CN116590308B - 马铃薯耐旱性相关热激蛋白基因hsp101及其应用 - Google Patents
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Abstract
本发明提供了一种马铃薯耐旱性相关热激蛋白基因HSP101,其核苷酸序列如SEQ IDNo.1所示,编码蛋白的氨基酸序列如SEQ ID No.2所示。该基因位于细胞质中,干涉该基因会明显降低马铃薯的耐旱性。这为解析马铃薯耐旱机制和耐旱改良育种提供了遗传材料和理论依据。
Description
技术领域
本发明涉及植物基因工程技术和马铃薯育种技术领域,具体涉及一种马铃薯耐旱性相关热激蛋白基因HSP101及其应用。
背景技术
马铃薯属于浅根系植物,其对干旱非常敏感。干旱会强烈抑制其生长发育过程,进而导致马铃薯块茎产量降低和品质下降。为应对干旱胁迫,马铃薯进化出一系列策略来抵御干旱。在生化层面,马铃薯植株在应对干旱胁迫时,可溶性物质积累增加使植物根细胞能够从土壤中吸收更多的水。在生理层面,马铃薯通过降低气孔传导率、减少叶子数目和叶片面积来最大限度地减少蒸腾失水,从而提高植株的水分利用率。在分子层面,马铃薯干旱相关基因在受到干旱刺激时快速反应诱导表达从而提高植株的耐受性。例如,干旱会导致脱落酸的合成,从而诱导干旱相关基因如ABI5、ABF1和ABF2的表达。
热休克蛋白(Heat Shock Proteins,HSPs)是在从细菌到哺乳动物中广泛存在一类热应激蛋白质。HSPs家族在多种植物的干旱胁迫响应机制中发挥了重要作用。番茄中,在干旱胁迫条件下,HSP70的沉默会导致植物细胞膜的高度损伤和渗漏、相对含水量降低、色素积累减少和抗氧化酶活性降低,表明HSP70基因在番茄响应和适应干旱过程中发挥了关键作用。在水稻中,OsHSP50.2是水稻耐旱性的正调节因子,可能参与干旱胁迫下活性氧的稳态的调节,并且可能作为分子伴侣在应激反应中发挥介导干旱胁迫信号传导的作用。另外,水稻OsHSP17.7还可能有助于在干旱胁迫和随后的渗透调节过程中保护质膜结构从而对抗干旱胁迫(Sato et al.,2008)。在辣椒中,CaHsp25.9的转录是由热、盐和干旱胁迫诱导的,它可以正向调节植物的耐热、耐盐和耐旱性。此外,CaHsp25.9可能通过减少活性氧ROS的积累和调节应激相关基因的表达来调节干旱胁迫的耐受性;CaHSP16.4作为辣椒中的一个小热休克蛋白基因,也可以参与植物耐热和耐旱性的调节。在玉米中,ZmHsf08可能通过增强植物内的ROS水平和丙二醛含量来增强其对盐和干旱胁迫的耐受性。在棉花中,GhHSP24.7可以介导线粒体蛋白乙酰化以调节棉花干旱胁迫下的气孔导度。由此可见,植物HSP家族在对抗干旱胁迫时发挥重要作用同时,它还调控着植物的生长发育过程。在马铃薯中,StHSP26.5被推测可能参与调控块茎发芽过程。细胞质小分子热激蛋白HSP17.5在玫瑰花发育期大量表达,表明在花发育中起着重要作用。在12℃至27℃的温度范围内,拟南芥中HSP70的表达与开花时间呈正相关;RNAi-HSP90转基因株系在正常生长条件下延迟拟南芥开花,并诱导开花相关基因的表达变化。
随着温室效应的加剧,极端干旱天气频发,马铃薯块茎形成过程受到这些极端环境的严重影响,严重威胁马铃薯的块茎的产量和品质。因此,研究马铃薯热激蛋白基因响应干旱胁迫的作用机制,挖掘和解析马铃薯的耐旱基因,可以为马铃薯耐旱性改良育种提供科学研究依据。
发明内容
基于此,本发明的目的在于提供一种马铃薯耐旱性相关热激蛋白基因HSP101及其应用。
为了实现上述目的,本发明提供了如下技术方案:
本发明提供了一种马铃薯耐旱性相关热激蛋白基因HSP101,其核苷酸序列如SEQID No.1所示,编码蛋白的氨基酸序列如SEQ ID No.2所示。
本发明还提供了上述马铃薯耐旱性相关热激蛋白基因HSP101在马铃薯耐旱性改良育种中的应用,干涉热激蛋白基因HSP101会降低马铃薯的耐旱性。
本发明还提供了上述马铃薯耐旱性相关热激蛋白基因HSP101的重组表达载体。
本发明还提供了上述马铃薯耐旱性相关热激蛋白基因HSP101的重组表达载体在马铃薯耐旱性改良育种中的应用。
本发明的有益效果在于:本发明提供了一种马铃薯耐旱性相关热激蛋白基因HSP10,其核苷酸序列如SEQ ID No.1所示,编码蛋白的氨基酸序列如SEQ ID No.2所示。该基因位于细胞质中,干涉该基因会明显降低马铃薯的耐旱性。这为解析马铃薯耐旱机制和耐旱改良育种提供了遗传材料和理论依据。
附图说明
图1为StHSP101的序列特征分析,其中,1A为StHSP101的CDS扩增后的琼脂糖凝胶电泳结果图,1B为StHSP101特征分析,1C为StHSP101保守结构域分析,1D、1E为***进化树分析;
图2为StHSP101表达模式分析,其中,2A为20% PEG6000模拟干旱处理诱导StHSP101表达的qRT-PCR鉴定;2B为20μM ABA诱导StHSP101表达的qRT-PCR鉴定;
图3为StHSP101转基因株系的qRT-PCR鉴定,其中3A干涉株系的qRT-PCR鉴定,3B为为超量表达株系的qRT-PCR鉴定;
图4为StHSP101转基因株系在甘露醇模拟干旱条件下的表型分析,其中,4A生长态势分析,4B为茎长分析,4C为地上鲜重分析,4D为分枝数分析,4E为地下鲜重分析,4F为根数分析;
图5为StHSP101转基因株系在干旱条件下的表型分析,其中,5A为干旱处理前的表型,5B为干旱处理10d的表型,5C为干旱处理14d后覆水后的表型。
具体实施方式
下面将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解,下列实施例仅为示例性地说明和解释本发明,而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。需要说明的是,本发明实施例中的实验材料均能商业获得,本发明实施例中未注明具体条件的实验方法,通常按照常规条件或按照实验材料厂商建议条件。另外,需要说明的是本发明中所述的马铃薯E3为鄂马铃薯3号。
实施例1StHSP101的克隆和序列特征分析
以马铃薯E3叶片的cDNA为模板,对StHSP101的CDS序列进行扩增,利用1%琼脂糖凝胶电泳检测到目的片段大小为2000-3000bp(图1A),与数据库预测的基因大小一致,登录号为PGSC0003DMG400024644,测序和分析结果表明,该基因位于细胞质中,基因开放阅读框为2739bp(174-2912bp),含有6个内含子和7个外显子,核苷酸序列如SEQ ID No.1所示,编码912个氨基酸(图1B),编码蛋白的氨基酸序列如SEQ ID No.2所示。利用SMART对StHSP101进行保守结构域分析(图1C),结果显示在StHSP101的200-345、598-740氨基酸序列区具有HSP100基因家族典型的AAA保守结构域(smart00382)。利用同源蛋白的氨基酸序列构建进化树(图1D、1E)。结果表明,马铃薯StHSP101的氨基酸序列与番茄(Solanum lycopersicum)、辣椒(Capsicum annuum)和烟草(Nicotiana attenuata)这些茄科植物的亲缘关系比较近,尤其与番茄的同源性最高、亲缘性最近。
实施例2StHSP101表达模式分析
以马铃薯E3为实验材料,分别进行20μM ABA和20% PEG6000处理0h、0.5h、1h、3h、6h、12h和24h,取叶片抽提RNA,利用qRT-PCR技术对StHSP101进行不同处理不同时间点的差异表达分析,结果显示,在20% PEG6000处理下,StHSP101基因的相对表达量在24h上升(图2A),说明StHSP101受PEG强烈诱导,为了进一步研究StHSP101是否通过ABA信号途径参与马铃薯干旱胁迫响应,使用20μM ABA对E3的组培苗处理一定时间,对处理前后的茎和叶取样进行qRT-PCR检测,实验结果显示在ABA处理StHSP101的转录丰度的变化趋势与干旱处理的结果基本一致,在24h达到最高(图2B)。以上结果表明StHSP101可能通过ABA信号途径参与马铃薯干旱胁迫响应。
实施例3StHSP101转基因株系筛选与鉴定
通过构建StHSP101超量载体和干涉载体,以E3为受体,采用农杆菌转化法进行遗传转化构建StHSP101超量表达株系和StHSP101干涉株系。其中,StHSP101超量载体和干涉载体的构建方法如下:根据目的基因的CDS序列,使用引物设计软件Vector NTI 10设计克隆所需引物,使用abm公司的高保真聚合酶MegaFiTM Fidelity 2×PCR MasterMix进行PCR扩增,获得目的基因片段后,使用abm公司的重组酶Pro Ligation-Free Cloning Kit将纯化的PCR目的片段与酶切载体进行重组,转化大肠杆菌后测序,最终使用WEIDI公司的农杆菌感受态细胞LBA4404进行农杆菌转化实验,经PCR鉴定获得超量表达株系和干涉株系。采用qRT-PCR对转化效率进行鉴定,超量表达株系qRT-PCR检测结果显示17个阳性超量株系中14个超量阳性株系表达上调明显(图3B),干涉株系qRT-PCR检测,结果显示19个阳性干涉株系中有19个株系与WT相比均下调十分明显(图3A),干涉效率较高。为了验证其结果的准确性,对其进行二次重复,两次结果一致。
实施例4表型鉴定
1.StHSP101增强马铃薯试管苗对甘露醇的耐受性
将野生型(WT)、干涉转基因(RNAi)、超量株系(OE)种植在含0.3mol/L的甘露醇的MS+3%蔗糖+0.42%卡拉胶培养基中,对照种植于正常MS+3%蔗糖+0.42%卡拉胶培养基中,进行长日照(22℃/18℃,16h/8h)培养,培养14d后,发现在甘露醇处理当中,与WT相比,超量植株生长状态相对较好,而干涉株系生长状况明显弱于野生型(图4A)。对其茎长、地上鲜重、分枝数、地下鲜重及根数进行测量,结果显示,正常条件下,WT与转基因株系在茎长、地上鲜重、分枝数及根数没有显著性差异,在甘露醇处理后,干涉转基因株系在茎长、地上鲜重、分枝数及根数均表现出低于野生型(图4B、C、D、F),但在正常条件下的地下鲜重,干涉株系明显低于野生型,并且在甘露醇处理后,加剧了这一表型(图4E)。以上结果可以说明,在正常条件下,StHSP101可能提高了马铃薯试管苗的抗性,且在甘露醇模拟的干旱条件下,干涉StHSP101明显降低了马铃薯试管苗的耐旱性。
2.StHSP101增强马铃薯幼苗抗旱性
将野生型、干涉转基因、超量株系种植在营养土中,在长日照(22℃/18℃,16h/8h)正常生长15d后,干旱组不再浇水,对照组正常浇水,此时各株系在干旱组及对照组中生长状况一致(图5A)。干旱处理10d后,干旱组中土壤含水量明显减少,土壤发生干涸,WT及超量株系生长状况良好,而干涉转基因株系发生明显萎蔫(图5B)。在干旱处理14d后对干旱组进行覆水,已经发生萎蔫的WT和超量株系均能恢复其表型,而干涉株系仍然为萎蔫状态(图5C)。整个过程中干涉转基因在干旱处理情况下相对于野生型和超量株系均表现出了较差的耐旱性,而在对照情况下,各株系并没有明显差异,以上结果说明在干旱条件下,干涉StHSP101会明显降低马铃薯的耐旱性。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所有的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (2)
1.马铃薯耐旱性相关热激蛋白基因HSP101在马铃薯耐旱性改良育种中的应用,其特征在于,干涉热激蛋白基因HSP101将降低马铃薯的耐旱性,马铃薯耐旱性相关热激蛋白基因HSP101的核苷酸序列如SEQ ID No.1所示,马铃薯耐旱性相关热激蛋白基因HSP101的编码蛋白的氨基酸序列如SEQ ID No.2所示。
2.马铃薯耐旱性相关热激蛋白基因HSP101的重组表达载体在马铃薯耐旱性改良育种中的应用,马铃薯耐旱性相关热激蛋白基因HSP101的核苷酸序列如SEQ ID No.1所示,马铃薯耐旱性相关热激蛋白基因HSP101的编码蛋白的氨基酸序列如SEQ ID No.2所示。
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