CN113461793B - 辣椒ERF转录因子CaERF102及其在提高辣椒素含量中的应用 - Google Patents
辣椒ERF转录因子CaERF102及其在提高辣椒素含量中的应用 Download PDFInfo
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Abstract
本发明属于分子生物学技术领域，本研究从辣椒中克隆出一个CaERF基因，并利用VIGS技术在辣椒中沉默该CaERF，沉默植株的辣椒果实胎座辣椒素类物质的含量及辣椒素生物合成相关结构基因的表达均显著降低，研究结果为创制高辣椒素辣椒材料提供新的方法和思路。
Description
技术领域
本发明属于分子生物学技术领域，更具体地，涉及辣椒ERF转录因子CaERF102及其在提高辣椒素含量中的应用。
背景技术
辣椒是源于拉丁美洲热带地区的茄科辣椒属一年生或多年生植物，它是全球广泛种植的最大调味料蔬菜。目前，在全球被驯化和栽培的品种分别为Capsicum frutescens、Capsicum pubescens、Capsicum chinense、Capsicum baccatum和Capsicum annuum。自1990年以来，全球的辣椒产业发展较快，其中，中国是发展最快的国家。根据粮农组织(Foodand Agriculture Organization of the United Nations，FAO)统计显示，目前全球辣椒种植总面积三百多万公顷，年产量约四千万吨。印度和中国是全球辣椒栽培最多的国家，但是印度的生产能力较低，而中国的栽培面积及产能逐年递增，所以我国逐渐成为辣椒生产最大的国家。到2018年为止，全国辣椒栽培总面积约3000万亩，占全世界的40％左右，年产量约占全世界的50％，创造的年产值1000亿元以上。
辣椒富含多种营养物质，如类胡萝卜、维生素C和钙铁矿质元素等，且果实、叶片均能作为蔬菜或调味品。辣椒果实具有独特的辛辣味，是绝大部分人日常生活中不可或缺的一味重要食材。辣椒素类物质是引起果实辛辣的源头，是胎座特异合成的生物碱。在植物中，次生代谢产物通常是长期适应环境和进化的结果。辣椒的辛辣味能抑制动物啃食果实破坏种子以免影响后代繁衍，但是鸟类对辣味不敏感，因其本身生理构造不易破坏和消化种子，同时，不同种类和含量的类胡萝卜素累积导致辣椒果实颜色鲜亮多样化，从而有助于吸引鸟类等传播者采食，达到成功传播种子的目的。在人们生活中，辣椒素类物质具有非常重要的生物学功能和应用价值，不仅能够促进食欲、改善消化、降血脂、抗氧化和治疗癌症等，还在军事、农业、化妆品等行业广泛应用。辣椒素类物质生物合成途径涉及多种酶的催化以及各种转录因子相互协调或拮抗作用。在该过程中转录因子调控生物合成的分子机制是一项有用的工具，可以用于生物工程生产有价值的次生代谢产物。目前辣椒素类物质生物合成通路网络已基本清楚，但是相关的转录调控仍报道较少。很多研究认为ERF转录因子不仅响应生物与非生物过程，还参与调控植物多种次生代谢产物的生物合成，尤其是生物碱。然而，ERF转录因子在调控辣椒素生物合成方面报道比较少。
发明内容
本发明所要解决的技术问题是克服现有技术中缺乏辣椒中辣椒素生物合成相关基因的缺陷，首先提供一种辣椒ERF转录因子CaERF102。
本发明的第二个目的是提供含有上述辣椒ERF转录因子CaERF102的生物材料。
本发明的第三个目的是提供上述辣椒ERF转录因子CaERF102的应用。
本发明的目的通过以下技术方案实现：
辣椒ERF转录因子CaERF102，其核苷酸序列如SEQ ID NO：1所示。
本发明以辣椒“59”自交系的cDNA为模板，扩增回收CaERF102的CDS全长序列(图1A)。通过TA克隆获得质粒后送去测序，测序结果显示，CaERF102的CDS全长序列含有576个碱基，编码191个氨基酸。
因此，本发明所述辣椒ERF转录因子CaERF102，其氨基酸序列如SEQ ID NO：2所示。
本发明还提供含有权利要求1所述辣椒ERF转录因子CaERF102的生物材料，所述生物材料包括但不限于载体、质粒、宿主细胞、植物。
本发明研究上述转录因子CaERF102的功能，在CaERF102沉默植株中果实胎座(16DPA)的辣椒素生物合成相关基因AT3、AMT、KasIa、Acl、BCKDH和FatA等转录水平也显著地下降。这些结果表明，沉默CaERF102可以显著降低辣椒素类物质的含量以及辣椒素类物质生物合成相关基因的表达。
因此本发明还提供所述辣椒ERF转录因子CaERF102在提高辣椒素含量中的应用。
本发明还研究在辣椒类物质生物合成途径中，AT3、AMT、KasIa基因发挥着非常重要的作用，由于它们的启动子存在ERF-bingding cis-element数量较多，且CaERF102基因沉默后，它们的转录水平极显著下降。因此，根据ERF-bingding cis-element的预测结果，在AT3、AMT、KasIa基因启动子-1bp至-1600bp区域，分别截取600-700bp序列(序列太长容易导致诱饵菌株自激活能力较强，AbA无法抑制)克隆至pAbAi载体后转化Y1HGold菌株，研究CaERF102蛋白是否与这些启动子结合。如图4B所示，与AD+pAbAi-ProAT3、AD+pAbAi-ProAMT和AD+pAbAi-ProKasIa酵母菌相比，转化AD-CaERF102质粒的诱饵菌株在无添加AbA的SD/-Leu平板上正常生长，且同时在添加AbA的SD/-Leu平板上也能生长，这说明CaERF102均与AT3、AMT、KasIa基因启动子相结合。
因此，优选的上述应用中，所述CaERF102均与AT3、AMT、KasIa基因启动子相结合，以提高辣椒素的生物合成。
本发明与现有技术相比，具有以下有益效果：
本研究从辣椒中克隆出一个CaERF基因，并利用VIGS技术在辣椒中沉默该CaERF，沉默植株的辣椒果实胎座辣椒素类物质的含量及辣椒素生物合成相关结构基因的表达均显著降低，研究结果为创制高辣椒素辣椒材料提供新的方法和思路。
附图说明
图1为CaERF102的克隆和进化树分析；图1A：CaERF102基因的扩增，图1B：CaERF102在辣椒“59”自交系的氨基酸序列与理论序列的比对，图1C：CaERF102进化树分析；通过MEGA-X软件，选择最大似然法构建进化树，bootstrap值1000，其它参数为默认，CaERF102(XM_016721537.1)，SlDREBA4(MN197531.1)，StTINY-like(XM_006356081.2)，NaDREB3-like(Nicotiana attenuata，XM_019384931.1)，NtDREB3-like(Nicotiana tabacum，XM_016635802.1)，NtDREB3-like(Nicotiana tabacum，XM_016630600.1)，At3g60490(NM_115913.3)，CaAIEF1(ARR75181.1)，CaPTI1(XP_016562047.1)CaERF53(NP_001311812.1)，CaPF1(AAP72289.1)；
图2为CaERF102的亚细胞定位和转录激活分析；图2A：胎座10-25DPA的发育期间，辣椒素和二氢辣椒素含量变化(左侧)，CaERF102在胎座不同发育时期的表达模式与辣椒素类物质的积累的相关性(右侧)；图2B：CaERF102的亚细胞定位模式。Marker：细胞核定位基因DsRed；标尺：50μm；
图3为CaERF102沉默植株辣椒素类物质含量和生物合成相关基因的表达；图3A：pTRV2-CaERF102菌液侵染植株的鉴定，以果实胎座(16DPA)cDNA为模板，VIGS体系中CP基因为引物，图3B：CaERF102沉默植株果实胎座(55DPA)的辣椒素类物质含量，图3C：CaERF102沉默植株果实胎座(16DPA)的辣椒素类物质生物合成相关基因的表达；显著性差异采用Duncan法检验，*和**分别表示P<0.05、P<0.01；
图4为CaERF102结合调控辣椒素生物合成相关基因启动子，图4A：辣椒素生物合成相关基因启动子ERF转录因子结合元件的预测；红色三角符号表示ERF转录因子特异结合的元件；图4B：CaERF102结合辣椒素生物合成相关基因启动子的鉴定，ProAT3-1：-1bp至-700bp；ProAMT-1：-1bp至-700bp；ProAMT-2：-701bp至-1400bp；ProKasIa-1：-1bp至-700bp；SD/-Leu表示培养基缺乏亮氨酸；SD/-Leu+AbA表示SD/-Leu培养基添加AbA；AbA使用浓度：ProAT3-1，350ng/mL；ProAMT-1和ProAMT-2，300ng/mL；ProKasIa-1，250ng/mL，绿色三角符号代表菌液稀释的倍数。
具体实施方式
下面对本发明的具体实施方式作进一步说明。在此需要说明的是，对于这些实施方式的说明用于帮助理解本发明，但并不构成对本发明的限定。此外，下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
下述实施例以及实验例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料；所用的设备，如无特殊说明，均为常规实验设备。
辣椒“59”自交系；大肠杆菌(E.coli)菌株DH5α和农杆菌(Agrobacteriumtumefaciens)菌株GV3101均购自上海唯地生物技术有限公司。
实施例1 CaERF102基因克隆和分析
以辣椒“59”自交系cDNA为模板，参照高保真酶和pMDTM 19-T VectorCloning Kit的说明书，将CaERF102序列进行分离且克隆至pMD19-T载体上，
引物序列为CaERF102：Forward sequence(5’-3’)-TCATCGTCATTTTCTATG，
Reverse sequence(5’-3’)-AATTCAACATTGAAAATC。
结果：以辣椒“59”自交系的cDNA为模板，扩增回收CaERF102的CDS全长序列(图1A)。通过TA克隆获得质粒后送去测序，测序结果显示，CaERF102的CDS全长序列含有576个碱基，编码191个氨基酸。通过NCBI进行比对发现，克隆的CaERF102与辣椒基因组对应转录因子的氨基酸序列完全相同(图1B)。为了了解CaERF102的进化和潜在功能，从拟南芥(https://www.arabidopsis.org/)、茄科(http://solgenomics.net/)和NCBI等数据库中找出与CaERF102同源性最高的转录因子，以及目前在辣椒中功能已被鉴定的ERF转录因子共同构建进化树。如图1C所示，CaERF102与番茄、土豆(StTINY-like)、烟草的ERF转录因子亲缘关系最近，它们之间的同源性高于90％，且CaERF102与SlDREBA4、StTINY-like聚类在同一个分支。其中，SlDREBA4已经被报道参与番茄果实的成熟，而在烟草和土豆中ERF转录因子的功能仍未清楚。在辣椒中，CaAIEF1、CaPTI1和CaPF1与CaERF102的亲缘关系较远，它们被报道主要参与生物和非生物胁迫以及激素响应过程；CaErf可能与辣椒辛辣水平有关，但是它与CaERF102的相似性极低，仅有21％。在拟南芥中，At3g60490被认为可能参与次生细胞壁的形成，且与CaERF102的相似性也仅有20％左右。这些结果表明，CaERF102可能与辣椒果实生长发育有关。
实施例2 CaERF102的表达特性与亚细胞定位分析
qRT-PCR分析：根据AceQ qPCR SYBR Green Master Mix试剂盒说明书，在Bio-RadCFX384/96Touch进行荧光定量PCR(Quantitative Reverse Transcription PCR，qRT-PCR)。反应体系：Mix-5μL，Primer-F-0.2μL，Primer-R-0.2μL，Template(cDNA)-1μL，RNase-free ddH2O-3.6μL；反应程序：95℃-5min；95℃-10s，55℃-30s，72℃-20s，40cycles。每个样品进行三次生物学重复和三次技术重复，采用CA00g52140和CA12g20490作为内参基因和2-ΔΔCt方法进行数据分析。
涉及引物序列如下：CA00g52140-F：GGTCGCTTGGTTATGGTTAT，CA00g52140-R：ACAGTAGGGTCTCGGTTTGA；
CA12g20490-F：GAAGACCCTGACGGGCAAGAC，CA12g20490-R：TTAGCACCACCACGGAGACGA；
CaERF102-F：CGCATTTGGCTTGGCACATA，CaERF102-R：GCTTTAGCCGCAGCATCTTG。
亚细胞定位分析：将目的基因的全长(去除终止密码子)克隆至pEAQ-EGFP载体上，由CaMV 35S启动子调控目的基因和EGFP基因在本氏烟草(Nicotiana benthamiana)叶片中融合表达，引物序列为pEAQ-CaERF102-EGFP：Forward sequence(5’-3’)-ctgcccaaattcgcgaccggtATGACGAAGCGAATAAGAGAGAGTG，Reverse sequence(5’-3’)-gcccttgctcaccataccggtAAAGCCAAACTCATTAAATTTAAATTCT。
结果：为了了解CaERF102在辣椒不同组织和胎座不同发育时期的表达特性，以辣椒59号自交系为试材，进行qRT-PCR分析。在胎座10-25DPA的发育期间，辣椒素和二氢辣椒素含量显著增加，之后趋于平缓(图2A)，CaERF102在胎座不同发育时期的表达模式与辣椒素类物质的积累相似且在胎座组织中转录水平较高(图2A)。以pEAQ载体为骨架，细胞核定位基因DsRed为marker，将CaERF102的CDS全长(去掉终止密码子)与EGFP基因串联在一起，在CaMV 35S启动子的驱动下使它们在烟草叶片细胞中融合表达。结果表明，空载体pEAQ-EGFP定位于整个烟草叶片细胞，而pEAQ-CaERF102-EGFP仅定位于细胞核(图2B)。
实施例3沉默CaERF102可以降低辣椒素类物质含量和辣椒素合成相关基因转录
病毒诱导基因沉默(VIGS)：将携带目的基因cDNA的病毒载体侵染寄主植物后，通过激活植物自身免疫系统促使病毒RNA降解，同时产生含内源目的基因的microRNA，这些microRNA与细胞质中同源RNA特异性互补结合，导致同源mRNA降解，从而发生转录后水平的基因沉默。该实验采用烟草脆裂病毒pTRV1和pTRV2组成的系统进行侵染辣椒叶片，引物序列如下：
pTRV2-CaERF102：Forward sequence(5’-3’)-gtgagtaaggttaccgaattcATATCCAACACCAGAAATGGCC，Reverse sequence(5’-3’)-cgtgagctcggtaccggatccTCATCCAAGTCAAAACTACCCTCC。
结果：在辣椒“59”自交系中，通过VIGS技术将CaERF102基因进行沉默以检测该基因对辣椒素生物合成的影响。如图3A所示，在注射过pTRV2-CaERF102农杆菌菌液辣椒植株中，以果实胎座(16DPA)cDNA为模板，利用VIGS体系特有病毒序列Coat protein(CP)基因的引物进行扩增，约获得20株阳性植株。其中，5棵辣椒植株果实胎座(55DPA)的二氢辣椒素和辣椒素含量比注射空载的降低50％-80％(图3B)。同时，通过qRT-PCR分析表明，与侵染空载菌液的材料相比，在该5棵辣椒植株果实胎座(16DPA)中CaERF102的表达被有效沉默，且下降30％-60％(图3C)。在CaERF102沉默植株中果实胎座(16DPA)的辣椒素生物合成相关基因AT3、AMT、KasIa、Acl、BCKDH和FatA等转录水平也显著地下降(图3C)。这些结果表明，沉默CaERF102可以显著降低辣椒素类物质的含量以及辣椒素类物质生物合成相关基因的表达。
实施例4 CaERF102结合调控辣椒素生物合成相关基因启动子活性的分析
酵母单杂交分析：通过酵母单杂交方法识别和分析蛋白质与DNA之间的相互作用，主要是将辣椒素类物质生物合成相关基因启动子(ProAT3、ProAMT、ProKasΙ)克隆至pAbAi载体，并整合至Y1HGold酵母菌株中，形成诱饵特异性报告菌株，而CaERF102、基因序列全长克隆至AD载体形成猎物蛋白，再转化至诱饵特异性报告菌株中。一旦猎物蛋白与诱饵序列结合，GAL4 AD就会激活AbAr的表达，从而能够在含有抗生素AbA的培养基上生长。该实验主要参照Clontech公司Gold Yeast One-Hybrid Library Screening System的说明书执行，涉及的引物见表1。
表1在酵母单杂交中CaERF以及辣椒素生物合成相关基因启动子的引物
辣椒素类物质的提取和测定：利用高效液相色谱法(High performance liquidchromatography，HPLC)测定辣椒素类物质的含量。烘干的样品，趁热研磨成粉末后，称取0.1g置于15mL离心管中；加入5mL提取液(液相色谱级甲醇：四氢呋喃＝1:1)；在超声波清洗机中超声30min，室温静置过夜；使用1mL一次性注射器吸取待测样品，通过0.22μm滤头注射到色谱瓶中；采用XSelect HSS C-18SB column分离柱，80％甲醇作为流动相，在WatersAlliance 2489高效液相色谱仪中测定待测样品和辣椒素以及二氢辣椒素的标准品；根据标准曲线进行计算辣椒素和二氢辣椒素的含量。
结果：ERF转录因子可以特异识别结合GCC-box、DRE/CRT等元件。基于CaERF102基因沉默后辣椒素生物合成相关基因的转录水平显著降低，通过在线数据库JASPAR(http://jaspar.genereg.net/)预测这些结构基因启动子可能存在的ERF-bingding cis-element。如图4A所示，AT3、AMT、KasIa、KR、Acl和ENRa基因启动子区域(-1bp至-2000bp)可能存在ERF-bingding cis-element的数量分别为17、13、18、15、7、20个；BCKDH和FatA启动子-1bp至-1800bp之间可能存在ERF-bingding cis-element分别为4、8个。
在辣椒类物质生物合成途径中，AT3、AMT、KasIa基因发挥着非常重要的作用，由于它们的启动子存在ERF-bingding cis-element数量较多，且CaERF102基因沉默后，它们的转录水平极显著下降。因此，根据ERF-bingding cis-element的预测结果，在AT3、AMT、KasIa基因启动子-1bp至-1600bp区域，分别截取600-700bp序列(序列太长容易导致诱饵菌株自激活能力较强，AbA无法抑制)克隆至pAbAi载体后转化Y1HGold菌株，研究CaERF102蛋白是否与这些启动子结合。如图4B所示，与AD+pAbAi-ProAT3、AD+pAbAi-ProAMT和AD+pAbAi-ProKasIa酵母菌相比，转化AD-CaERF102质粒的诱饵菌株在无添加AbA的SD/-Leu平板上正常生长，且同时在添加AbA的SD/-Leu平板上也能生长，这说明CaERF102均与AT3、AMT、KasIa基因启动子相结合。
以上对本发明的实施方式作了详细说明，但本发明不限于所描述的实施方式。对于本领域的技术人员而言，在不脱离本发明原理和精神的情况下，对这些实施方式进行多种变化、修改、替换和变型，仍落入本发明的保护范围内。
序列表
<110> 华南农业大学
<120> 辣椒ERF转录因子CaERF102及其在提高辣椒素含量中的应用
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atgacgaagc gaataagaga gagtgccaac actggaaaca aacacccaat ttacagagga 60
gttcgcatgc gtagttgggg aaaatgggtg tcagaaatac gcgaaccgcg taaaaagtca 120
cgcatttggc ttggcacata tccaacacca gaaatggccg ctcgagcaca tgatgtcgca 180
gcattgagta tcaaaaagga ctcatcaata ttaaattttc cacatcttat cgactcattg 240
cctcgtccaa tttcactttc tcctagagat gtacaagatg ctgcggctaa agcagctgca 300
atggaggaac taaattctgc ctcttcttca atatcttcgt cttctgttaa atcgattgag 360
aaaataacgt ctgcatcgga tgagttatgt gaaattattg agcttcctag cttggagggt 420
agttttgact tggatgaatc gaaaaccgag ttgaagttga gcgacacagt tgacgggtgg 480
ctgtacccac cgtggtgggc atcagataaa gacttcgatg ggtattttct cgctgagaca 540
gatttagaat ttaaatttaa tgagtttggc ttttaa 576
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Met Thr Lys Arg Ile Arg Glu Ser Ala Asn Thr Gly Asn Lys His Pro
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Ile Tyr Arg Gly Val Arg Met Arg Ser Trp Gly Lys Trp Val Ser Glu
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Ile Arg Glu Pro Arg Lys Lys Ser Arg Ile Trp Leu Gly Thr Tyr Pro
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Thr Pro Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Leu Ser Ile
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Lys Lys Asp Ser Ser Ile Leu Asn Phe Pro His Leu Ile Asp Ser Leu
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Ser Ser Ser Val Lys Ser Ile Glu Lys Ile Thr Ser Ala Ser Asp Glu
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Leu Tyr Pro Pro Trp Trp Ala Ser Asp Lys Asp Phe Asp Gly Tyr Phe
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Leu Ala Glu Thr Asp Leu Glu Phe Lys Phe Asn Glu Phe Gly Phe
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Claims (1)
1.氨基酸序列如SEQ ID NO：2所示的转录因子CaERF102在降低辣椒中辣椒素含量中的应用。
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