CN116200424B - 叶斑艺兰花CcMYB24基因的应用 - Google Patents
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Abstract
本发明属于植物基因工程技术领域,具体公开叶斑艺兰花CcMYB24基因的应用,所述叶斑艺兰花CcMYB24基因的CDS序列如SEQ ID NO:1所示,所述性状为:转基因植株的表现为更健壮;转基因植株的莲座叶数增加;转基因植株的侧枝数增加;转基因植株的叶绿素a增加;转基因植株的叶绿素b增加,本发明是利用简并引物设计扩增全长的方法从叶斑艺兰花中克隆出CcMYB24基因,在拟南芥中进行过量表达,并通过农杆菌介导花序浸润法遗传转化拟南芥,实验结果表明在过表达转基因拟南芥中CcMYB244基因能够正常表达,转入过表达CcMYB24基因的生长情况比对照植株表现为更健壮,过量表达CcMYB24基因能够很大程度的提高增加转基因植株的莲座叶数、侧枝数、叶绿素a、叶绿素b和总叶绿素含量。
Description
技术领域
本发明属于植物基因工程技术领域,具体涉及叶斑艺兰花CcMYB24基因的应用。
背景技术
兰科(Orchidaceae)植物是单子叶植物第一大科,有5个亚科801属28237种,其中兰属(Cymbidium)、兜兰属(Paphiopedilum)、石斛属(Dendrobium)等是世界级的花卉名品。兰科兰属小花型地生种,俗称国兰,在我国有悠久的栽培历史,其花形、叶形、株型优美,花色变化丰富,具有重要的观赏价值。兰花经过长期的选育栽培,出现了很多的变异品种,根据观赏部位的不同,分为花艺、叶艺两大类型,其中的叶斑艺兰花起源于中国,在近些年中逐渐被人们认识。“叶艺”在园艺学上称为叶部彩斑(variegation),是观赏植物叶部变异的重要组成部分。其中,兰花叶斑艺品种观赏价值很高,是“精品兰花”的重要特征。叶艺兰(线艺兰)指兰科兰属植物(Cymbidium)的叶片上镶嵌着金黄色、银白色、浓绿色、朱红色、或墨黑的嘴、边、点、线、斑的中国兰统称。明朝咏兰诗中就有看叶胜看花的佳句,人们在追求兰花花艺的同时也在追求兰花植株外形美,叶艺兰凭借其古朴、厚重、矮小的株型以及多样奇异的叶片形态,自古受到人们追捧,其独特的叶斑性状受人们重视并被选为培育的重点。目前,叶艺兰多来自野生兰科植物的天然杂交后代,或在长期培育过程中的自然变异植株,因此,叶艺兰育种存在数量极为稀少,选育时间较长等瓶颈问题,并且艺象也通常不稳定,导致了叶艺兰定向培育极为困难。近年来随着叶艺兰新品种的培育以及公众对叶艺兰深入的鉴赏,出现了不同颜色和形状变异的新类型。
针对叶斑艺兰花中CcMYB24基因的功能探究以及应用,目前尚未见相关报道。
发明内容
本发明的主要目的是提供一个可显著影响拟南芥的基因片段叶斑艺兰花CcMYB24基因CDS序列如SEQ ID NO:1所示,该基因在拟南芥中正常表达,且促进拟南芥莲座叶数、叶侧枝数、植株叶绿素a、叶绿素b和总叶绿素合成。
为实现以上目的,本发明提供以下技术方案:
本发明提供叶斑艺兰花CcMYB24基因在植物性状改良中的应用,所述叶斑艺兰花基因的名称为CcMYB24 CDS序列如SEQ ID NO:1所示,所述性状为以下(1)、(2)、(3)、(4)或(5)性状中的至少一种:
(1)转基因植株的表现为更健壮;
(2)转基因植株的莲座叶数增加;
(3)转基因植株的侧枝数增加;
(4)转基因植株的叶绿素a增加;
(5)转基因植株的叶绿素b增加。
进一步的,所述转基因植株的获得方法为:
(1)叶斑艺兰花CcMYB24基因的扩增;
(2)构建重组载体;
(3)农杆菌介导的遗传转化;
(4)转基因植物培养、检测。
优选的,步骤(1)中,叶斑艺兰花CcMYB24基因的扩增所用的引物核苷酸序列如SEQID NO:2、SEQ ID NO:3所示。
优选的,步骤(2)中,用于构建所述重组载体的起始载体为pCAMBIA1301,含有35S启动子,其后紧接CcMYB24基因。
本发明是利用简并引物设计扩增全长的方法从叶斑艺兰花中克隆出CcMYB24基因,在拟南芥中进行过量表达,并通过农杆菌介导花序浸润法遗传转化拟南芥,实验结果表明在过表达转基因拟南芥中CcMYB244基因能够正常表达,转入过表达CcMYB24基因的生长情况比对照植株表现为更健壮,过量表达CcMYB24基因能够很大程度的提高增加转基因植株的莲座叶数、侧枝数、叶绿素a、叶绿素b和总叶绿素含量。
附图说明:
图1: CcMYB24基因扩增前后凝胶电泳显示图(M:maker2000bp; 1:MYB24基因全长);
图2:过表达载体PCR验证凝胶电泳显示图;
图3:过表达转化铁皮石斛原球茎显示图(A:农杆菌介导花序浸润,B:T0代植株,C:T0种子收集及消毒,D:T0种子播种于筛选培养,E:筛选培养基中的小苗,F:移栽到基质土中的小苗);
图4: PCR检测转基因阳性植株情况显示图(M:DL2000,PC:阳性对照,NC:阴性对照);
图5 转基因和野生型植株形态显示图(A、B:转基因植株,C、D:野生型植株);
图6:野生型和转基因拟南芥植株莲座叶侧枝数与莲座叶数比较对比图;
图7:野生型和转基因拟南芥植株光合色素含量对比图;
图8:CcMYB24在野生型和转基因拟南芥植株中茎、叶、花的表达水平效果图;
图9重组载体pCAMBIA1300-35S -CcMYB24结构显示图。
具体实施方式
为了能够更加清楚地理解本发明的技术实质和有益效果,申请人在下面以实施例的方式作详细说明,但是对实施例的描述均不是对本发明方案的限制,任何依据本发明构思所做出的仅仅为形式上的而非实质性的等效变换都应视为本发明的技术方案范畴。
为了避免过多不必要的细节,在以下实施例中对属于公知技术将不进行详细描述。除有定义外,以下实施例中所用的技术和科学术语具有与本发明所属领域技术人员普遍理解的相同含义。
以下实施例中所用的试验试剂耗材,如无特殊说明,均为常规生化试剂;所述实验方法,如无特殊说明,均为常规方法。
实施例1
叶斑艺兰花CcMYB24基因, ORF Finder分析表明CcMYB24基因具有序列较长且完整的开放阅读框,共1390bp,核苷酸序列如SEQ ID NO:4所示,起始密码子为ATG,终止密码子为TAG,CDS序列906 bp,如SEQ ID NO:1所示,共编码301个氨基酸。经ExPASy分析蛋白质的总平均亲水系数为-0.483,表明该蛋白质是亲水性蛋白。
一、重组载体pCAMBIA1300-35S -CcMYB24(图9)由下述方法构建而成:
(1)叶斑艺兰花CcMYB24基因的扩增
设计引物,并以叶斑艺兰花突变体基因组DNA作为扩增模板,获得叶斑艺兰花CcMYB24基因全长序列,图1为CcMYB24基因全长序列电泳图。
① PCR引物
设计引物:
CcMYB24-BamHI F:GCAGGTCGACTCTAGAGGATCCATGGGGAGGGCTCCTTGC
CcMYB24-SacI R:GGAATTCGAGCTGGTCAGAGCTCTCAAAAAGTGCTCATACTCTTCCA
②用上述反转录产物进行PCR,体系如下:10xPCR buffer 5.0 ul、dNTP mixture(10 mM) 1.0ul、Primer F (10μM) 1.0 ul、Primer R (10μM) 1.0ul、DNA 1.0ul、KOD (1U/μL) 1.0 ul和ddH2O 40.0 ul,总容量为50ul;
③ PCR反应程序如下:94℃预变性5min;98℃变性30 sec,56℃退火30 sec,68℃延伸2min,32个循环;68℃延伸5min,4℃终止反应。
(2)CcMYB24基因植物过表达载体pCAMBIA1300-35S-CcMYB24构建
用SacI与BamHI双酶切pCAMBIA1300-35S载体并回收纯化,37℃反应30 min。酶切后载体与CcMYB24的PCR扩增产物混合,使用无缝克隆方法构建pCAMBIA1300-35S-CcMYB24载体,37℃反应30 min。
① 载体酶切体系如下:pCAMBIA1300-35S 16 ul、10×FastDigest buffer 2 ul、FastDigest BamHI 1 ul、FastDigest SalI 1ul,总容量为20 ul;
② 用上述PCR产物和酶切载体进行无缝连接,反应体系如下:PCR产物 4 ul、酶切载体 4 ul、Assembly Enzyme 1 ul、10×Assembly Buffer 1 ul,总容量为10 ul;
将植物过表达载体转化大肠杆菌,通过酶切鉴定筛选出重组子,构建起了植物表达的重组载体pCAMBIA1300- 35S - CcMYB24,之后获得重组质粒。目的基因片段清晰,大小与预期结果一致(1390bp),无非特异性扩增带,过表达载体成功构建;图2为过表达载体图。
三、农杆菌的遗传转化和转化子的检测
制备农杆菌的感受态细胞,用电脉冲法将上述构建好的植物表达载体pCAMBIA1300-35S-CcMYB24转入农杆菌(EHA105)中,在加有潮霉素的平板上筛选转化子菌落。以农杆菌菌落的裂解液作为PCR反应的模板,用CcMYB24基因的特异引物CcMYB24-BamHIF和CcMYB24-SalI R做PCR检测,经菌落PCR确认的转化子菌落用于转化植物。
二、用含有CcMYB24基因的植物过表达的农杆菌转化植物
采用农杆菌介导拟南芥花序浸润法
挑取携带有质粒pCAMBIA1300-35S -CcMYB24的农杆菌单菌落接种于液体培养基中培养,离心收集菌体,选择主花序开始结荚,次生花序开始开花的拟南芥侵染,侵染前一天拟南芥浇透水并剪去植株上已结实的所有果荚,向重悬液中加入200-600 μL/L的Silwet-77搅匀,将拟南芥种植盆倒置于装有重悬液的倒三角玻璃杯中使花序完全浸泡于重悬液,浸泡2-6 min,注意避免重悬液滴入拟南芥的莲座。侵染后套上黑色塑料袋气候箱中黑暗湿润培养24 h,后去除黑色塑料袋调整至正常培养条件培养20 d后减少浇水,按前述方法收集种子并保存。过表达遗传转化流程见图3,获得了阳性植株。
五、CcMYB24基因在转基因植物中的插入情况及表达量的检测
为了确认用抗生素(卡那霉素)筛选获得的转基因植物确实含有CcMYB24基因,用利用2XT5 Direct PCR Kit (Plant)试剂盒对筛选到的转基因植物做进一步的鉴定。首先提取转基因植物的基因组DNA,然后以植物基因组DNA为模板,用CcMYB24特异性引物做PCR检测,鉴定是否为阳性植株,鉴定结果如图4所示。
此外,CcMYB24基因在T1代转基因拟南芥中茎、叶、花的表达分别显著高于对照植株(未转基因的野生型),并且在转基因拟南芥中茎、叶、花表达水平有差异,其中叶片表达量最高,其次是花和茎段,检测结果如图5所示。
六、转基因植株形态和生理指标变化的实验
将转基因以及对照野生型在光照培养箱中培养成苗,对过表达CcMYB24基因的植物拟南芥进行各项生理指标的测定,得到了图6、图7、图8的测定结果,实验结果表明在转基因过表达拟南芥中CcMYB24基因能够正常表达,转入拟南芥基因的生长情况相比对照植株(未转基因的野生型),转基因植株表现为莲座叶数,莲座叶侧枝数、叶绿素a、叶绿素b和总叶绿素含量增加;而类胡萝卜素、总黄酮、可溶性糖和可溶性蛋白含量降低,以上结果表明叶斑艺兰花的CcMYB24转录因子与调控光合色素合成以及叶片、叶芽的生长发育有关。
Claims (5)
1.叶斑艺兰花CcMYB24基因在植物性状改良中的应用,其特征在于,所述叶斑艺兰花CcMYB24基因的CDS序列如SEQ ID NO:1所示,所述性状为以下(1)、(2)、(3)或(4)性状中的至少一种:
(1)转基因植物的莲座叶数增加;
(2)转基因植物的侧枝数增加;
(3)转基因植物的叶绿素a增加;
(4)转基因植物的叶绿素b增加
所述植物为拟南芥。
2.根据权利要求1所述的叶斑艺兰花CcMYB24基因在植物性状改良中的应用,其特征在于,所述转基因植物的获得方法为:
(1)叶斑艺兰花CcMYB24基因的扩增;
(2)构建重组载体:所述重组载体是通过引物扩增叶斑艺兰花CcMYB24 基因后酶切连接到植物表达载体中构建获得的;
(3)农杆菌介导的遗传转化:将构建好的重组载体转入农杆菌后转化拟南芥最终筛选获得过表达CcMYB24 基因的拟南芥转基因植物;
(4)转基因植物培养、检测。
3.根据权利要求2所述的叶斑艺兰花CcMYB24基因在植物性状改良中的应用,其特征在于,步骤(2)中,叶斑艺兰花CcMYB24基因的扩增所用的引物核苷酸序列如SEQ ID NO:2、SEQID NO:3所示。
4.根据权利要求2所述的叶斑艺兰花CcMYB24基因在植物性状改良中的应用,其特征在于,步骤(2)中,用于构建所述重组载体的起始载体为pCAMBIA1301,含有35S启动子,其后紧接CcMYB24基因。
5.根据权利要求2所述的叶斑艺兰花CcMYB24基因在植物性状改良中的应用,其特征在于,步骤(3)中,所述农杆菌为EHA105。
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