CN116554291B - 一种梨bZIP类转录因子PubZIP914及其应用 - Google Patents
一种梨bZIP类转录因子PubZIP914及其应用 Download PDFInfo
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Abstract
本发明公开了一种梨bZIP类转录因子PubZIP914及其应用，属于植物分子基因工程技术领域，本发明从梨中分离得到编码PubZIP914转录因子的完整cDNA，连接到植物过表达载体上，利用农杆菌介导转化植物，通过烟草瞬时表达证明PubZIP914定位于植物细胞核内；利用农杆菌注射获得瞬时转化梨果实，分别利用叶盘转化法和组织侵染法获得转基因番茄和转基因梨愈伤，结果表明过表达PubZIP914能够提高植物中挥发性脂肪酸衍生物的合成。本发明为bZIP转录因子促进植物挥发性脂肪酸衍生物合成的研究提供了研究基础和基因资源。
Description
技术领域
本发明属于植物分子基因工程技术领域，具体涉及一种梨bZIP类转录因子PubZIP914及其应用。
背景技术
梨(蔷薇科梨属)是温带地区最重要的水果作物之一，具有很高的经济价值，目前在50多个国家均有种植。国内种植较为广泛的亚洲梨品种可分为四种：白梨(P.bretschneideri Rehd.)、秋子梨(P.ussuriensis Max.)、砂梨(P.pyrifolia Nakai.)和新疆梨(P.sinkiangensis Yu.)。大多数亚洲梨果实的果肉偏脆，汁水清甜，香气偏淡。然而秋子梨中的‘南果’梨，果实经后熟会释放出非常浓郁的果香和酒香，因其香味宜人，口感特别而受到广泛关注，是我国主要栽培的秋子梨品种之一。‘南果’梨成熟果实中的主要香气成分是己酸乙酯，乙酸乙酯和乙酸己酯。然而市场上最为流行的梨品种主要是白梨、砂梨和新疆梨，挥发性化合物种类较少且浓度较低。
香气浓郁的梨果实更容易吸引消费者前来购买，南果梨是香气机理研究的良好材料。因此，利用基因工程技术针对挥发性化合物的关键调控因子进行遗传改良，对于改善淡香梨品种的香气含量具有重要意义。
发明内容
针对现有技术的不足，本发明的目的在于一种梨bZIP类转录因子PubZIP914及其应用。
本发明解决其技术问题采用的技术方案是：
第一方面，本发明保护一种梨bZIP类转录因子PubZIP914，其选自如下a)、b)或c)：
a)其核苷酸序列如SEQ ID NO.1所示；
b)SEQ ID NO.1所示的核苷酸序列经取代、缺失和/或增加一个或几个核苷酸；
c)在严格条件下与1)限定的DNA序列杂交的核苷酸序列。
第二方面，本发明保护前文所述的转录因子PubZIP914基因编码的蛋白。
在具体的实施方案中，所述蛋白选自如下A)或B)：
A)如SEQ ID NO.2所示的氨基酸序列；
B)SEQ ID NO.2所示的氨基酸序列经取代、缺失和/或增加一个或多个氨基酸且具有相同活性的由1)衍生的蛋白。
第三方面，本发明保护扩增前文所述的转录因子PubZIP914基因全长或任一片段的引物对。
在具体的实施方案中，所述引物对包括引物对A和/或引物对B，其中引物对A如SEQID No.3～4所示；引物对B如SEQ ID No.5～6所示。
第四方面，本发明还保护含有前文所述的转录因子PubZIP914基因的重组表达载体、表达盒、转基因细胞系、或基因工程菌。
在具体的实施方案中，本发明提供两种重组表达载体的构建方法：
方法一：将前文所述的基因PubZIP914插入到pCAMBIA1301的XbaI和BamHI位点之间得到重组表达载体35S-PubZIP914-GFP。
方法二：将前文所述的基因PubZIP914插入到PSAK277的HindⅢ和XbaⅠ位点之间得到重组表达载体PSAK277-PubZIP914。
重组表达载体的方法不限于本发明提供的两种，任何利用现有技术得到的本发明的重组表达载体都在本发明的保护范围内。
第五方面，本发明还保护前文所述的转录因子PubZIP914和/或前文所述的蛋白和/或前文所述的重组表达载体、表达盒、转基因细胞系或基因工程菌在如下(1)-(5)中的应用：
(1)在调节果实香气中的应用；
(2)在制备调节果实香气的产品中的应用；
(3)在培育座果园艺作物中的应用；
(4)在植物育种中的应用；
(5)在促进植物挥发性脂肪酸衍生物合成中的应用。
在具体的实施方案中，所述果实为番茄或梨愈伤或梨果实。
在具体的实施方案中，所述植物为番茄或梨。
第六方面，本发明保护一种提高果实香气含量的方法，将前文所述的转录因子PubZIP914基因转入目的植物即可实现。
有益效果
本发明相比现有技术具有以下优点：本发明从梨中分离得到编码PubZIP914转录因子的完整cDNA，连接到植物过表达载体上，利用农杆菌介导转化植物，通过烟草瞬时表达证明PubZIP914定位于植物细胞核内；利用农杆菌注射获得瞬时转化梨果实，分别利用叶盘转化法和组织侵染法获得转基因番茄和转基因梨愈伤，结果表明过表达PubZIP914能够提高植物中挥发性脂肪酸衍生物的合成。本发明为bZIP转录因子促进植物挥发性脂肪酸衍生物合成的研究提供了研究基础和基因资源。
附图说明
图1为南果梨果实五个发育过程中bZIP基因家族的FPKM值；
图2为八个bZIP基因在南果梨果实、花瓣叶片中的组织定量分析；A:Pbr005914.1；B:Pbr027818.1；C:Pbr009262.1；D:Pbr002981.1；E:Pbr002622.1；F:Pbr009654.1；G:Pbr022685.1；H:Pbr040390.1。
图3为PubZIP914的亚细胞定位结果图；
图4为转基因番茄及梨愈伤阳性PCR鉴定结果图；A:梨愈伤；B:番茄。
图5为转基因番茄及梨愈伤表观图；
图6为瞬时超表达的南果梨挥发性脂肪酸衍生物含量及PubZIP914的定量分析结果图；
图7为转基因番茄中挥发性脂肪酸衍生物总含量及具体成分含量分析图；
图8为转基因梨愈伤中挥发性脂肪酸衍生物总含量及具体成分含量分析图。
具体实施方法
下面将结合本发明中的实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动条件下所获得的所有其它实施例，都属于本发明保护的范围。
实施例1梨bZIP基因家族的筛选：
基于已发表数据，获得梨中92条bZIP基因；结合南果梨果实五个发育期的转录组数据，发现20个在南果梨发育后期表达量显著升高的bZIP基因(图1)；
对上述20个南果梨基因进行南果梨不同组织中荧光定量结果分析，发现PubZIP914基因在果实中特异性高表达(图2)；因此PubZIP914被列为研究的候选基因。
实施例2南果梨中PubZIP914基因序列的获得：
取南果梨果实置于液氮预冷的研钵中进行研磨，参照多糖多酚植物总RNA提取试剂盒(Tiangen)说明书提取植物RNA，提取完成后使用凝胶电泳检测RNA完整性，NanoDrop仪器检测RNA浓度，经检测合格的RNA样品使用RNA反转录试剂盒(Transgen)进行反转录，根据产品说明书合成cDNA；
根据白梨基因组中PubZIP914的CDS序列设计克隆引物(F：ATGGCTTCTTCAAGCGGA；R：ATAGTATTGGTGAAGCAT)；通过PCR扩增目的基因，纯化回收，使用TA克隆将目的基因连到T载体上，转化至大肠杆菌DH5α感受态，挑取单克隆用M13引物进行菌液PCR验证，将阳性克隆菌液送至天津安升达生物科技有限公司测序获得基因序列，如序列表SEQ ID NO.1所示。
实施例3
设计载体构建引物(pCAMBIA1300-GFP-PubZIP914-F:gagaacacgggggactctagaATGGCTTCTTCAAGCGGA,pCAMBIA1300-GFP-PubZIP914-R:gcccttgctcaccatggatccATAGTATTGGTGAAGCAT,PSAK277-PubZIP914-F:tccaaagaattcaaaaagcttATGGCTTCTTCAAGCGGA PSAK277-PubZIP914-R:tcattaaagcaggactctagaATAGTATTGGTGAAGCAT)，将载体菌液活化后参照质粒提取试剂盒说明提取质粒，利用XbaⅠ、BamHⅠ和HindⅢ、XbaⅠ分别酶切质粒pCAMBIA1300-GFP和PSAK277形成线性片段，酶切产物纯化后用于后续的载体构建；
使用诺唯赞一步克隆试剂盒构建重组表达载体，转化至大肠杆菌感受态，挑取单克隆进行菌液PCR验证。提取阳性菌液中的重组质粒，使用热激法将重组质粒转入农杆菌GV1301中。
实施例4 PubZIP914在烟草中的亚细胞定位
将含有重组载体的GV1301在LB培养基中培养至0.8OD左右，室温下5000rmp离心，倒去上清液，菌体用加入乙酰丁香酮的MES缓冲液液等比例重悬，并在28℃，120rmp的条件下诱导4h，用1mL注射器吸取菌液从15天的烟草叶片背面注射。注射完成后将烟草置于黑暗中培养24h，随后转入光照培养箱培养2-3天。剪取注射区的烟草，并置于加入DAPI染料的超纯水中抽成真空状态，于超高分辨率激光共聚焦显微镜下观察荧光并拍照，以转入未构建载体的烟草叶片作为对照；
观察发现，在转入未构建载体的烟草叶片中，细胞核被DAPI染料染成蓝色，整个烟草细胞均观察到绿色荧光，而在转入pCAMBIA1300-GFP-PubZIP914融合表达载体的烟草细胞中，仅在细胞核中观察到绿色荧光蛋白，因此PubZIP914定位在细胞核(图3)。
实施例5 PubZIP914在南果梨中瞬时超表达
将含有PSAK277-PubZIP914重组质粒的菌液培养后重悬诱导，用1mL注射器吸取菌液注射进入成熟南果梨中，每个梨注射四个孔，八个梨为一个组，共注射三组。注射完成后将南果梨置于黑暗中培养24h，随后转入光照培养箱培养4天。南果梨去皮并切取菌液注射的果实，于液氮中冻样并置于-80℃保存。
实施例6PubZIP914转化梨愈伤
在超净工作台内将野生型梨愈伤切成碎末，置于含有pCAMBIA1300-GFP-PubZIP914过表达载体的GV1301农杆菌侵染液(OD值为0.4-0.5)中侵染10min，侵染条件为25℃，120rmp；侵染后用无菌滤纸吸干梨愈伤表面的液体，薄铺于共培养培养基上，2天后转入加有抗生素(150mg/L cephalothin,20mg/L Hygromycin B)和生长激素(1mg/L 2,4-Dand 0.5mg/L 6-BA)的MS培养基上，15天换一次培养基，直至长出新的愈伤。将新长出的愈伤置于新的抗生素培养基上扩大培养。
实施例7PubZIP914转化番茄
将野生型“小汤姆”番茄种子种植在无菌MS培养基上，待生长至13-15天，两片子叶展开后，切掉子叶的四边放置在MS培养基上暗培养1天。将含有pCAMBIA1300-GFP-PubZIP914过表达载体的GV1301农杆菌在水中重悬(OD值调至0.8)后加入200mM乙酰丁香酮，将子叶放在含有农杆菌的水中侵染10min，侵染条件为25℃，120rmp；侵染后用纱布滤去农杆菌并用无菌水清洗3遍子叶，用无菌滤纸吸干表面液体，叶片背面朝下转入共培养基，于25℃黑暗下培养48h；暗培养后将外植体转入分化培养基诱导愈伤和发芽，待分化的不定芽形成苗体后，切下苗体并转移至生根培养基以诱导生根。待植株生长稳定后，把番茄幼苗从组培瓶中取出，洗净其根部残留的培养基，栽培至营养基质土，在25℃，16h光照/8h黑暗的环境下培养。
实施例8转基因梨愈伤和转基因番茄验证
分别称取50mg转基因梨愈伤和1片转基因番茄嫩叶于液氮下研磨至粉末状态，置于2.0mL Rnase离心管中，做好标记，使用思科捷植物基因组DNA提取试剂盒提取植物基因组DNA。使用35S通用检测引物进行PCR验证转基因梨愈伤和番茄，以野生型梨愈伤和番茄作为阴性对照。PCR产物经琼脂糖凝胶电泳检测，发现转基因样本中扩增得到的目的基因与目的基因的条带大小一致，而野生型植株中没有扩增出目的条带，表明pCAMBIA1300-GFP-PubZIP914过表达载体成功转入梨愈伤和番茄中(图4)。图中WT为野生型梨愈伤阴性对照，line为阳性转基因植物。
实施例9阳性转基因梨愈伤和转基因番茄的收集
将鉴定为阳性的梨愈伤切成碎末，置于新的抗性培养基上扩大培养。取部分阳性梨愈伤液氮冻存，并于-80℃冰箱保存备用。将鉴定为阳性的转基因番茄在光照培养室培养至结果，待番茄果实转红后9天，摘取阳性转基因番茄的果实，取出成熟果实的种子，将果实的果肉置于液氮中冻存，并于-80℃冰箱保存备用(图5)。
实施例10挥发性成分萃取与分离：
分别称取-80℃保存的南果梨果实、转基因梨愈伤和转基因番茄果实各2g，研成粉末后转移至20mL螺纹口透明顶空瓶中，加入3mL 0.32g/mL的饱和NaCl溶液，并加入5μl内标溶液(0.82g/L 3-壬酮/甲醇混合溶液)，，用聚四氟乙烯丁基合成橡胶隔片密封；
利用顶空固相微萃取(HS-SPME)技术用于分离萃取挥发性成分。将顶空瓶放置在固相微萃取自动上样器(ThermoFisher,USA)中，于40℃加热震荡平衡20mi，并使用65μmPDMS/DVB萃取头(Supelco Co.,Bellefonte,PA,USA)萃取30min。萃取完成后萃取头被自动插入GC-MS(ThermoFisher TRACE 1310GC-TSQ 9000MS)，于250℃解吸1min。
色谱条件：色谱柱为TG-5MS毛细管柱，采取分流进样，进样口温度为250℃；升温程序，柱子起始温度为40℃，保持8min，以5℃/min的速率升至140℃，保持2min，再以10℃/min的速率，升至270℃，保持5min。载气为高纯氦气，流速为1.5mL/min，检测器温度为250℃，连接杆温度为280℃。质谱条件:电子轰击(electron impact，EI)电离源，电子能量70eV，灯丝电流为0.25mA，电子倍增器电压为1200V，扫描范围为33～370μ，离子源温度为230℃，传输线温度为280℃。
挥发性成分定性定量分析：
定性方法：未知化合物谱图经计算机检索同时与NIST 2017谱库相匹配，并结合人工图谱解析及保留指数比对进行初步分析。
定量方法：按峰面积归一化法求得各成分相对含量，并选择3-壬酮为内标进行定量。3-壬酮质量浓度为0.82g/L，用量为5μL。各组分的含量(μg/g)＝[各组分的峰面积÷内标的峰面积*内标质量浓度(g/L)×5μL]÷样品量(g)。
经分析结果如图，过表达PubZIP914能够显著提高南果梨果实、梨愈伤和番茄中的挥发物总含量，其中含量显著增加的挥发物成分主要是脂肪酸衍生物，如挥发性短链酯类和醛类。
在瞬时过表达的南果梨中，含量显著增加的挥发物以短链酯类为主，包括乙酸乙酯，丁酸乙酯，己酸乙酯，乙酸己酯等，还有少量的醛类化合物，包括己醛和反式-2-己烯醛(图6)。在超表达的转基因梨愈伤中，含量显著增加的挥发物以乙酸乙酯为主(图7)。在超表达的转基因番茄中，含量显著增加的挥发物以短链醛类为主，包括己醛，反式-2-己烯醛，反式-2-庚烯醛等(图8)。以上结果表明，过表达PubZIP914能够促进植物中挥发性脂肪酸衍生物的合成。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。
Claims (2)
1.梨bZIP类转录因子PubZIP914，和/或梨bZIP类转录因子PubZIP914编码的蛋白，和/或含有梨bZIP类转录因子PubZIP914的重组表达载体、表达盒、转基因细胞系或基因工程菌在如下（1）-（3）任一中的应用：
（1）在增加果实香气中的应用；
（2）在培育增加果实香气的座果园艺作物中的应用；
（3）在促进植物挥发性脂肪酸衍生物合成中的应用；
其中，所述果实、植物和园艺作物为番茄或梨；
所述bZIP类转录因子PubZIP914核苷酸序列如SEQ ID NO.1所示；
所述蛋白氨基酸序列如SEQ ID NO.2 所示。
2. 一种提高果实香气含量的方法，其特征在于，将梨bZIP类转录因子PubZIP914基因转入目的植物即可实现；所述果实和植物为番茄或梨；所述bZIP类转录因子PubZIP914核苷酸序列如SEQ ID NO.1所示。
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