模板,3.4μL ddH2O。反应程序:95℃预变性30s,接下来95℃~5s、55℃~30s和72℃~30s共40个循环;循环结束后绘制溶解曲线:65℃加热至95℃,每0.5℃收集一次荧光信号;每次反应要重复三次。
如图2所示,芍药PlMYB103基因在茎尤其是维管束中具有最高表达,随着芍药花茎发育,PlMYB103基因在茎维管束中的表达水平先上升,在木质部次生细胞壁加厚过程中达到最大值,后缓慢下降。
实施例3芍药PlMYB103的亚细胞定位观察
芍药PlMYB103蛋白融合eGFP绿色荧光蛋白的表达载体构建:设计含有酶切位点Sal I用于扩增PlMYB103序列的引物(Forward Primer:5'-cggggatcctctagagtcgacATGGGTCATCACTCTTGCTGCA-3',Reverse Primer:5'-caccatggtactagtgtcgacCAGAGATGAAGGATAAGAAGATAACATAGTT-3')。PCR扩增体系:12.5μL 2×Phanta Flash Master Mix(Vazyme)、1μLForward Primer、1μL Reverse Primer、2μL DNA模板(采用MiniBEST Plant Genomic DNAExtraction Kit(TakaRa)试剂盒提取的芍药茎秆DNA作为模板)、8.5μL ddH2O。反应程序:98℃预变性30s;98℃变性10s,52℃退火10s,72℃延伸10s,共35个循环;72℃延伸1min。反应结束后将PCR反应液进行1%琼脂糖凝胶电泳分析,并采用DNA Gel Extraction Kit(Tsingke)凝胶回收试剂盒回收含酶切位点的PlMYB103大片段。取双元表达载体pCAMBIA2300质粒用SalI进行单酶切,反应体系为:2.0μL 10×CutSmart Buffer、7μLpCAMBIA2300质粒、0.4μLSal I(20000U/mL)、10.6μL ddH2O;37℃反应1h。单酶切产物经过琼脂糖凝胶电泳分析,采用DNA Gel Extraction Kit(Tsingke)凝胶回收试剂盒回收纯化质粒pCAMBIA2300大片段。用plus One step PCR Cloning Kit(Novoprotein)试剂盒采用同源重组的方法连接两个回收的产物,反应体系为:2μL 5×反应缓冲液、plus重组酶(Novoprotein NR005-02B)、4μL pCAMBIA2300大片段、1μLPlMYB103大片段;在50℃金属浴连接15min后置于冰上冷却,连接产物转化TreliefTM5α感受态细胞(Tsingke),而后在LB平板上(含有Kan 50mg/L)37℃过夜培养,挑取阳性单克隆扩大培养,进行测序验证,直至pCAMBIA2300-PlMYB103-eGFP表达载体构建成功。
芍药PlMYB103蛋白融合eGFP绿色荧光蛋白的表达载体瞬时侵染本氏烟草叶片:取5μL pCAMBIA2300-PlMYB103-eGFP表达载体质粒转化GV3101(pSoup-p19)感受态细胞(TOLOBIO),而后在YEB平板上(含有Rif 50mg/L和Kan 50mg/L)28℃培养2d,挑取阳性单克隆于YEB液体培养基中(含有Rif 50mg/L和Kan 50mg/L),28℃,200rpm培养过夜。取所摇菌液2mL加入50mL含有相同抗生素的液体YEB(含有Rif50mg/L和Kan 50mg/L)中,在相同条件下培养至OD600=0.7。将摇好的菌倒入50mL离心管中,室温离心5000rpm、10min,弃上清,再用灭菌水清洗两次(清除多余的培养基和抗生素),随后用缓冲液(含有MES 10mmol/L、MgCl2 10mmol/L和乙酰丁香酮200μmol/L)重悬至OD600=0.7,静置1h备用。选用培养35d~45d的本氏烟草,利用1mL注射器侵染,随后暗培养2~3d,取注射部位下表皮制片,在超高分辨率激光共聚焦显微镜(TCS SP8 STED,徕卡公司,德国)激发光488nm下观察绿色荧光信号。结果发现,PlMYB103蛋白定位在本氏烟草叶片表皮细胞的细胞核中(图3),表明芍药PlMYB103能在细胞核中发挥作用。
实施例4芍药PlMYB103的转录激活活性分析
芍药PlMYB103的GAL4-BD酵母表达载体构建:设计含有酶切位点Nde I用于扩增含有完整开放阅读框(不含终止密码子)的PlMYB103序列的引物(Forward Primer:5'-tcagaggaggacctgcatatgATGGGTCATCACTCTTGCTGCA-3',Reverse Primer:atggccatatgCAGAGATGAAGGATAAGAAGATAACATAGTT-3')、仅含有N端(包括R2R3保守结构域)的PlMYB103序列的引物Forward Primer:5'-tcagaggaggacctgcatatgATGGGTCATCACTCTTGCTGCA-3',ReversePrimer:5'-ttcggcctccatggccatatgAGTTGCTGGAGGCACCAAGTT-3')和仅含有C端(不包括R2R3保守结构域)的PlMYB103序列的引物(Forward Primer:5'-tcagaggaggacctgcatatgGTTACGCCTAGCAGCACTGATCA-3',Reverse Primer:5'-ttcggcctccatggccatatgCAGAGATGAAGGATAAGAAGATAACATAGTT-3')。PCR扩增体系:12.5μL 2×Phanta Flash Master Mix(Vazyme)、1μL Forward Primer、1μL Reverse Primer、2μL DNA模板(采用MiniBEST PlantGenomic DNA Extraction Kit(TakaRa)试剂盒提取的芍药茎秆DNA作为模板)、8.5μLddH2O。反应程序:98℃预变性30s;98℃变性10s,52℃退火10s,72℃延伸10s,共35个循环;72℃延伸1min。反应结束后将PCR反应液进行1%琼脂糖凝胶电泳分析,并采用DNA GelExtraction Kit(Tsingke)凝胶回收试剂盒回收含酶切位点的PlMYB103、PlMYB103-N和PlMYB103-C大片段。取GAL4-BD酵母表达载体pGBKT7(BD7)质粒(简称为pGBKT7-BD)用NdeI进行单酶切,反应体系为:2.0μL 10×CutSmart Buffer、7μLBD7质粒、0.4μLNde I(20000U/mL)、10.6μL ddH2O;37℃反应1h。单酶切产物经过琼脂糖凝胶电泳分析,采用DNA GelExtraction Kit(Tsingke)凝胶回收试剂盒回收纯化质粒BD7大片段。用plusOne step PCR Cloning Kit(Novoprotein)试剂盒采用同源重组的方法连接两个回收的产物,反应体系为:2μL 5×反应缓冲液、plus重组酶、4μLBD7大片段、1μLPlMYB103/PlMYB103-N/PlMYB103-C大片段;在50℃金属浴连接15min后置于冰上冷却,连接产物转化TreliefTM5α感受态细胞(Tsingke),而后在LB平板上(含有Kan 50mg/L)37℃过夜培养,挑取阳性单克隆扩大培养,进行测序验证,直至BD7-PlMYB103、BD7-PlMYB103-N和BD7-PlMYB103-C表达载体构建成功。
芍药PlMYB103的GAL4-BD酵母表达载体在酵母Y2HGold菌株(上海唯地生物技术有限公司)中表达:取5μL BD7-PlMYB103、BD7-PlMYB103-N和BD7-PlMYB103-C酵母表达载体质粒转化Y2HGold感受态细胞(上海唯地生物技术有限公司),而后在SD/-Trp平板上30℃培养3d,挑取阳性单克隆于SD/-Trp液体培养基中,30℃,200rpm培养过夜至OD600=0.5。取适量菌液用SD/-Trp液体培养基梯度稀释101、102、103、104倍,点于SD/-Trp、SD/-Trp-Ade-His和含有X-α-gal的SD/-Trp-Ade-His培养基平板上,根据菌落生长情况及酵母是否变蓝来验证转录激活活性。
结果发现,含有BD77-PlMYB103和BD77-PlMYB103-C的质粒的酵母能在含有X-α-gal的SD/-Trp-Ade-His平板上正常生长并变蓝,表明PlMYB103的转录激活结构域能够在酵母中发挥功能,并且其转录激活结构域位于C端的调控结构域(图4)。
实施例5芍药PlMYB103基因过量表达载体在烟草中的表达
芍药PlMYB103基因过量表达载体构建:设计含有酶切位点BamHI和Kpn I用于扩增PlMYB103序列的引物(Forward Primer:5'-caggtcgactctagaggatccATGGGTCATCACTCTTGCTGCA-3',Reverse Primer:5'-ttcgagctcagatctggtaccCAGAGATGAAGGATAAGAAGATAACATAGTT-3')。PCR扩增体系:12.5μL 2×Phanta Flash Master Mix(Vazyme)、1μL ForwardPrimer、1μL Reverse Primer、2μL DNA模板(采用MiniBEST Plant Genomic DNAExtraction Kit(TakaRa)试剂盒提取芍药茎秆DNA作为模板)、8.5μL ddH2O。反应程序:98℃预变性30s;98℃变性10s,52℃退火10s,72℃延伸10s,共35个循环;72℃延伸1min。反应结束后将PCR反应液进行1%琼脂糖凝胶电泳分析,并采用DNA Gel Extraction Kit(Tsingke)凝胶回收试剂盒回收含酶切位点的PlMYB103大片段。取双元表达载体pCAMBIA1301质粒用BamHI和Kpn I(NEB)进行双酶切,反应体系为:2.0μL 10×CutSmartBuffer、7μL pCAMBIA1301质粒、0.4μLBamH I(20000U/mL)、0.4μLKpn I(20000U/mL)、10.2μL ddH2O;37℃反应1h。双酶切产物经过琼脂糖凝胶电泳分析,采用DNA Gel ExtractionKit(Tsingke)凝胶回收试剂盒回收纯化质粒pCAMBIA1301大片段。用plus Onestep PCR Cloning Kit(Novoprotein)试剂盒采用同源重组的方法连接两个回收的产物,反应体系为:2μL 5×反应缓冲液、0.5plus重组酶、4μL pCAMBIA1301大片段、1μLPlMYB103大片段、2.5μL ddH2O;在50℃金属浴连接15min后置于冰上冷却,连接产物转化TreliefTM5α感受态细胞(Tsingke),而后在LB平板上(含有Kan 50mg/L)37℃过夜培养,挑取阳性单克隆扩大培养,进行测序验证,直至pCAMBIA1301-PlMYB103过量表达载体构建成功。
芍药PlMYB103基因过量表达载体转化烟草:取5μL pCAMBIA1301-PlMYB103过量表达载体质粒转化GV3101(pSoup-p19)感受态细胞(TOLOBIO),而后在发根农杆菌培养基(YEB)平板上(含有Rif 50mg/L和Kan 50mg/L)28℃培养2d,挑取阳性单克隆于YEB液体培养基中(含有Rif 50mg/L和Kan 50mg/L),28℃,200rpm培养过夜。取所摇菌液2mL加入50mL含有相同抗生素的液体YEB(含有Rif 50mg/L和Kan 50mg/L)中,在相同条件下培养至OD600=0.7。将摇好的菌倒入50mL离心管中,室温离心5000rpm、10min,弃上清,再用灭菌水清洗两次(清除多余的培养基和抗生素)备用。先在灭菌后的小三角瓶中加入适量乙酰丁香酮(20mg/mL),向离心管中加入5mL MS0(MS0液体基本培养基,无琼脂和蔗糖)将溶解菌体,用枪打匀,倒入加有适量乙酰丁香酮(20mg/mL)的小三角瓶中,然后加MS0至50mL。向另一个灭菌小三角瓶中加入50mL MS0(MS0液体基本培养基,无琼脂和蔗糖)备用。取烟草的无菌苗叶片,切成小块(约1cm×1cm),放入上述小三角瓶中,共切100片叶片放入瓶中,将叶片倒入覆有纱布的烧杯,取经过滤后的叶片加入含有50mL MS0+100μL乙酰丁香酮(100μmol/mL)的小三瓶中,侵染8min,侵染时不断轻轻摇动;侵染完成后,滤去菌液,取出叶片,用无菌滤纸吸干叶片表面多余的菌液,接种于共培养培养基[MS+3.0mg/L 6-BA+0.1mg/L NAA+30g/L蔗糖+6.66%琼脂]中,暗培养3d;共培养结束后,转入抗性芽筛选分化培养基[MS+3.0mg/L 6-BA+0.1mg/L NAA+30g/L蔗糖+6.66%琼脂+100mg/L Cb+25mg/L Hyg]中进行选择培养,两周继代一次,直到分化出芽;当分生不定芽达2cm以上时,切下不定芽,转入生根筛选培养基[1/2MS+0.3mg/L IBA+30g/L蔗糖+6.66%琼脂+50mg/L Cb+8mg/L Hyg]进行生根筛选。经过4-6个月的培养,可获得转芍药PlMYB103基因烟草。
实施例6转芍药PlMYB103基因烟草的植株鉴定
转芍药PlMYB103基因的烟草植株的PCR鉴定:采用MiniBEST Plant Genomic DNAExtraction Kit(TakaRa)试剂盒提取烟草叶片DNA。在此基础之上,以烟草NtActin(AB158612)基因为内参(Forward Primer:5'-TCCTCATGCAATTCTTCG-3'、Reverse Primer:5'-ACCTGCCCATCTGGTAAC-3'),同时设计PlMYB103基因的特异性引物(Forward Primer:5'-ATGGGTCATCACTCTTGC-3'、Reverse Primer:5'-CTTTCTCAGGGACTTCAC-3')进行PCR扩增。反应体系:12.5μL 2×Rapid Taq Master Mix(Vazyme)、1μL Forward Primer、1μL ReversePrimer、2μL DNA模板、8.5μL ddH2O。反应程序:95℃预变性3min;95℃变性15s,52℃退火15s,72℃延伸15s,共35个循环;72℃延伸5min。反应结束后将PCR反应液进行凝胶电泳检测。
从图5可以看出,野生型烟草和转芍药PlMYB103基因烟草中均可检测到单一而明亮的NtActin条带,而就扩增PlMYB103条带而言,仅在转芍药PlMYB103基因烟草中检测到单一、明亮、清晰的条带,在野生型烟草中检测到双条带,且较转芍药PlMYB103基因烟草存在长度差,表明芍药PlMYB103基因已成功转入烟草中。
转芍药PlMYB103基因的烟草植株的qRT-PCR鉴定:采用MiniBEST Plant RNAExtraction Kit(TaKaRa)试剂盒提取总RNA,并采用HiScript III RT SuperMix for qPCR(+gDNA wiper)(Vazyme)试剂盒将总RNA反转录成cDNA,反应体系为:1.0μL RNA、4.0μL 4×gDNA wiper Mix、11.0μL RNase Free dH2O;反应条件为:42℃反应2min。反应完成后,再在第一步的反应液中加入4.0μL 5×HiScript III qRTSuperMix;反应条件为:37℃反应15min,85℃反应5s。将反转录获得的cDNA用SYBR qPCR SuperMix Plus(Novoprotein)试剂盒进行qRT-PCR检测。在此基础之上,以烟草NtActin(AB158612)基因为内参(Forward Primer:5'-TCCTCATGCAATTCTTCG-3'、Reverse Primer:5'-ACCTGCCCATCTGGTAAC-3'),同时设计PlMYB103基因的特异性引物(Forward Primer:5'-ATGGGTCATCACTCTTGC-3'、Reverse Primer:5'-CTTTCTCAGGGACTTCAC-3')进行qRT-PCR检测。反应体系:0.8μL cDNA、SYBR qPCR SuperMix、0.4μL ForwardPrimer、0.4μL Reverse Primer、3.4μL ddH2O。反应程序:95℃预变性3min;95℃变性5s,55℃退火30s,72℃延伸30s,共40个循环;72℃延伸10min。反应结束后采用2-△△Ct方法进行基因相对表达水平的分析。
如图6所示,qRT-PCR鉴定结果表明,芍药PlMYB103基因能够在转芍药PlMYB103基因烟草中表达。
实施例7转芍药PlMYB103基因的烟草植株表型观察
将实施例5中的烟草植株培养5个月后,比较野生型和转芍药PlMYB103基因的烟草植株表型。如图7所示,转芍药PlMYB103基因烟草植株与野生型相比,转芍药PlMYB103基因烟草植株的茎秆相对较为直立,尤其上部茎秆较野生型更加为挺直。
实施例8转芍药PlMYB103基因的烟草植株茎秆相关形态指标测定
利用游标卡尺、电子天平(BSA224S)以及茎秆强度测定仪(NK-2)对野生型和转芍药PlMYB103基因烟草植株上部茎秆相关形态指标包括直径、鲜重和强度进行测定。如图8所示,转芍药PlMYB103基因的烟草植株与野生型相比,转芍药PlMYB103基因的烟草植株上部茎秆的直径、鲜重和强度显著提高,分别提高了23.10%、31.93%和53.38%,表明转芍药PlMYB103基因烟草植株具有更加粗壮的茎秆。
实施例9转芍药PlMYB103基因的烟草植株茎秆微观结构与木质素沉积水平观测
利用石蜡切片通过组织化学染色法进行观察,结合ImageJ统计进行分析。石蜡切片样品的制备流程:取出固定于FAA固定液中的样品,用50%无水乙醇和甘油混合物(1/1;v/v)进行软化;随后进行乙醇梯度脱水,再分别用二甲苯和100%无水乙醇混合物(1/3、1/1、3/1;v/v)脱水,每次30min;之后将样品浸入二甲苯和石蜡混合物中分别在42℃、52℃和62℃中浸泡24h、3h和3h,并浸入纯石蜡中在62℃浸泡2h,重复3次;最后用石蜡包埋机(EG1160,徕卡公司,德国)进行包埋;切片使用石蜡切片机(RM2235,徕卡公司,德国)进行,厚度8μm;切片展片和烘片使用石蜡展片烘干一体机(PHY-III,常州市中威电子仪器有限公司,中国)42℃进行展片和60℃进行烘片;使用二甲苯浸泡2次,每次20min,随后用二甲苯和100%无水乙醇混合液(1/1;v/v),10min进行脱蜡;最后分别使用100%、95%、80%、50%、30%乙醇和蒸馏水梯度水化,每次10min。采用间苯三酚-盐酸染色法进行组织化学染色,切片先用5%间苯三酚染色2min,然后再用18%盐酸染色,盖上盖玻片后立即在光学显微镜(CX31RTSF,奥林巴斯公司,日本)下观察。切片利用ImageJ测量木质部细胞壁厚度进行分析。
如图9和图10所示,转芍药PlMYB103基因烟草植株与野生型相比茎秆木质部更加发达,表现为木质部的径宽和细胞壁厚度的显著增加,分别增加了113.31%和24.23%,木质素沉积水平也有明显提高。表明转芍药PlMYB103基因烟草植株具有更加发达木质化程度高的木质部。
综上所述,本发明提供了一种芍药PlMYB103基因在增强植物茎秆强度方面的应用,通过将构建的PlMYB103基因过量表达载体转化到烟草中进行表达,提高了茎秆强度,促进了茎秆的直径、木质部径宽和细胞壁厚度的增加以及木质化,创制了高茎秆强度的烟草新种质。
Claims (10)
1.一种芍药PlMYB103基因,其特征在于,所述芍药PlMYB103基因的核苷酸序列如SEQID NO.1所示。
2.权利要求1所述的芍药PlMYB103基因编码的蛋白,其特征在于,所述蛋白的氨基酸序列如SEQ ID NO.2所示。
3.表达盒、重组载体、重组菌或细胞,其特征在于,含有权利要求1所述的芍药PlMYB103基因。
4.根据权利要求3所述的重组载体,其特征在于,所述重组载体是将芍药PlMYB103基因导入载体质粒。
5.根据权利要求3所述的重组菌,其特征在于,所述重组菌是将权利要求4所述重组载体导入宿主菌,筛选鉴定后保留阳性菌株即得。
6.权利要求1所述的芍药PlMYB103基因、权利要求2所述的芍药PlMYB103蛋白、权利要求3所述的表达盒、重组载体、重组菌或细胞在增强植物茎秆强度中的应用。
7.一种获得具有高茎秆强度植物的方法,其特征在于,包括以下步骤:
1)使植物包含芍药PlMYB103基因;或
2)使植物表达芍药PlMYB103基因编码的蛋白。
8.根据权利要求7所述的方法,其特征在于,所述方法还包括转基因、杂交、回交或无性繁殖。
9.鉴定权利要求7所述的方法获得的具有高茎秆强度植物的方法,其特征在于,包括以下步骤:
(1)测定所述植物是否包含芍药PlMYB103基因;或
(2)测定所述植物是否表达芍药PlMYB103基因编码的蛋白。
10.权利要求6所述的应用、权利要求7所述的方法或权利要求8所述的鉴定方法,其特征在于,所述植物包括烟草。
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