CN116590328A - 一种植物表达载体及恢复Ogura CMS青花菜育性的方法 - Google Patents
一种植物表达载体及恢复Ogura CMS青花菜育性的方法 Download PDFInfo
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Abstract
本发明公开了一种植物表达载体及恢复Ogura CMS青花菜育性的方法，涉及植物基因工程技术领域。植物表达盒包括：Rfo基因启动子、Rfo基因的编码序列和终止子序列，Rfo基因启动子的核苷酸序列如SEQ ID NO.1所示，Rfo基因的编码序列如SEQ ID NO.2所示。本发明提供的表达盒中Rfo基因及其启动子全部来自于萝卜，未加入其他作物基因片段。本发明提供的植物表达盒可以用于快速、高效的创制青花菜Ogura CMS恢复系材料。此外，创制的Ogura CMS恢复系材料，通过和其他性状优良的Ogura CMS青花菜材料进行回交转育，可以将目标性状分离出来加以再利用。
Description
技术领域
本发明涉及植物基因工程技术领域，具体而言，涉及一种植物表达载体及恢复Ogura CMS青花菜育性的方法。
背景技术
青花菜(Brassica oleracea L.var.italica)又名西兰花，原产于地中海地区。近二十年，作为外来蔬菜的青花菜慢慢被认识和接受，并开始在国内扎根发展，现在已作为营养健康型蔬菜在全国各地推广种植。
目前，我国育种家掌握的材料大多是进口品种经自交分离而来，类同性明显、种质材料间遗传多样性低，难以育成有突破性的品种。另外，目前市场上的商品种全部采用Ogura CMS细胞质雄性不育技术生产杂交一代。因为Ogura CMS不育基因位于细胞质内，杂交后代均为100％不育，所以优良性状无法被分离出来进行再利用，对青花菜种质资源的创新和优良新品种的培育具有极大的影响。
Ogura CMS属于质核互作雄性不育类型，育性是由线粒体不育基因orf138和细胞核育性恢复基因Rfo相互作用控制的。当Rfo恢复基因和orf138基因同时在不育植株中表达时，Rfo基因编码的一种PPR蛋白，包含687个氨基酸，能够使产生的orf138蛋白稳定性降低、蛋白质量减少，从而导致雄性不育系育性恢复。
到目前为止，还没有发现甘蓝类作物中有Ogura CMS育性恢复系的存在，使得青花菜不能通过杂交转育的方法获得Ogura CMS恢复系材料。尽管甘蓝型油菜中已经成功创制Ogura CMS恢复系，通过远缘杂交可以将甘蓝型油菜的育性恢复基因导入青花菜中，但是远缘杂交由于存在生殖隔离，授粉后易出现杂交不亲、胚败育以及后代遗传变异复杂等问题，并且需要多年的转育才能获得可以用于育种的Ogura CMS恢复系材料。而通过基因工程技术将Rfo基因直接转入青花菜中可快速获得Ogura CMS恢复系，实现优良性状的分离和再利用。
鉴于此，特提出本发明。
发明内容
本发明的目的在于提供一种植物表达载体及恢复Ogura CMS青花菜育性的方法以高效的创制青花菜Ogura CMS恢复系材料。
本发明是这样实现的：
第一方面，本发明提供了一种植物表达盒，其包括：Rfo基因启动子、Rfo基因的编码序列和终止子序列，Rfo基因启动子来自萝卜Ogura CMS恢复系，其核苷酸序列如SEQ IDNO.1所示，Rfo基因的编码序列如SEQ ID NO.2所示；
终止子序列如SEQ ID NO.3所示。
本发明提供的表达盒中的Rfo基因及其启动子全部来自于萝卜，未加入其他作物基因片段。未加入其他作物基因片段有助于Rfo基因表达更加稳定，不会由于加入其他作物基因从而影响Rfo基因的正常表达。本发明提供的植物表达盒可以用于快速、高效的创制青花菜Ogura CMS恢复系材料，具体通过基因工程技术将Rfo基因直接转入青花菜中可快速获得Ogura CMS恢复系，实现优良性状的分离和再利用。此外，上述植物表达盒的提供，还有助于缩短育种时间，可以通过创制Ogura CMS恢复系材料，通过和其他种质材料进行回交转育，使得其他优质品种获得目标性状。
第二方面，本发明提供了一种植物表达载体，其包括上述的植物表达盒。
在本发明应用较佳的实施方式中，植物表达载体在植物表达盒的上游还插入有筛选标记基因表达元件，筛选标记基因是BAR基因(除草剂草丁膦抗性基因)、NPTII(卡拉霉素抗性基因)、PAT基因(除草剂草铵膦抗性基因)、CP4-EPSPS(除草剂草甘膦抗性基因)、GAT基因(除草剂草甘膦抗性基因)、GR79-epsps基因(除草剂草甘膦抗性基因)或hpt(潮霉素抗性基因)。
在本发明应用较佳的实施方式中，筛选标记基因表达元件中的启动子为组成型启动子或诱导型启动子。
在本发明应用较佳的实施方式中，组成型启动子选自NOS启动子或CaMV35S。诱导型启动子选自光诱导型启动子、胁迫诱导型启动子、伤诱导型启动子和激素诱导型启动子中的任意一种。
光诱导型启动子例如选自捕光叶绿素a/b蛋白复合体(CAB)基因启动子、核酮糖1,5-二磷酸羧化酶小亚基基因(RBCS)启动子或茄啶半乳糖基转移酶(SGT1)基因启动子。
胁迫诱导型启动子例如选自生物胁迫型启动子或环境胁迫型启动子。环境胁迫型启动子例如选自干旱胁迫型、温度胁迫型或盐胁迫型诱导启动子。温度胁迫型启动子例如选自热诱导表达启动子、低温诱导表达启动子。
激素诱导型启动子例如选自水杨酸诱导型启动子、脱落酸诱导型启动子、赤霉素诱导型启动子、生长素诱导型启动子、茉莉酸和乙烯诱导型启动子。
在一种可选的实施方式中，植物表达载体还包括增强子。
在一种可选的实施方式中，植物表达载体选自pBI121、pBI101、pBI 221、pBI121-GFP、pBI221-GFP、pCAMBIA 1300、pCAMBIA 1301,pCAMBIA 1302、pCAMBIA 1303、pCAMBIA1304、pCAMBIA 1305、pCAMBIA 2300、pCAMBIA 2301、pCAMBIA 3300、pCAMBIA 3301、pCAMBIA1380、pCAMBIA 1390、pCAMBIA super1300或pCAMBIA super1300-GFP。
本发明还提供了一种重组菌或重组细胞，其包括上述的植物表达载体。
第三方面，本发明提供了植物表达盒、植物表达载体、重组菌或重组细胞在制备Ogura CMS细胞质雄性不育恢复系中的应用。
重组菌或重组细胞例如选自大肠杆菌。
第四方面，本发明提供了一种培育Ogura CMS细胞质雄性不育恢复系的方法，其包括如下步骤：将上述的植物表达盒或上述的植物表达载体转化Ogura CMS青花菜。
利用本发明提供的载体转化后所获得的任何甘蓝类Ogura CMS细胞质恢复系，均可通过普通的有性杂交方式(无需远缘杂交胚抢救手段)将其恢复位点导入其他甘蓝类型植物如芥蓝、白花菜、青花菜、紫花菜、球茎甘蓝、孢子甘蓝、羽衣甘蓝、紫甘蓝等甘蓝类蔬菜，快速获得上述甘蓝类蔬菜的Ogura CMS细胞质不育专用恢复系。
转化的方法包括不限于农杆菌介导基因转化法，基因枪转化法、花粉管通道法。
第五方面，本发明提供了一种利用基因工程恢复Ogura CMS青花菜育性的方法，其包括：以上述的方法培育获得的Ogura CMS细胞质雄性不育恢复系为父本，与待恢复育性的Ogura CMS青花菜植株进行杂交，获得种子后，筛选可育的且具有目标优势性状的青花菜。
在本发明应用较佳的实施方式中，上述目标优势性状为抗根肿病、黑腐病、黑斑病、耐旱、耐寒和低温不紫中的至少一种。
在本发明应用较佳的实施方式中，上述方法还包括将获得的可育且含有目标性状的植株与其他种质材料进行回交转育。
利用本发明提供的植物载体转化后所获得的Ogura CMS细胞质雄性不育恢复系，可作为父本与其他含有目标优异性状的材料杂交，利用载体中生物筛选标记基因，例如通过除草剂膦丝菌素(Phosphinothricin,PPT)的选择，获得的杂交后代含有转基因成分的个体可以被去除，并最终筛选出具有目标优异性状的非转基因个体供后续育种利用。
本发明具有以下有益效果：
本发明提供的表达盒中的Rfo基因及其启动子全部来自于萝卜，未加入其他作物基因片段。未加入其他作物基因片段有助于Rfo基因表达更加稳定，不会由于加入其他作物基因从而影响Rfo基因的正常表达。本发明提供的植物表达盒可以用于快速、高效的创制青花菜Ogura CMS恢复系材料，具体通过基因工程技术将Rfo基因直接转入青花菜中可快速获得Ogura CMS恢复系，实现优良性状的分离和再利用。此外，上述植物表达盒的提供，还有助于缩短育种时间，可以通过创制Ogura CMS恢复系材料，通过和其他种质材料进行回交转育，使得其他优质品种获得目标性状。
利用本发明提供的植物表达载体转化后所获得的Ogura CMS细胞质雄性不育恢复系，可作为父本与其他含有目标优异性状的材料杂交，利用载体中生物筛选标记基因，例如通过除草剂膦丝菌素(Phosphinothricin,PPT)的选择，获得的杂交后代含有转基因成分的个体可以被去除，并最终筛选出具有目标优异性状的非转基因个体供后续育种利用。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本发明的Rfo基因表达载体结构示意图；
图2为Ogura CMS青花菜的Rfo基因检测示意图，A：为pRfo-FT和pRfo-RT引物检测结果；B为Rfo-FT和Rfo-RT引物检测结果；其中字母“M”为DNA marker；数字“1-33”为抗卡那霉素植株；“+”为表达载体质粒作阳性对照；“—”为未转化的青花菜DNA做阴性对照；
图3为转基因Ogura CMS青花菜育性恢复后的实物图及显微图；
图4为转基因Ogura CMS青花菜自交授粉结籽情况实物图；
图5为Ogura CMS恢复系的应用流程图。
具体实施方式
现将详细地提供本发明实施方式的参考，其一个或多个实例描述于下文。提供每一实例作为解释而非限制本发明。实际上，对本领域技术人员而言，显而易见的是，可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如，作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中，来产生更进一步的实施方式。
除非另有定义，否则本文使用的所有技术和科学术语具有与本公开内容所属领域的普通技术人员通常理解的含义相同的含义。尽管与本文描述的那些方法和材料类似或等同的任何方法和材料都可用于本文的制剂或单位剂量的实践或测试，但现在描述一些方法和材料。除非另有说明，否则本文采用或考虑的技术是标准方法。材料、方法和实例仅是说明性而非限制性的。
除非另外指明，否则实践本发明将采用植物生理学、植物分子遗传学、细胞生物学、分子生物学(包含重组技术)、微生物学、生物化学和免疫学的常规技术，所述常规技术在本领域技术人员的能力范围内。文献中充分解释了这种技术，如《分子克隆：实验室手册(Molecular Cloning:A Laboratory Manual)》，第二版(Sambrook等人，1989)；《寡核苷酸合成(Oligonucleotide Synthesis)》(M.J.Gait编，1984)；《植物生理学》(苍晶等人，2017)；《酶学方法(Methods in Enzymology)》(学术出版社有限公司(Academic Press，Inc.)；《实验免疫学手册(Handbook of Experimental Immunology)》(D.M.Weir和C.C.Blackwell编)；《当代分子生物学方法(Current Protocols in Molecular Biology)》(F.M.Ausubel等人编，1987)；《植物分子遗传学》(Monica A.Hughes等人著)；《PCR:聚合酶链反应(PCR:The Polymerase Chain Reaction)》(Mullis等人编，1994)，所述文献中的每个文献均通过引用明确并入本文中。
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例1
本实施例提供了一种培育Ogura CMS细胞质雄性不育恢复系的方法，具体包括植物表达载体的构建、转化。
(1)
设计Linker片段引物：
L-F：5’-
AGCTTTCAGGCGCGCCGGCTCTAGAGCCGCGGATCCGCGC G-3’和L-R:5’
-TTAACGCGCGGATCCGCGGCTCTAGAGCCGGCGCGCCTG AA-3’构建双链Linker片段。
上述Linker片段构建具体步骤为：按表1所示，将Linker引物对加ddH2O混匀后，95℃变性5min，然后以0.2℃/s降温至25℃；反应产物用ddH2O稀释250倍。
表1linker片段构建反应体系
将获得Linker片段连接到植物表达载体pBI121质粒的HindⅢ和EcoRⅠ酶切位点之间，以获得含有AscⅠ、XbaⅠ、BamHⅠ等特异酶切位点的改良质粒pBI121-linker。
上述连接的具体步骤为：按表2所示，将pBI121载体质粒用限制性内切酶HindⅢ和EcoRⅠ进行双酶切，酶切产物经琼脂糖凝胶电泳后切胶回收线性化的质粒；再按表3所示，将linker片段和pBI121线性化载体质粒在T4连接酶的作用下4℃连接过夜；再将连接产物转化大肠杆菌DH5α感受态细胞，涂布在50μg/mL卡那霉素的LB固体培养基上37℃培养过夜，筛选阳性克隆，提取质粒。
表2载体质粒酶切反应体系
表3连接反应体系
(2)利用十二烷基磺酸钠(SDS)法提取萝卜Ogura CMS恢复系的基因组DNA，采用引物pRfo-F和pRfo-R扩增萝卜的Rfo基因启动子，将Rfo启动子片段(SEQ ID NO.1)克隆到pClone007 simple vector(购于擎科生物公司)，并对扩增片段进行测序，验证Rfo基因启动子序列正确。
按表2酶切反应体系用HindⅢ和AscⅠ酶分别对pClone007-pRfo和pBI121-linker载体进行双切，琼脂糖凝胶电泳后切胶回收目的条带，再按表3连接反应体系将扩增的Rfo启动子片段插入到步骤(1)获得的pBI121-linker载体中。
pRfo-F引物序列：5’-
CAAGCTTCATATTCATAGATTTTGTTT-3’；pRfo-R引物序列：5’-AGGCGCGCCTTTATTTTTGTTTCGCCTAA-3’。
(3)以萝卜cDNA为模板，用引物对Rfo-F和Rfo-R进行PCR扩增，将获得Rfo基因(包括终止子，包括SEQ ID NO.2和SEQ ID NO.3)片段克隆到pClone007 simple vector，并对扩增片段进行测序，验证Rfo基因序列正确。
按表2酶切反应体系用AscⅠ和EcoRⅠ酶分别对pClone007-Rfo和pBI121-pRfo载体进行双切，琼脂糖凝胶电泳后切胶回收目的条带，再按表3连接反应体系将扩增的Rfo基因片段插入到步骤(2)获得的载体中，构建成可以表达Rfo基因的表达载体(载体结构示意图参照图1所示)。
Rfo-F引物序列：
5’-AGGCGCGCCATGTTGGCTAGGGTTTGTGG-3’；Rfo-R引物序列：5’-GGAATTCGAAAAGCATTGAAGTATTGT-3’。
(4)利用冻融法将获得的Rfo基因表达载体质粒转化到EHA105农杆菌感受态细胞中。
(5)通过农杆菌介导转化法对Ogura CMS青花菜的下胚轴外植体进行侵染，经过抗性筛选培养，获得具有抗卡那霉素的青花菜植株。
(6)利用SDS法提取抗卡那霉素植株的基因组DNA，用引物对pRfo-FT和pRfo-RT、Rfo-FT和Rfo-RT进行PCR扩增，琼脂糖凝胶电泳检测结果表明获得的33株抗卡那霉素植株全部含有Rfo基因(图2)。
实施例2
将实施例1获得的33株转化株进行低温诱导开花，观察发现有12株出现了明显的花粉，进一步取转化株的花粉用0.5％ TTC(氯化三苯基四氮唑)染色法对花粉活力进行检测，其花粉数量和花粉活力都和正常可育植株无明显差异(图3)。
将获得有花粉的转化株进行人工剥蕾授粉自交，经过50-60天的生长，能够正常可育株自交一样获得饱满的种子(参照图4所示)。
实施例3
本实施例提供了青花菜Ogura CMS恢复系的应用。
将实施例2中出现了明显的花粉的转化株作父本和含有目标优势性状(抗根肿病、黑腐病、黑斑病、耐旱、耐寒和低温不紫中的至少一种)的Ogura CMS青花菜植株进行杂交，同样能够获得饱满的种子。然后将获得种子再进行种植，根据自交分离原则通过pRfo-FT和pRfo-RT、Rfo-FT和Rfo-RT等载体特异引物可以筛选出可育且含有目标性状的植株。获得的可育且含有目标性状的植株可以和其他种质材料进行回交转育(Ogura CMS恢复系的应用流程图参照图5所示)，其他材料就可以含有目标性状。在转育过程中还可以利用载体特异引物将转基因成分完全剔除，获得的材料不受转基因的影响。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (10)
1.一种植物表达盒，其特征在于，其包括：Rfo基因启动子、Rfo基因的编码序列和终止子序列，所述Rfo基因启动子来自萝卜Ogura CMS恢复系，其核苷酸序列如SEQ ID NO.1所示，所述Rfo基因的编码序列如SEQ ID NO.2所示；
优选地，所述终止子序列如SEQ ID NO.3所示。
2.一种植物表达载体，其特征在于，其包括权利要求1所述的植物表达盒。
3.根据权利要求2所述的植物表达载体，其特征在于，所述植物表达载体在所述植物表达盒的上游还插入有筛选标记基因表达元件，所述筛选标记基因是BAR基因(除草剂草丁膦抗性基因)、NPTII(卡拉霉素抗性基因)、PAT基因(除草剂草铵膦抗性基因)、CP4-EPSPS(除草剂草甘膦抗性基因)、GAT基因(除草剂草甘膦抗性基因)、GR79-epsps基因(除草剂草甘膦抗性基因)或hpt(潮霉素抗性基因)。
4.根据权利要求2所述的植物表达载体，其特征在于，所述筛选标记基因表达元件中的启动子为组成型启动子或诱导型启动子；
优选地，所述组成型启动子选自NOS启动子或CaMV35S；
优选地，所述植物表达载体还包括增强子；
优选地，所述植物表达载体选自pBI121、pBI101、pBI 221、pBI121-GFP、pBI221-GFP、pCAMBIA 1300、pCAMBIA 1301,pCAMBIA 1302、pCAMBIA 1303、pCAMBIA 1304、pCAMBIA1305、pCAMBIA 2300、pCAMBIA 2301、pCAMBIA 3300、pCAMBIA 3301、pCAMBIA 1380、pCAMBIA1390、pCAMBIA super1300或pCAMBIA super1300-GFP。
5.一种重组菌或重组细胞，其特征在于，其包括权利要求2-4任一项所述的植物表达载体。
6.如权利要求1所述的植物表达盒、权利要求2-4任一项所述的植物表达载体或权利要求5所述的重组菌或重组细胞在制备Ogura CMS细胞质雄性不育恢复系中的应用。
7.一种培育Ogura CMS细胞质雄性不育恢复系的方法，其特征在于，其包括如下步骤：将权利要求1所述的植物表达盒或权利要求2-4任一项所述的植物表达载体转化Ogura CMS青花菜。
8.一种利用基因工程恢复Ogura CMS青花菜育性的方法，其特征在于，其包括：以权利要求7所述的方法培育获得的Ogura CMS细胞质雄性不育恢复系为父本，与待恢复育性的Ogura CMS青花菜植株进行杂交，获得种子后，筛选可育的且具有目标优势性状的青花菜。
9.根据权利要求8所述的利用基因工程恢复Ogura CMS青花菜育性的方法，其特征在于，所述目标优良性状为抗根肿病、黑腐病、黑斑病、耐旱、耐寒和低温不紫中的至少一种。
10.根据权利要求8所述的利用基因工程恢复Ogura CMS青花菜育性的方法，其特征在于，所述方法还包括将获得的可育且含有目标性状的植株与种质材料进行回交转育。
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