CN117625603A - 一种创制耐冷茄子种质的方法及其应用 - Google Patents

一种创制耐冷茄子种质的方法及其应用 Download PDF

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CN117625603A
CN117625603A CN202311421552.5A CN202311421552A CN117625603A CN 117625603 A CN117625603 A CN 117625603A CN 202311421552 A CN202311421552 A CN 202311421552A CN 117625603 A CN117625603 A CN 117625603A
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eggplant
sgrna
cold
germplasm
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杨艳
周晓慧
庄勇
刘军
刘松瑜
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Jiangsu Academy of Agricultural Sciences
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Abstract

本发明涉及植物基因工程领域，具体涉及一种创制耐冷茄子种质的方法及其应用。本发明首先提供了用于创制耐冷茄子种质的sgRNA，能够特异性靶向茄子基因SmWRKY4。针对这个sgRNA构建的CRISPR/Cas9敲除载体可以对茄子基因SmWRKY4进行基因编辑，有效促进茄子叶片光合作用，降低茄子叶片的相对电导率，增强茄子植株的耐冷性，从而能快速创制耐冷茄子种质。

Description

一种创制耐冷茄子种质的方法及其应用
技术领域
本发明涉及一种创制耐冷茄子种质的方法及其应用，属于植物基因工程领域。
背景技术
随着多种蔬菜作物基因组数据的公开，蔬菜基因功能的研究和蔬菜现代分子育种得到了快速发展。例如，病毒诱导的基因沉默、RNAi、过量表达等基因功能研究手段以及分子标记育种、转基因育种和分子设计育种为代表的现代分子育种手段在蔬菜研究中占据了一定地位。但这些手段存在一些缺点：得到的材料不能稳定遗传，非定点敲除，育种周期长等。
基因编辑技术已成为基因功能研究和精准分子育种的重要手段。相对于其他分子育种手段，基因编辑技术具有操作简便、编辑效率高、支持多靶点编辑等优点，可克服传统育种周期长、工作量大、成本高、效率低等缺点，还可以通过自交纯化获得不含有外源DNA的材料。CRISPR/Cas9编辑技术已迅速被被应用在模式植物拟南芥、农作物水稻、小麦、棉花以及蔬菜作物番茄中。
茄子是重要的茄科蔬菜作物之一，起源于东南亚热带地区，属于喜温作物。与其他茄科作物相比，茄子对冷害更加敏感。冷害对茄子种子萌发、幼苗生长和开花结果等各个生长过程均能产生不良的影响，进而严重威胁茄子的产量和品质，制约冬春季茄子生产的可持续发展。因此，利用现代生物技术培育适于设施栽培的耐冷品种对茄子优质高效生产具有重要的意义。
发明内容
本发明利用基因编辑技术提供了一种用于创制耐冷茄子种质的方法，该方法利用基因编辑技术靶向茄子基因SmWRKY4，快速创制耐冷茄子种质。
具体而言，用于创制耐冷茄子种质的sgRNA，包括：
序列如SEQ ID NO.1所示的特异性靶向茄子SmWRKY4的sgRNA。
本发明进一步提供含有所述sgRNA的CRISPR/Cas9基因编辑载体。
作为优选，所述CRISPR/Cas9基因编辑载体为含有sgRNA的pSmP 1C载体。
本发明进一步提供用于创制耐冷茄子种质的试剂盒，包括以下任一种：
1)权利要求1所述的sgRNA；
2)所述sgRNA的编码DNA分子；
3)所述的CRISPR/Cas9基因编辑载体；
本发明进一步提供所述的sgRNA或所述的CRISPR/Cas9基因编辑载体或所述的试剂盒在敲除茄子基因WRKY4中的应用。
本发明进一步提供所述的sgRNA或所述的CRISPR/Cas9基因编辑载体或所述的试剂盒在创制耐冷茄子种质中的应用；
本发明进一步提供所述的sgRNA或所述的CRISPR/Cas9基因编辑载体或所述的试剂盒在促进茄子叶片光合作用中的应用；
本发明进一步提供所述的sgRNA或所述的CRISPR/Cas9基因编辑载体或所述的试剂盒在降低茄子叶片相对电导率中的应用。
本发明进一步提供一种创制耐冷茄子种质的方法，包括：
将含有所述sgRNA的CRISPR/Cas9基因编辑载体利用农杆菌介导的遗传转化法转入茄子中。
本发明还提供用于检测与耐冷茄子种质相关的SmWRKY4基因的突变效果的引物组合，包括：序列如SEQ ID NO.2-3所示的引物。
基于上述技术方案，本发明的有益效果如下：
本发明所提供的sgRNA和CRISPR/Cas9敲除载体可以对茄子基因SmWRKY4进行基因编辑，有效降低茄子叶片的电导率，促进茄子光合作用，增强茄子耐冷性，从而能快速创制耐冷茄子种质。
附图说明
图1为本发明实施例的基因编辑载体。
图2为茄子基因SmWRKY4突变体鉴定。
图3为茄子基因SmWRKY4突变后耐冷性鉴定。
具体实施方式
为了使本发明的目的、技术方案以及优点更加清楚明白，下面结合具体实施例对本发明进一步进行描述。此处所描述的具体实施例仅用于解释本发明，并不用于限定本发明。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例
1 CRISPR/Cas9编辑载体的构建
在茄子基因组网站(http：//eggplant-hq.cn/Eggplant/home/index)中下载SmWRKY4基因序列，利用在线软件CRISPRdirect(https：//crispr.dbcls.jp/)设计SmWRKY4的sgRNA序列，选择第一个外显子上的特异性序列为靶位点序列，用于后续CRISPR/Cas9编辑载体的构建。sgRNA序列如SEQ ID NO.1所示。
针对SmWRKY4的sgRNA序列设计引物oligo R，序列为GCTATTTCTAGCTCTAAAACGTACGGCTGATTTGGAGCTGcGAACTCATTACTTCGCTA。利用高保真酶，以载体pSmP1C为模板，用载体引物pSmP1C-F和Oligo R进行PCR扩增获得sgRNA克隆框。载体引物pSmP1C-F为CAGGAAACAGCTATGACCATATTCGACAACATCTGCCATTGG。
利用EcoRI和XbaI对载体pSmP1C进行双酶切，凝胶电泳，割胶回收，获得大小约14kb的酶切产物。
利用同源重组酶将sgRNA克隆框和线性化的pSmP1C载体进行同源重组，将重组载体进行热击转化至大肠杆菌感受态细胞，并用pSmP1C-F和Oligo R为引物进行菌液PCR检测，将获得的阳性菌液送测序鉴定获得最终基因编辑载体。测序引物为pSmP1C-F。
2含有基因编辑载体的根癌农杆菌的获得
将获得的基因编辑载体利用冻融法转化至根癌农杆菌EHA105，PCR鉴定获得阳性重组菌，进行茄子遗传转化，鉴定引物为载体引物pSmP1C-F和Oligo R。
3‘三月茄’遗传转化
‘三月茄’种子经75％的乙醇浸泡30s，10％NaClO消毒20min，无菌水冲洗5次后接种于1/2MS培养基中。待种子萌发长至子叶完全展开，用刀片将子叶切成4mm×4mm小段，置于共培养培养基中光照预培养1d。
将含有基因编辑载体的农杆菌在含卡那霉素(50mg/L)和利福平(25mg/L)的YEP培养基上划板，挑取单菌落于液体培养基中过夜培养至OD600为1.0。对培养好的菌液4000r/min离心10min，弃上清，用含有200μM乙酰丁香酮的液体培养基重悬至OD600为0.2-0.3。
将外植体浸泡于悬浮液中暗下侵染5min后，倒掉悬浮液并将外植体表面的液体吸干，将其背面朝上置于共培养培养基，避光共培养2d。将外植体转接至愈伤诱导培养基上进行愈伤诱导。约2周后将诱导出的愈伤组织转入芽诱导培养基上进行芽诱导。待芽伸长成小苗接至生根培养基进行生根。芽诱导和伸长期间每2周进行一次继代。待小苗生根后，打开培养瓶驯化两三天后，移至基质中。
4编辑植株靶位点鉴定
从成活植株上取样，利用CTAB法提取其基因组DNA，以Cas9-F：AAGCCCATCAGAGAGCAGG和Cas9-R：TGTCGCCTCCCAGCTGAG为引物，进行PCR扩增检测Cas9元件是否插入‘三月茄’。PCR程序包括95℃预变性3min；30个循环的95℃变性15s，60℃退火15s和72℃延伸30s；最后72℃延伸5min。
根据靶位点在基因组中的位置设计特异性引物进行PCR扩增和测序，引物序列如SEQ ID NO.2-3所示。通过分析植株基因组中靶位点的序列特征，进行靶位点突变分析。如图2所示，本次编辑事件获得一个突变事件，即wrky4-3存在2bp缺失。
5突变植株耐冷性鉴定
具体处理方法如下：挑选饱满的野生型和突变体wrky4-3的种子，并播种于基质中，于16h光照/8h黑暗的光周期下培养至四叶一心期。将大小和长势一致的野生型植株和突变体植株同时转移至4℃生长箱中进行处理7天。随后放置在正常生长条件下恢复生长3天进行性状调查，并测定各个植株的叶绿素荧光和相对电导率。同时设置1个对照组进行平行实验(不做冷胁迫处理)。
冷胁迫处理实验结果如图3a所示，正常生长条件下的野生型和突变体植株无明显差异。冷处理恢复3天后，野生型植株表现为几乎所有的叶片萎蔫失绿，生长点坏死。突变体植株表现为相对较轻的症状，即下部叶片坏死，上部新生叶片出现少量坏死斑和失绿症状。
利用Imaging-PAM型叶绿素荧光成像系统(Walz，德国)对植株的叶绿素荧光进行检测。
相对电导率测定如下：从植株叶片上打孔取叶圆片，洗净擦干后放入含有25mlddH2O的50ml离心管中，于室温下200rpm摇晃2h后测定电导率E1，然后沸水浴15min，冷却至室温测定电导率E2。根据相对电导率公式进行计算：REC＝E1/E2*100。
正常生长条件下，各植株叶绿素荧光参数值没有差异。与常温相比，冷胁迫下植株的Fv/Fm都有所降低。突变体植株的Fv/Fm显著高于野生型(图3b)。
正常生长条件下，野生型和突变体植株的电导率保持在比较低的水平，且没有差异。而受到冷害胁迫后，各个植株的渗透性增强，电导率增大。但突变体植株的电导率显著低于野生型(图3c)。
上述实验结果说明SmWRKY4基因的突变可以提高茄子植株的耐冷性。以上所述仅为本发明专利的具体实施案例，但任何参照本发明申请专利所作的变化或修饰皆涵盖在本发明的专利范围之中。

Claims (8)

1.用于创制耐冷茄子种质的sgRNA，其特征在于，包括：序列如SEQ ID NO.1所示的特异性靶向茄子SmWRKY4的sgRNA。
2.含有权利要求1所述的sgRNA的CRISPR/Cas9基因编辑载体。
3.根据权利要求2所述的CRISPR/Cas9基因编辑载体，其特征在于，包含权利要求1所述的sgRNA的pSmP1C载体。
4.用于创制耐冷茄子种质的试剂盒，其特征在于，包括以下任一种：
1)权利要求1所述的sgRNA；
2)所述sgRNA的编码DNA分子；
3)权利要求2或3所述的CRISPR/Cas9基因编辑载体。
5.权利要求1所述的sgRNA或要求2或3所述的CRISPR/Cas9基因编辑载体或权利要4所述的试剂盒在敲除茄子基因WRKY4中的应用。
6.权利要求1所述的sgRNA或要求2或3所述的CRISPR/Cas9基因编辑载体或权利要求4所述的试剂盒在以下任一方面的应用：
1)在创制耐冷茄子种质中的应用；
2)在促进茄子叶片光合作用中的应用；
3)在降低茄子叶片相对电导率中的应用。
7.一种创制耐冷茄子种质的方法，其特征在于，包括：
将权利要求2或3所述的CRISPR/Cas9基因编辑载体利用农杆菌介导的遗传转化法转入茄子中。
8.用于检测与耐冷茄子种质相关的SmWRKY4基因的突变效果的引物组合，其特征在于，包括：序列如SEQ ID NO.2-3所示的引物。
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