CN116462744A - 牡丹响应光信号调控花果性状的蛋白及编码基因PsCIP7与应用 - Google Patents
牡丹响应光信号调控花果性状的蛋白及编码基因PsCIP7与应用 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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Abstract
本发明公开了牡丹响应光信号调控花果性状的蛋白及编码基因PsCIP7与应用。本发明提供了一种蛋白,是如下a)或b)的蛋白质:a)由序列表中序列1所示的氨基酸序列组成的蛋白质;b)将序列表中序列1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与响应光信号调控花果性状相关的由a)衍生的蛋白质。本发明所提供的响应光信号调控花果性状的蛋白及编码基因PsCIP7是从牡丹品种‘日暮’(P.suffruticosa‘Higurashi’)中获得的。构建表达载体并异源转化烟草进行稳定表达,发现该基因能够响应光照调控花青苷和叶绿素的积累,影响植物生长发育。
Description
技术领域
本发明涉及生物技术领域,特别地涉及牡丹响应光信号调控花果性状的蛋白及编码基因PsCIP7与应用。
背景技术
近年来,国内外围绕牡丹花色和花色素的研究,主要集中在一些野生种和各个栽培品种群花色素成分分析。至今,在牡丹不同野生种和不同居群中已经检测到30多种类黄酮成分,包括花青苷和多种形式的黄酮/黄酮醇糖苷。
前人研究发现,除少数绿色牡丹品种的花瓣中含有叶绿素外,绝大多数牡丹的花色素主要是花青苷、黄酮和黄酮醇。目前从牡丹花瓣中共检测出6种花青苷(Hosoki T,Hamada M,Kando T,et al.Comparative study of anthocyanin in tree peony flowers[J].Journal of the Japanese Society for Holticultural Science,1991,60:395-403.;Sakata Y,Toki K,Tsunematsu S,etal.Petal coloration and pigmentationoftree peony bred and selected in Daikon Island(Shimane Prefecture).JournalofThe Japanese Society for Horticultural Science,1995,64(2):351-357.;Wang LS,Hashimoto F,Shiraishi A.Chemical taxonomy of the Xibei tree peony fromChina by floral pigmentation[J].The Journal of Plant Research,2004,117:47-55.),3种黄酮和3种黄酮醇(Wang L S,Hashimoto F,Shiraishi A,et al.Phenetics intree peony species from China by flower pigment cluster analysis[J].TheJournal of Plant Research,2001a,114:213-221.)。
随着类黄酮生物合成途径及其调控机制研究的不断深入,对牡丹花色形成分子基础的认识逐渐清晰的同时,科研人员从不同角度陆续开展了牡丹花色形成分子机理的研究。目前,利用同源克隆和转录组高通量测序的方法,已分离到多个牡丹类黄酮生物合成结构基因,并对这些基因在不同品种中的表达模式进行分析,从分子水平上解析了牡丹黄色花(Shi Q Q,Zhou L,Wang Y,et al.Transcriptomic Analysis of Paeonia delavayiWild Population Flowers to Identify Differentially Expressed Genes Involvedin Purple-Red and Yellow Petal Pigmentation[J].PLoS ONE,2017,10(8).;Zhou L,Wang Y,Ren L,et al.Overexpression of Ps-CHI1,a homologue of the chalconeisomerase gene from tree peony(Paeonia suffruticosa),reduces the intensityofflowerpigmentation in transgenic tobacco[J].Plant Cell Tissue and Organ,2014,116:285-295.)、红色花(Zhao F,Lim S,Igori D,et al.Development of tobaccoringspot virus-based vectors for foreign gene expression and virus-inducedgene silencing in a variety ofplants[J].Virology,2016,492:166-178.)、紫色花(DuH,Wu J,Ji K X,Zeng Q Y,et al.Methylation mediated by an anthocyanin,O-methyltransferase,is involved in purple flower coloration in Paeonia[J].Journal of experimental botany,2015,66(21).)、紫色花斑(Zhang X,Xu Z,Yu X,etal.Identification ofTwo Novel R2R3-MYB Transcription factors,PsMYB114L andPsMYB121L,Related to Anthocyanin Biosynthesis in Paeonia suffruticosa[J].International Journal ofMolecular Sciences,2019,20(5).)以及切花瓶插期间花瓣褪色的分子机理。如周琳等人较早探究了牡丹花瓣的呈色机理,通过克隆牡丹花青苷合成途径中的关键基因,并在不同品种和不同组织中分析其表达模式的不同,推定出F3’H和DFR两个基因可能是影响红色牡丹呈色的关键基因(Zhou L,Wang Y,Ren L,etal.Overexpression ofPs-CHI1,a homologue ofthe chalcone isomerase gene fromtree peony(Paeonia suffruticosa),reduces the intensity offlower pigmentationin transgenic tobacco[J].Plant Cell Tissue and Organ,2014,116:285-295.;ZhouL,Wang Y,Peng Z.Molecular characterization and expression analysis ofchalcone synthase gene during flower development in tree peony Paeoniasuffruticosa[J].African Journal of Biotechnology,2011,10.)。
在MBW转录调控研究方面,目前仅通过分析转录组表达差异基因,筛选到一些与牡丹花青素合成相关的转录因子,但其与结构基因启动子间的调控关系以及各转录因子间的互作等深入研究较少报道。
目前对花青苷光响应合成的研究多集中在模式植物和果树上,对观赏植物的研究还比较薄弱。对牡丹花青苷光响应合成的研究多集中在表型和生物化学分析层面,从分子水平上解析光照如何诱导牡丹花青苷生物合成研究还未见报道。
发明内容
有鉴于此,本发明提供了牡丹响应光信号调控花果性状的蛋白及编码基因PsCIP7与应用。
本发明的技术方案如下:
一种蛋白,是如下a)或b)的蛋白质:
a)由序列表中序列2所示的氨基酸序列组成的蛋白质;
b)将序列表中序列2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与响应光信号调控花果性状相关的由a)衍生的蛋白质。即,从取代和/或缺失和/或添加这三种中取一种或几种进行处理。
所述蛋白的编码基因也属于本发明的保护范围。
所述编码基因为如下1)或2)或3)所示∶
1)其核苷酸序列是序列表中序列1所示DNA分子;
2)在严格条件下与1)限定的DNA分子杂交的DNA分子;
3)与1)或2)限定的DNA分子具有90%以上的同源性的DNA分子。
该编码基因含有2667个核苷酸,如序列表中序列1所示;其编码含有888个氨基酸的蛋白,如序列表中序列2所示,将该基因命名为PsCIP7,将其编码的蛋白命名为PsCIP7。
含有所述编码基因的表达盒、重组表达载体或重组菌也属于本发明的保护范围。
本发明还提供了一种制备转基因植物的方法。
本发明所提供的制备转基因植物的方法,包括如下步骤:将所述编码基因导入出发植物中,得到转基因植物;与出发植物相比,转基因植物花色和叶色变浅、果实数量减少、果实变小以及株高降低;遮光后,花色和叶色均加深。
所述编码基因是通过重组表达载体导入的,所述重组表达载体是将所述编码基因插入出发载体pSuper1300的多克隆位点得到的。
所述植物为烟草。
扩增所述编码基因全长或其任一片段的引物对也属于本发明的保护范围,所述引物对中,一条引物序列如序列表中序列3所示,另一条引物序列如序列表中序列4所示。
所述蛋白在响应光信号调控植物花色和/或果实大小中的应用也属于本发明的保护范围。
所述编码基因在响应光信号调控植物花色和/或果实大小中的应用也属于本发明的保护范围。
本发明所提供的响应光信号调控花果性状的蛋白及编码基因PsCIP7是从牡丹品种‘日暮’(P.suffruticosa‘Higurashi’)中获得的。随后构建表达载体并异源转化烟草进行稳定表达,发现该基因能够响应光照调控花青苷和叶绿素的积累,影响植物生长发育。
本发明通过转录组测序解析牡丹花青苷光响应关键调节基因,为深入理解观赏植物花青苷光响应合成的分子机制奠定理论基础,并为通过调节光照条件进行牡丹花色改良的分子育种研究提供参考。
附图说明
为了说明而非限制的目的,现在将根据本发明的优选实施例、特别是参考附图来描述本发明,其中:
图1是牡丹品种‘日暮’花朵发育的5个阶段。
图2是RNA琼脂糖凝胶电泳图。
图3是PCR产物凝胶电泳检测图像。
图4是PsCIP7的亲/疏水性、跨膜性结构、二级及三级结构预测;其中,a:亲/疏水性预测分析;b:跨膜性预测分析;c:信号肽预测分析;d:二级结构预测;e:三级结构预测分析;f:保守结构域分析。
图5是重组载体pSuper1300-PsCIP7构建图。
图6是重组载体pSuper1300-PsCIP7PCR产物的琼脂糖凝胶电泳检测结果。
图7是pSuper1300-PsCIP7转化烟草结果;其中,a:共培养阶段;b:愈伤组织阶段;c:筛选培养阶段;d:诱导生根;f:生根;g:阳性植株。
图8是PsCIP7转基因烟草的PCR检测结果。
图9是黑暗和光照条件下PsCIP7转基因烟草不同株系各器官表型结果。
图10是PsCIP7转基因烟草不同株系整株表型结果。
图11是野生型和PsCIP7转基因烟草成熟果实对比分析结果。
图12是黑暗和光照条件下野生型和PsCIP7转基因烟草颜色表型对比分析。
图13是黑暗和光照条件下野生型和PsCIP7转基因烟草色素含量对比分析结果。
具体实施方式
植物材料:
以牡丹品种‘日暮’(P.suffruticosa‘Higurashi’)为实验材料,花色为红色,栽植于青岛农业大学园林与林学院实验基地,购自山东省菏泽市曹州百花园。将花朵的发育阶段分为5个时期(图1),分别命名为S1(花苞紧实,花瓣未着色)、S2(花苞膨大,可以看到已着色的花瓣)、S3(花朵即将开放)、S4(花朵初开,花心可见)和S5(花朵盛开)。
野生型(WT)烟草Nc89(Nicotiana tabacum cv.Nc89)(记载过野生型(WT)烟草Nc89(Nicotiana tabacum cv.Nc89)的非专利文献是:Physiol Mol Biol Plants,2021,27(2):237-249)种子来自中国科学院植物研究所牡丹组舒庆艳老师馈赠。WT烟草种子均匀接种于已倒有MS培养基的组培瓶中,置于28℃,相对湿度为60%,光照强度为2500lx,光周期为16/8h的环境下培养,待植株长出4-5片真叶时移栽至青岛农业大学园林与林学院智能温室,置于相对湿度80%,光强250μmol m-2s-1,25℃光照16h/18℃黑暗8h的光周期下培养。
菌株与载体:
本发明所用到的Transl-T1大肠杆菌感受态、Fast-T1大肠杆菌化学感受态细胞(Fast-T1 competent cell)、pMD18-T载体(pMD18-T Vector)购自南京诺唯赞生物科技有限公司;GV3101农杆菌感受态细胞(GV3101Chemically Competent Cell)购自上海唯地生物技术有限公司。
酶及化学试剂:
植物总RNA提取试剂盒(RNA prep Pure plant kit)、植物基因组DNA提取试剂盒、实时荧光定量PCR试剂盒(ChamQ Universal SYBR qPCR Master Mix)、实时荧光定量反转录试剂盒(RT SuperMix for qPCR(+gDNA wiper))、pTaq Plus Master MixⅡ(Dye Plus)、Ultra GelRed、高保真酶2酶/>Max Master Mix(Dye Plus)、同源重组酶/>Ultra One Step Cloning Kit、T4DNA连接酶、DNA凝胶回收试剂盒、高纯度质粒DNA小量试剂盒均购自南京诺唯赞生物科技有限公司;限制性内切酶、MatchmakerInsert CheckPCR MixⅠ、Genome Walking Kit购自宝日医生物技术(北京)有限公司;高纯度低电渗琼脂糖、DL 2000DNA Marker购自青岛擎科梓熙生物技术有限公司;抗生素Kan、Rif、Cef、Hyg和激素NAA、6-BA购自北京索莱宝科技有限公司。
引物:
引物合成及测序由青岛擎科梓熙生物技术有限公司完成。
实施例1、牡丹响应光信号调控花果性状的蛋白及编码基因PsCIP7与应用
一、PsCIP7基因的编码区克隆
采用诺唯赞多糖多酚植物总RNA试剂盒提取牡丹品种‘日暮’(P.suffruticosa‘Higurashi’)S3期花瓣总RNA。电泳检测显示具有28S和18S两条带(图2),紫外分光光度计显示OD260/280均在1.8-2.0之间,表明RNA质量符合后续试验要求,保存于-80℃或立即反转录获得cDNA模板后保存于-20℃,用于后续试验。
由转录组数据Unigene(c82586_c0)得到4174bp的全长序列,并于ORF Finder在线软件找查询其开放阅读框(open reading frame,ORF)2667bp。在其两端设计引物PsCIP7-F/R(表1),以牡丹品种‘日暮’(P.suffruticosa‘Higurashi’)的cDNA为模板进行PCR扩增,对PCR产物进行琼脂糖凝胶电泳检测,通过凝胶成像仪观察条带,结果显示在2667bp附近获得了单一特异性条带(图3)。
目的基因的PCR扩增。目的基因的PCR扩增体系(50μL):0.5μLTaKaRa LA Taq(5U/μL),5μL 10/TaqTaq BufferⅡ(Mg2+Plus)(20mM),8μL dNTP Mixture(2.5mM),2μL cDNA,2μL上游引物(10μM),2μL下游引物(10μM)和30.5μL灭菌水。扩增程序为:95℃预变性,5min;35个循环(94℃变性30s,55℃退火30s,72℃延伸1min);72℃,5min。
回收产物连接转化。将PCR产物于1%的琼脂糖凝胶中进行电泳检测,选择长度大小正确的条带进行切胶,参照DNA凝胶回收试剂盒(诺唯赞)说明书回收目的DNA。将回收的目的条带克隆到pMD18-T载体上测序。将测序结果与转录组数据进行多序列比对,结果显示,获得的基因序列与转录组序列的重叠区域一致性达到100%。
表1克隆基因所用引物序列
获得的基因序列为2667bp,如序列表中序列1所示,将该基因命名为PsCIP7,其编码含有888个氨基酸的蛋白,如序列表中序列2所示,将该蛋白命名为PsCIP7。
二、PsCIP7蛋白的生物信息学分析
使用ProtParam在线分析工具预测蛋白质理化性质,PsCIP7基因编码蛋白的相对分子质量99513.76,分子式为C4295H6740N1238O1435S26。原子总数为13734个,理论等电点PI值为5.49。不稳定指数为44.94,由此推测该蛋白为不稳定蛋白。
利用TMHMM在线工具预测蛋白跨膜结构域,依据膜内部分与膜外部分有无交汇点来判断是否属于跨膜蛋白。结果显示PsCIP7没有蛋白跨膜结构域,不属于膜蛋白(图4中a)。是否存在信号肽决定了新生肽链能否被分泌到细胞外,用Signal P预测信号肽,结果显示,PsCIP7没有信号肽,属于非分泌性蛋白(图4中b)。这说明其蛋白质不能跨膜运输,在合成处即开始行使功能,可能是一种在细胞质或者细胞器里的蛋白质。利用NCBI保守结构域分析显示,该序列无典型的保守结构域(图4中c)。利用ProtScale对蛋白质的疏水性进行分析,PsCIP7的亲水性/疏水性的最大值为1.767,最小值为-3.367,在整个肽链中亲水性氨基酸残基比疏水性氨基酸残基多。同时Y轴上正值越大表明疏水性越强,负值越大表明亲水性越强,说明该蛋白具有非常明显的亲水和疏水区域交替排列,PsCIP7总体为偏亲水性蛋白(图4中d)。PsCIP7蛋白质二三级结构预测用NetSurfP-3.0和SWISS-MODEL进行预测。PsCIP7的二级结构预测表明其氨基酸组成中α-螺旋(Hh)占25.68%,β-折叠(Tt)占1.35%,延伸链(Ee)占9.12%,无规卷曲占(Cc)占63.85%,属于不规则结构(图4中e)。对PoMYB1蛋白进行三级结构预测,其模型相似度与3hqi.1.B Speckle-type POZ蛋白达到16.67%(图4中f)。
三、基因功能的验证
1、PsCIP7基因过表达载体的鉴定
将PsCIP7基因ORF区片段、N端、C端分别与植物表达载体pSuper1300(记载过植物表达载体pSuper1300的非专利文献是:Plant Science,2022,317:111189,由本实验室保存)连接,构建重组载体pSuper1300-PsCIP7(图5)。
具体方法如下:
植物RNA提取和定量反转录。以1μg S2时期花瓣总RNA为模板,参照IIIRT SuperMix for qPCR(+gDNA wiper)的说明书进行反转录,合成cDNA置于-20℃保存备用。
目的基因的PCR扩增。目的基因的PCR扩增体系(50μ0):0.5μLTaKaRa LA Taq(5U/μL),5μL 10/TaqTaq BufferⅡ(Mg2+Plus)(20mM),8μL dNTP Mixture(2.5mM),2μLcDNA,2μL上游引物(10μ0),2μL下游引物(10μM)和30.5μL灭菌水。扩增程序为:95℃预变性,5min;35个循环(94℃变性30s,55℃退火30s,72℃延伸1min);72℃,5min。
回收产物连接转化。将PCR产物于1%的琼脂糖凝胶中进行电泳检测,选择长度大小正确的条带进行切胶,参照DNA凝胶回收试剂盒(诺唯赞)说明书回收目的DNA。将4.0μL回收产物与1.0μL pEASY-T1 Cloning Vector轻柔吸打混匀,室温反应5min。将连接好的5μL重组载体与半融化状态的50μL Transl-T1大肠杆菌感受态细胞轻弹混匀,冰浴30min;42℃热激45s;立即置于冰上2min。加入500μl LB液体培养基混匀,200rpm,37℃孵育1h。5000rpm,6min离心后,吸取100μL复苏液均匀涂布到含有50Mg/L卡那霉素(Kan)的LB固体培养基上,将平板倒置于37℃培养箱中培养12-14h。
阳性重组质粒鉴定及提取。挑取单克隆菌落至500μL含有50mg/LKan的LB液体培养基中,200rpm,37℃孵育5h后进行菌液PCR鉴定,体系(15.0μ5)为:7.5μL 25Taq PCRMasterMix,0.6μL M13F(10μ0),0.6μ.M13R(10μ0),5.7μL ddH2O,0.6μL菌液。扩增程序为:94℃预变性,3min;30个循环(94℃,30s;55℃,30s;72℃,1min);72℃,10min。经电泳检测后选择条带大小正确的菌液进行测序(擎科)。采用高纯度质粒DNA小量试剂盒(诺唯赞)提取质粒,-20℃保存备用。
载体双酶切和同源重组。使用限制性内切酶HindⅢ和Kpn I双酶切Super1300载体,反应体系如表2所示:
表2限制性内切酶HindⅢ和Kpn I双酶切Super1300载体反应体系
37℃恒温器酶切5h,得到Super1300的线性化载体。
将目的片段质粒再次进行目的片段PCR扩增(同上)。测量以上浓度后,根据公式:最适克隆载体使用量=[0.02×克隆载体碱基对数]ng(0.03pmol)最适插入片段使用量=[0.04×插入片段碱基对数]ng(0.06pmol)计算重组反应所需DNA量。为了确保加样的准确性,在配制重组反应体系前可将线性化载体与插入片段适当稀释,各组分加样量不低于1μL。于冰上配制以下反应体系(表3):
表3片段重组反应的反应体系
片段重组反应,50℃,10min;降至4℃或立即置于冰上冷却。
重组反应转化。以重组反应的连接产物为模板,使用Fast-T1化学感受态细胞,参照C505-02/03试剂盒说明书进行高效转化:
a.将Fast:T1感受态细胞从-80℃拿出,迅速置于冰上融化,加入目的DNA(质粒或连接产物),轻弹管壁混匀(避免用枪吸打),冰上静置30min;
b.42℃水浴热激30sec后,迅速置于冰上静置2min,勿摇动离心管;
c.向离心管中加入900μL LB液体培养基(不含抗生素),混匀后置于37℃,200rpm摇床中复苏1h;
d.2,500xg,离心3min,弃掉900μL上清,用剩余培养基将菌体吹打混匀后,均匀涂布在含Kan抗生素的LB固体培养基平板上。
e.将平板正置于37℃培养箱10min,待菌液被完全吸收后,倒置平板,过夜培养。
阳性重组质粒鉴定及提取。将鉴定正确的重组产物菌液样品扩大振荡培养过夜并按照7:3的比例加入50%甘油,混匀后于-80℃保存备用;重组产物质粒于-20℃保存备用。
引物合成及测序由青岛擎科梓熙生物技术有限公司完成,引物序列如下表4所示:
表4PsCIP7基因克隆所用引物序列
以pSuper1300-PsCIP7的重组质粒为模板,进行PCR验证(图6),PCR产物的琼脂糖凝胶电泳检测结果表明在约2667bp处有PsCIP7的正确条带。对阳性质粒测序结果比对分析表明与原序列一致性达到99%以上,成功构建过表达重组载体pSuper1300-PsCIP7。
2、PsCIP7过表达转基因烟草的筛选
转化农杆菌获得阳性克隆和验证。参照GV3101 Chemically Competent Cell说明书,用冻融法转化农杆菌GV3101感受态细胞,获得阳性单克隆农杆菌。转化完成后加入400μL含有50mg/L Spec的LB液体培养基,200rpm,28℃孵育2-3h。涂布于含有100mg/L Spec和50mg/L利福平(Rif)的LB固体培养基上,置于28℃培养箱中倒置培养2-3d。挑取单克隆菌落在含有100mg/L Spec和50mg/L Rif的LB液体培养基中28℃、200rpm孵育8h,进行PCR检测阳性重组子。将鉴定正确的菌液样品振荡培养过夜并加入50%甘油,混匀后于-80℃保存备用。
烟草无菌苗的培养。取适量野生型种子(野生型(WT)烟草Nc89(Nicotianatabacum cv.Nc89)种子来自中国科学院植物研究所牡丹组舒庆艳老师馈赠)于2mL离心管中,加1mL 75%乙醇,上下颠倒混匀浸泡2min,弃乙醇溶液;加1mL 2%次氯酸钠,上下颠倒混匀浸泡5min,弃次氯酸钠溶液;灭菌水漂洗种子3-5次后置于无菌滤纸上,自然吸干水分。烟草种子均匀接种于已倒有MS培养基的组培瓶中,置于28℃,相对湿度为60%,光照强度为2500lx,光周期为16/8h的环境下培养,待植株长出4-5片真叶时用于侵染。
根癌农杆菌介导的叶盘法转化烟草,具体步骤如下:
(1)活化保存的阳性农杆菌菌液,按1:100的比例取活化菌液加入含100mg/L Kan和50mg/L Rif的LB液体培养基中,28℃,180rpm振荡培养OD600=0.6;
(2)室温5000rpm离心10min,收集菌体沉淀,用MS0液体培养基(MS培养基+蔗糖30g/L,PH为5.8)重悬菌体,以备浸染叶片;
(3)取生长良好的无菌烟草苗的叶片(第2-4片叶),切掉叶尖、叶缘及主叶脉,将剩余部分切成1cm2左右的小块;
(4)将切好的烟草叶片小块置入MS0悬浮液中,浸染10-15min,叶背面向下,轻轻摇晃利于菌液完全接触叶缘伤口;
(5)取出侵染好的叶片置于无菌滤纸上吸干残留于表面菌液;
(6)将叶片接种至固体共培养基MS0(MS培养基+蔗糖20g/L+琼脂7.5g/L,PH为5.8)上,置于28℃避光共培养3d;
(7)共培养结束后,用含有500mg/L头孢霉素(Cef)的无菌水清洗叶片表面残留的农杆菌,用无菌滤纸吸干水分后,将叶片转移至含有Kan的分化诱导及筛选培养基MS1(MS培养基+6-BA(1.0mg/L)+NAA(0.2mg/L)+蔗糖20g/L+琼脂7.5g/L+Kan(100mg/L)+Cef(200mg/L),PH为5.8)上,28℃、相对湿度为50%-60%、光照强度为2500lx、光周期为16/8h静置培养,每十天更换一次MS1培养基;
(8)分化出再生芽后,待分化苗长到2-3cm高时将其从愈伤上分离,转入MS2生根培养基(1/2MS培养基+蔗糖20g/L+琼脂7.5g/L+Kan(100mg/L)+Cef(200mg/L),PH为5.8)中诱导生根,经1-2周后阳性转基因烟草可生根。
(9)待根系发达后,取出烟草组培苗,用无菌水洗净其根部所带琼脂。将其定植于草炭土:蛭石=3:1(V/V)的栽培基质中,置于上述同样环境下覆膜培养1周左右,之后揭膜正常培养,直至种子成熟。收集种子储存于4℃冰箱,低温春化备用。
营养生长和生殖生长指标测定。烟草筛选到T2代即认为转基因株系纯和,可以用于生物学功能验证。对光照和黑暗条件下野生型和转基因株系烟草花瓣进行荧光定量PCR,验证目的基因和花青苷生物合成途径中相关结构基因的相对表达量。选择目的基因表达量高的三个转基因烟草株系的T2代植株用以表型验证。比较苗龄为12周的野生型和转基因烟草植株光照和黑暗条件下的营养和生殖生长状况,对二者叶色、花色、萼片颜色、株高、果实长度、宽度、重量和整株果实数量等数据进行测量,并拍照记录器官表型。
颜色表型和花瓣花青素含量的测定方法如下:
使用便携式色差仪(Hunter Associates Laboratory Inc.,USA)测定全部样本的L*(明度)、a*(红度)、b*(黄度)值,并依据公式C*=(a*2+b*2)1/2(McGuire,1992)转换为C*(彩度)值。每组样本进行五次随机测量,取平均值和标准差作为分析数据。
采用有机溶剂萃取法提取总黄酮和总花青素,紫外分光光度计UH 5300(HITACHI,Tokyo,Japan)测定。总花青素的提取:称取样本0.2g在液氮中研磨成粉末,用10mL 1%的盐酸-甲醇溶液(1mol/L盐酸:1mol/L甲醇=1:99,v/v)进行低温浸提至花瓣完全变为白色。总花青素含量(TA)的测定:用1%的盐酸-甲醇溶液作为参比液,在分光光度计上测得提取液在530、620、650nm下的光密度。通过公式计算出提取液花青素含量:Aλ=(A530-A620)-0.1(A650-A620),TA=(Aλ/ξ)×(v/m)×1000000(Ma and Cheng,1984)。总黄酮的提取:用1mol/l甲醇溶液代替1%的盐酸-甲醇溶液提取总黄酮。采用AlCl3显色法测定总黄酮含量(TF),用芦丁作标准曲线,紫外分光光度计测定510nm处的吸光度值。通过公式计算出提取液总黄酮含量:TF=(C×V3×V1)/(V2×M×10)。
使用便携式叶绿素仪测量叶片SPAD值来比较叶片相对叶绿素含量。果实成熟后分别进行采收,并记录成熟果实的长度、宽度和重量。
利用根癌农杆菌介导的叶盘法将PsCIP7基因转入野生型烟草中,烟草叶片经过共培养、筛选培养、芽诱导、生根、移栽培养后获得Hyg抗性再生苗(图7,其中,a:共培养阶段;b:愈伤组织阶段;c:筛选培养阶段;d:诱导生根;f:生根;g:阳性植株)。使用植物基因组DNA提取试剂盒(诺唯赞,南京)提取获得的转基因烟草的DNA,进行PCR检测(同上),以野生型烟草为阴性对照,以pSuper1300-PsCIP7重组质粒为阳性对照,转PsCIP7基因烟草均可扩增得到一条大小与预期一致的特异条带(图8),而对照组未扩增出任何片段,初步表明外源PsCIP7基因已经在转基因烟草株系中正常表达。
3、PsCIP7过表达转基因烟草表型验证
选择目的基因表达量高的三个转基因烟草株系的T2代植株用以表型验证,分别命名为OE-1、OE-2和OE-3。比较苗龄为12周的野生型和转基因烟草植株光照和黑暗条件下的营养和生殖生长状况,对二者叶色、花色、萼片颜色、株高、果实长度、宽度、重量和整株果实数量等数据进行测量,并拍照记录器官表型。比较野生型和T1代PsCIP7转基因烟草植株的营养和生殖生长状况,发现PsCIP7转基因植株与野生型植株已经存在显著差异。过表达转基因烟草幼苗移栽至盆土14d左右叶片开始出现黄化,生长速度较野生型明显缓慢,植株明显偏小(图9;图10中a和c);在开花、结实之后,其花瓣对比野生型的红色呈现出更浅的粉红色,萼片颜色更浅,果实更小(图9);每株果实数减少(图10中b),成熟果实的大小和重量均显著减小(图11);与野生型对照呈鲜明的对比。鉴于PsCIP7过表达能使不同组织器官褪色或缩小,初步推测其是一个能够负调控植株花青苷和叶绿素积累,影响植株生长发育的转录因子。
为了探究光照对PsCIP7转基因烟草的影响,在现蕾阶段,对野生型和PsCIP7转基因植株用锡箔纸进行100%遮光的黑暗处理。10d后发现,野生型烟草的叶片、萼片和花瓣在黑暗处理后出现明显褪色,花瓣红度呈极显著下降和叶片黄度显著上升(图9;图12),对应的叶绿素和总花青素含量均呈极显著下降(图13)。而黑暗处理PsCIP7转基因植株,与野生型对照呈完全相反的结果。PsCIP7转基因株系的叶片黄度呈极显著下降(图12中b),代表叶绿素含量的SPAD值极显著上升(图13中a);花瓣的红度呈极显著上升(图12中a),总花青素含量显著增加(图13中b);萼片颜色变化不显著,但仍有不同程度的加深(图9)。PsCIP7在黑暗条件下能使花瓣和叶片颜色加深,能够响应光照调控花青苷和叶绿素的积累,影响植生长发育。
上述具体实施方式,并不构成对本发明保护范围的限制。本领域技术人员应该明白的是,取决于设计要求和其他因素,可以发生各种各样的修改、组合、子组合和替代。任何在本发明的精神和原则之内所作的修改、等同替换和改进等,均应包含在本发明保护范围之内。
Claims (10)
1.一种蛋白,是如下a)或b)的蛋白质:
a)由序列表中序列2所示的氨基酸序列组成的蛋白质;
b)将序列表中序列2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与响应光信号调控花果性状相关的由a)衍生的蛋白质。
2.权利要求1所述蛋白的编码基因。
3.根据权利要求2所述的编码基因,其特征在于∶所述编码基因为如下1)或2)或3)所示∶
1)其核苷酸序列是序列表中序列1所示DNA分子;
2)在严格条件下与1)限定的DNA分子杂交的DNA分子;
3)与1)或2)限定的DNA分子具有90%以上的同源性的DNA分子。
4.含有权利要求2或3所述编码基因的表达盒、重组表达载体或重组菌。
5.一种制备转基因植物的方法,包括如下步骤:将权利要求2或3所述的编码基因导入出发植物中,得到转基因植物;与出发植物相比,转基因植物花色和叶色变浅、果实数量减少、果实变小以及株高降低;遮光后,花色和叶色均加深。
6.根据权利要求5所述的方法,其特征在于∶
所述编码基因是通过重组表达载体导入的,所述重组表达载体是将所述编码基因插入出发载体pSuper1300的多克隆位点得到的。
7.根据权利要求5所述的方法,其特征在于:所述植物为烟草。
8.扩增权利要求2或3所述编码基因全长或其任一片段的引物对,所述引物对中,一条引物序列如序列表中序列3所示,另一条引物序列如序列表中序列4所示。
9.权利要求1所述的蛋白在响应光信号调控植物花色和/或果实大小中的应用。
10.权利要求2或3所述编码基因在响应光信号调控植物花色和/或果实大小中的应用。
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