CN103146709A - 云南红梨PybHLH基因及其原核表达载体和应用 - Google Patents
云南红梨PybHLH基因及其原核表达载体和应用 Download PDFInfo
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- CN103146709A CN103146709A CN2013100857034A CN201310085703A CN103146709A CN 103146709 A CN103146709 A CN 103146709A CN 2013100857034 A CN2013100857034 A CN 2013100857034A CN 201310085703 A CN201310085703 A CN 201310085703A CN 103146709 A CN103146709 A CN 103146709A
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Abstract
本发明公开了一种云南红梨花青素生物合成、毛状体发生发育及盐胁迫调控蛋白PybHLH基因及其原核表达载体，本发明利用RT-PCR技术从云南红梨云红梨1号红色果皮中克隆PybHLH基因，然后构建了原核表达载体pGEX-4T-PybHLH，pGEX-4T-PybHLH载体在大肠杆菌Rosetta(DE3)中进行表达PybHLH蛋白，纯化后，获得云南红梨PybHLH纯化蛋白，本发明还将PybHLH基因原核表达载体应用在制备PybHLH特异性抗体中，获得的PybHLH特异性抗体还用于研究蛋白质与蛋白质以及蛋白质与DNA的相互作用以及对PybHLH转基因植物的检测，本发明获得的PybHLH特异性抗体中含有谷胱甘肽S转移酶（GST），获得的PybHLH特异性抗体运用于FarWesternblotting和免疫共沉淀实验研究蛋白质相互作用以及准确分离获得与PybHLH相互作用的蛋白质和DNA片段。
Description
技术领域
本发明属于基因工程领域，具体涉及一种云南红梨花青素生物合成、毛状体发生发育及盐胁迫调控蛋白基因PybHLH及其原核表达和原核表达载体在制备PybHLH蛋白和特异性抗体中的应用。
背景技术
红色果皮是梨分子育种的重要性状指标。云南省因其独特的地理和气候条件而拥有较多的红皮梨种质资源，1986年以来先后已选育出早白蜜、美人酥、满天红和云红梨1号四个栽培品种。但生产中除云红梨1号外，其它3个品种都会因气候变化而导致着色较差甚至不着色，因此需要研究红梨果皮的着色机理。已有研究表明植物花青素的合成是由转录复合物MYB/bHLH/ WD40调控的。前人对MYB研究较多，而对植物中的bHLH和WD40蛋白研究较少。在拟南芥中LDOX基因启动子直接包括EGL3和TT8在内的不同的MYB-BHLH-TTG1转录复合物的调控，egl3、tt8和egl3 tt8功能缺失突变体试验也表明了egl3和tt8是拟南芥幼苗花青素累积的必要条件。在拟南芥中JAZ（Jasmonate ZIM-domain）蛋白与WD-repeat/bHLH/MYB 转录复合物中的bHLH（Transparent Testa8, Glabra3 [GL3]和 Enhancer of Glabra3 [EGL3]）和R2R3 MYB 转录因子(MYB75和Glabra1)互作，抑制了JA调控的花青素的累积和毛状体的发生，而JA诱导的JAZ蛋白的降解能够解除这种抑制。在大丽花(Dahlia variabilis)中，bHLH转录因子DvIVS通过调控DvCHS1, DvF3H, DvDFR和DvANS的转录来参与其花青素的生物合成。在葡萄中，bHLH转录因子MYCA1可能通过调控ANR和UFGT基因的表达来调控其花青素的生物合成。在龙胆花（G. triflora）中，GtMYB3和GtbHLH参与花青素的生物合成。Zhang等人通过酵母双杂交和植物过表达技术发现EGL3、GL3能够与TTG1(WD40)和MYB蛋白（GL1, PAP1、PAP2、CPC、TRY）组合，形成MYB/bHLH/WD40复合物进行包括花青素生物合成和毛状体形成的多功能的调控。Baudry等人通过遗传和分子的方法发现TT2 (MYB)、TT8 (bHLH)和TTG1(WDR) 能够形成四元复合物直接调控BAN在植物中的表达。Payne等通过酵母双杂交证实了在拟南芥中GL3/GL1/TTG1复合物调控毛状体的发育。Bouyer通过克隆分析、误表达和微注射试验提出了毛状体形成的捕获-耗尽机制：在高浓度的毛状体促进蛋白GL3细胞中， GL3通过结合毛状体形成蛋白TTG1来耗尽临近细胞中的TTG1，从而导致毛状体形成，而周围的细胞因TTG1的减少而不能形成毛状体。而Zhao等人通过离子轰击试验证明了TTG1，GL3，GL1和GL2并不在邻近细胞间转移，而R3-MYB，CPC则可在邻近细胞间转移，提出TTG1复合物直接调节激活子和抑制子，而抑制子的运动影响拟南芥叶上毛状体的类型。另外，植物中的bHLH转录因子也参与植物抗逆性机制，拟南芥中过表达OrbHLH2或OrbHLH001基因增强拟南芥的抗盐和抗冷的特性。
在云南红梨着色机理研究方面，本实验室研究了光照对云南红梨着色的影响、构建了光诱导果皮差异表达基因的SSH文库，进行了着色相关基因的分离和表达分析。前期研究结果表明云南红梨果皮花青素的生物合成是由MYB/bHLH/WD40调控的。前期分离克隆的PybHLH基因的部分片段（△PybHLH）构建的原核表达载体，由于该载体只含有PybHLH基因的一段序列而不是全长序列，获得的PybHHL抗体特异性不是很理想，不能准确的用于分离与PybHLH蛋白相互作用的蛋白质和DNA片段；在前人研究基础上，针对云南红梨栽培品种早白蜜、满天红、美人酥着色不稳定、在国内外尚未见有关云南红梨果皮红色着色机理方面研究报道，本研究选择着色最好且稳定的‘云红梨1号’作为实验材料，克隆了‘云红梨1号’中PybHLH基因的全长序列，并且进行了序列分析、原核表达和蛋白纯化抗体制备的研究，这将为研究云南红梨转录因子PybHLH的结构和功能奠定了基础，并为作物花卉育种提供有利的科学依据。
发明内容
本发明的目的在于提供一种云南红梨PybHLH基因，该基因主要是对云南红梨花青素生物合成、毛状体发生发育及盐胁迫调控蛋白起作用，PybHLH基因来源于云红梨1号，云南红梨PybHLH基因含有两个相连的基本亚区，即富含碱性氨基酸BASIC区域及其下游的HLH区域，其中碱性氨基酸基序与DNA的结合有关，对基因的转录发挥调控作用。
本发明另一目的是提供云南红梨PybHLH基因的原核表达载体pGEX-4T-PybHLH，该载体含有PybHLH基因，上游有Ptac启动子和细菌核糖体结合位点RBS，Ptac启动子的下游有可被IPTG诱导的操作子序列，PybHLH基因的N端含有GST标签。
本发明另一目的是将原核表达载体应用在制备云南红梨色素合成特异性调控蛋白PybHLH中。
本发明另一目的是将原核表达载体应用在制备PybHLH特异性抗体中，PybHLH特异性抗体应用在检测其在转基因植物中的表达情况、PybHLH蛋白的亚细胞定位检测、PybHLH蛋白的纯化并且在蛋白相互作用分析以及用于Far Western Blot和免疫沉淀（IP）及染色质免疫共沉淀（ChIP）中的应用。
为了实现本发明的上述目的，本发明提供了如下的技术方案：
1、原核表达载体的构建
（1）根据苹果MdbHLH33基因（GenBank登录号为DQ266451.1）编码框，设计1对特异引物PybHLH-F：5’-GGATCCatggctcagaatcatgagagggtg-3’和PybHLH-R：3’：CTCGAGgcacttaccagcaattttccaaagc，在上下游引物的5′端分别加入BamHI和XhoI酶切位点（下划线部分为酶切位点）。以云红梨1号总RNA为模板，用M-MLV反转录酶合成cDNA后使用PybHLH-F和PybHLH-R进行扩增；
（2）PybHLH基因全长片段的回收；
（3）PybHLH基因的T/A克隆和测序；
（4）PybHLH基因的生物信息学分析及GenBank登录号的获得；
（5）构建原核表达载体：使用BamHI和XhoI对pMD-18T-PybHLH质粒和pGEX-4T-1进行双酶切，分别获得5’和3’末端带有分别带有BamHI和XhoI酶切位点的PybHLH基因片段和pGEX-4T-1载体，琼脂糖凝胶电泳后分别进行回收，然后在16℃ 连接16 h，获得原核表达载体pGEX-4T-PybHLH；
2、PybHLH基因的原核表达
使用热刺激法将pGEX-4T-PybHLH质粒转入大肠杆菌Rosetta（DE3）中，在IPTG诱导下摸索最佳表达条件，在最适条件下进行大量表达；
3、PybHLH蛋白纯化及抗体制备
收集菌体，进行超声破碎，过GST琼脂糖亲和层吸柱进行纯化，收集纯化后的蛋白，纯化后的蛋白用于制备PybHLH蛋白特异性抗体、PybHLH蛋白表达检测。
本发明的有益效果：
本发明获得了PybHLH基因的全长序列以及重组原核表达载体pGEX-4T-PybHLH，通过表达获得纯化蛋白，制备特异性较强的PybHLH抗体，与前期制备的抗体相比，本次发明获得载体与抗体的优势更强，减少了在GST pull down和免疫共沉淀实验中出现的非特异性条带，使实验结果更加精确，并且得出更好的结论。本发明制备的抗体可用于检测PybHLH蛋白，弥补了目前没有专门检测PybHLH蛋白、蛋白相互作用分析的缺陷，另外，本发明的抗体还含有谷胱甘肽S转移酶（GST）标签，该抗体还可用于GST pull down、Far Western Blot和免疫沉淀（IP）及染色质免疫共沉淀（ChIP）中，研究与PybHLH蛋白相互作用的蛋白质和DNA片段，并且，该抗体含有的GST标签，也方便了后续实验。
附图说明
图1为本发明中云红梨1号的总RNA电泳示意图；
图2为本发明PybHLH基因的PCR结果示意图，图中：1是DNA MarkerIII；2和3为PybHLH基因PCR的结果；
图3为本发明中原核表达载体pMD18T-PybHLH的酶切结果示意图；图中：1是DNA MarkerIII；2、4、6为pMD18T-PybHLH质粒；3、5、7分别为泳道2、4、6中pMD18T-PybHLH质粒的BamHI和XhoI双酶切结果；
图4为本发明中 PybHLH蛋白在16℃、IPTG终浓度为1 mM条件诱导下的表达情况示意图，图中：1是转化pGEX-4T-1空载体的Rosetta菌体总蛋白；2-6是工程菌在16℃、IPTG终浓度为1 mM条件下诱导0、2、4、6和8 h后的表达情况；M是蛋白质分子量标准M0431；7-8是工程菌在16℃、IPTG终浓度为1 mM条件下诱导8 h，总蛋白破碎后目的蛋白在上清和沉淀中的表达情况；
图5为本发明中 PybHLH蛋白的割胶纯化示意图，图中：1的割胶回收后的PybHLH蛋白样品；2是蛋白质分子量标准M0441；
图6为本发明中PybHLH蛋白的Western Blot检测结果示意图；图中：1是是PybHLH蛋白，2是GST蛋白负对照；
图7为本发明中使用PybHLH抗体检测35S:: PybHLH转基因烟草叶片PybHLH蛋白表达结果示意图，图中：1，2，3，4是转基因株系，5是野生型对照；
图8为本发明中免疫共沉淀分析结果示意图，图中：1-3是免疫沉淀的PybHLH蛋白；4是用anti-GST进行免疫沉淀的负对照；
图9为本发明中Far Western blotting分析结果示意图，图中：1和3是PybHLH蛋白，2是GST为负对照；
图10为原核表达载体pGEX-4T-PybHLH的构建策略示意图。
具体实施方式
下面通过实施例对本发明作进一步详细说明，但本发明的内容并不局限于此，本实施例中方法如无特殊说明的均按常规方法操作，所用试剂如无特殊说明的采用常规试剂或按常规方法配置的试剂。
实施例中使用的试剂主要为分子生物学试验试剂：各种限制性内切酶、Pfu DNA聚合酶、RNA酶抑制剂、dNTP等为宝生物工程有限公司（大连，Takara）产品，反转录酶购于Promaga公司的，PCR产物纯化试剂盒、质粒提取试剂盒购自上海生工生物技术有限公司，大肠杆菌Rosetta (DE3)菌株和大肠杆菌DH5α感受态细胞为北京Transgene公司产品，其余试剂均为国产分析纯。
使用的仪器为GST琼脂糖凝胶柱子，其它仪器均为分子生物学以及基因工程实验室常用仪器设备。
所有引物序列合成在上海杰瑞生物科技股份有限公司进行，所有基因测序在北京六合华大基因科技股份有限公司进行。
实施例1：本发明云南红梨PybHLH基因的克隆、原核表达、纯化和抗体制备，具体步骤如下：
（1）引物设计
根据苹果MdbHLH33基因（GenBank登录号为DQ266451.1）编码框，设计1对特异引物PybHLH-F：5’-GGATCCatggctcagaatcatgagagggtg-3’和PybHLH-R：5’-CTCGAGgcacttaccagcaattttccaaagc-3’，在上下游引物的5′端分别加入BamHI和XhoI酶切位点（下划线部分为酶切位点）。
（2）总RNA的提取
a、用液氮将0.1 g云红梨1号红色果皮充分研磨成粉末以后，加入1 ml RNA提取缓冲液（4 M异硫氰酸胍，25 mM柠檬酸钠，0.5 % (w/v) 十二烷基肌氨酸钠，2 %(w/v) PVP，β-巯基乙醇），转入2 ml离心管中，再研磨均匀后，再加入1/10体积2 M醋酸钠(pH 4.2)；
b、颠倒离心管数次，混匀之后，接着加入等体积的氯仿：异戊醇(24:1)，盖紧离心管盖，剧烈摇动5 min，冰上静置15 min 后，4℃离心15 min (12000 g/min)；
c、离心结束后取上清于新离心管中，加入等体积的氯仿，盖紧离心管盖，剧烈摇动5 min，冰上静置15 min 后，4 ℃离心15 min (12000 g/min)；
d、取上清，加入等体积异丙醇，在-20℃沉淀RNA 30 min，再次4℃离心15 min (12 000 g/min)，让RNA沉淀在管壁；
e、RNA沉淀经75%乙醇清洗两次后抽真空干燥，然后加入20μl无RNase的DEPC处理水彻底溶解RNA；
f、使用1.2%的琼脂糖凝胶电泳检测RNA（见图1）。
（3）RT-PCR
以云红梨1号总RNA为模板，用M-MLV反转录酶进行反转录合成cDNA，具体步骤如下：
a、在EP 管中加入以下组分：总RNA 2μg；oligo(dT)18 0.5μg；无 RNase的水补足15 μl；
b、70℃ 5 min后，迅速冰上静置2 min；
c、离心数秒使模板 RNA/引物的变性溶液聚集离心底部；
d、再向离心管中加入以下组分：5×M-MLV反应缓冲液 5 μl、dNTP (10mM)、1.25 μl RNase 抑制剂0.5 μl、RTase M-MLV(200 U) 1 μl，无RNase的水补足25 μl；
e、42℃保温60 min，-20 ℃保存备用；
以反转录合成的云红梨1号cDNA后使用PybHLH-F和PybHLH-R进行PCR扩增，反应体系如下：
反应体系：ddH2O 17.8 μl，Taq Plus DNA Polymerase 10×Buffer 2.5μl，10 mM dNTP 0.4μl，10μM的5’引物1μl，10 μM的3’引物1 μl，cDNA 模板2 μl，5 U/μl的Taq Plus DNA Polymerase 0.3 μl；反应条件为：94 ℃ 2 min，(94℃ 30 s， 59℃ 30 s，72℃ 2 min) 30 个循环，72℃10 min（见图2）。
（4）PybHLH基因片段的回收：对PCR产物进行1 %琼脂糖凝胶电泳后，切下目的片段；使用胶回收试剂盒，按照试剂盒说明书对目的片段进行回收。
（5）PybHLH基因的T/A克隆和测序；将回收的PCR产物，按照pMD18T说明书，进行T/A克隆；转化大肠杆菌感受态DH5α后，涂固体LB + Amp平板，挑取白色菌落，接种到液体LB + Amp培养基中，37℃、200 rpm摇床过夜，提取质粒，进行酶切检测，然后送北京六合华大基因科技股份有限公司进行测序。
（6）PybHLH基因测序和生物信息学分析及GenBank登录号的获得：PybHLH基因的读码框为1947 bp，该基因编码648个氨基酸，分子量为72925.8 Da，等电点为5.54。DNAMAN比对结果表明，PybHLH与苹果、矮牵牛、拟南芥和玉米等调控花青素生物合成的bHLH转录因子具有很高的同源性，尤其是与苹果中的MdbHLH33在同一进化枝，故将该序列命名为PybHLH基因。
（7）原核表达载体的构建（构建策略见图10）：使用BamHI和XhoI对pMD18T-PybHLH和载体pGEX-4T-1按照说明书进行双酶切（见图3），分别获得5’端带有BamHI和3’端带有XhoI的PybHLH基因片段和pGEX-4T-1载体，跑电泳后分别进行胶回收，按照摩尔比目的基因：载体=3：1加样，然后加入Ligation Solution I，16℃连接16 h；将感受态大肠杆菌E.coli DH5α100μl加入6 μl连接体系中混匀；混合液冰浴30 min，42℃热刺激45s后， 冰浴2 min；加入900μl液体培养基SOC，37℃摇床200 rpm 60min 使菌体复苏；培养结束后，常温8000 rpm离心1 min收集菌体；在超净台上吸去上清，剩余约0.1 ml时，使用移液枪混匀，接入带有Amp抗性的LB固体平板上，用无菌三角棒涂布均匀；37℃过夜培养；挑取白色菌落，接种于LB液体 + Amp的培养基，37℃、180 rpm培养12 h后，提取质粒；进行酶切验证后，再送至北京六合华大基因科技股份有限公司测序进行测序验证插入基因的正确性，最后获得原核表达载体pGEX-4T-PybHLH。
（8）PybHLH蛋白的原核表达：使用热刺激法将重组质粒pGEX-4T-PybHLH转化入大肠杆菌Rosetta（DE3）感受态细胞中，涂LB+Amp固体平板，挑取pGEX-4T-PybHLH的重组菌落于LB + Amp液体培养基中37℃、200 rpm振荡培养过夜，按1：100的比例接种于相同的LB培养基上培养至OD600为0.6-0.8，加IPTG至终浓度为1 mM，在16℃下培养0、2、4、6和8 h后收集菌液用于分析总蛋白；4℃、12 000 rpm离心1 min，弃上清液，沉淀用100 μl SDS凝胶加样缓冲液(Tris-HCl 50 mM，pH 6.8；SDS 2 %；DTT 100 mM；溴酚蓝0.1 %；甘油10 %)重悬，煮沸5 min后，12 000 rpm离心1 min，取20 μl上清液进行SDS-PAGE检测。用考马斯亮蓝R-250染色液(0.1 %考马斯亮蓝R-250，40 %甲醇，10 %冰乙酸)染色，脱色液(25 %甲醇，6 %冰乙酸)进行脱色后，使用凝胶成像系统进行拍照；结果显示插入有外源片段的重组质粒pGEX-4T-PybHLH经IPTG诱导后，在预期的蛋白分子量约98.9 kD（包括载体上GST蛋白）左右有1条蛋白条带，而未诱导的转化重组质粒和pGEX-4T-1对照质粒未出现这条蛋白带（见图4），表明重组质粒pGEX-4T-PybHLH在大肠杆菌Rosetta（DE3）中诱导表达了PybHLH蛋白，超声破碎后，SDS-PAGE检测后，发现该蛋白主要在包涵体表达。
（9）PybHLH蛋白纯化：包涵体蛋白的割胶纯化，具体内容如下：
1、重组蛋白的大量表达及包涵体的洗涤
a、最优条件大量表达重组蛋白，超声波破碎；
b、离心12000rpm，4℃，30分钟，收集沉淀；
c、3M尿素洗涤沉淀，离心12000rpm，4℃，20分钟，加入适量8M尿素冰上溶解沉淀。
2、透析袋电洗脱法纯化蛋白
a、透析袋的预处理：剪取适当长度(10-20 cm)的透析袋(截留范围为8 000-12 000 Da)；置于大体积的2 %(W/V)碳酸氢钠和1 mM EDTA(pH8.0)中，煮沸10 min；蒸馏水彻底清洗透析袋；置于1mM EDTA(pH8.0)溶液中再次煮沸10 min；冷却后4 ℃保存；
b、取已溶解的蛋白沉淀，加1/5体积5×蛋白上样缓冲液进行SDS-PAGE电泳；
c、电泳结束后将胶放在0.25 M预冷的KCl中，4℃浸泡5分钟使蛋白显白色，蒸馏水冲洗蛋白胶，用刀片从SDS-PAGE电泳凝胶上切下目的蛋白，切成1 mm × 1 mm × 1 mm的碎片，放入预处理好的透析袋中，注入2 ml SDS-PAGE电泳缓冲液, 将透析袋前后封口；
d、电泳回收：在水平核酸电泳槽中加入适量SDS-PAGE电泳缓冲液，将装有凝胶的透析袋置入其中，100 V电压，4 ℃电泳4-5 h，直至凝胶变透明，说明目的蛋白从凝胶中洗脱出来；
e、透析：洗脱完毕，吸出透析袋内溶液，SDS-PAGE 检测（以10 μl 的BSA 标准蛋白定量）电泳结果（见图5），鉴定后正确的蛋白溶液装入干净的透析袋中，用预冷的 1×PBS溶液（1 × PBS（1L）KCl 0.2 g、NaCl 8 g、Na2HPO4 1.42 g、KH2PO4 0.27 g）添加尿素【8 M(96 g)、6 M(72 g)、4 M(48 g)、2 M(24 g)、0 M】于4 ℃下分别透析 3-5 h，其间换透析液2-3次；纯化后的蛋白溶液送抗体制备公司制备PybHLH蛋白特异性抗体，可用于PybHLH蛋白表达水平检测及进行免疫沉淀（IP）、Far Western blotting和染色质免疫共沉淀（ChIP）试验。
实施例2：PybHLH蛋白特异性抗体的Western blotting和转基因烟草的检测
为检测PybHLH蛋白特异性抗体的有效性，使用PybHLH蛋白抗体检测诱导的PybHLH原核表达蛋白和转基因烟草，具体过程如下：
本实施例使用的仪器为垂直蛋白电泳仪、PVDF膜（millipore），Semi-Dry Transfer Cell(BIO-RAD)转膜系统；
试剂为丙烯酰胺、二甲叉丙烯酰胺，10 %APS，TEMED，1×甘氨酸Buffer，1×PBS，1×PBT，脱脂奶粉，Whitman 3MM滤纸等SDS-PAGE和Western试验试剂。】、
A、PybHLH原核表达蛋白的Western blotting检测
蛋白样品制备：蛋白样品和1/5体积5×蛋白上样缓冲液（Tris-Cl（pH 6.8）250 mM，SDS 10 %，溴酚蓝0.5 %，甘油 50 %，β-巯基乙醇5 %），95℃加热5 min后置于冰上5-10 min，常温，13 000 rpm离心1 min；
一、SDS-PAGE
1、75 %酒精清洗玻璃胶板，组装电泳装置；按表1配制分离胶和浓缩胶，倒胶，小心用正丁醇或异丙醇覆盖；
表1： 分离胶和浓缩胶的配方
2、0.5 h胶凝固后，倒掉异丁醇，水冲洗，滤纸吸干；
3、按表1进行浓缩胶配制，倒胶，插上梳子，保证浓缩胶厚度(梳子底端与分离胶间距)达到5 mm以上；0.5-1 h凝固后即可电泳；
4、电泳：恒流，浓缩胶30 mA，分离胶40 mA。
二、Western操作
1、转膜
（1）剪刀剪取分离胶大小的PVDF膜、Whatman 3MM滤纸6张（8.2 cm × 5.8 cm）；
（2）将PVDF膜浸入甲醇约5秒，立即转移至20 ml Western blotting buffer（39 mM 甘氨酸，48 mM Tris，0.037 % SDS）中，同时放入分离胶，摇床20 min；
（3）用平头镊子夹3层滤纸用Western blotting buffer（转膜液）浸润后放置于Semi-Dry Transfer Cell（半干转膜仪），取出PVDF膜置于滤纸上，后将胶置于PVDF膜上；
（4）3层滤纸用Western blotting buffer浸润后置于分离胶上；
（5）设定电流为2.0 mA/cm2(100 mA)转膜，转膜1 h。
2、洗膜
（1）转膜完毕，将PVDF膜（8.2×5.8 cm2）转至20 ml的1×PBS，摇床5 min；
（2）弃PBS，加入20 ml封闭液液（1×PBT + 1 g脱脂奶粉），37℃摇床2 h；
（3）弃封闭液，加20 ml 1×PBT，室温摇床10 min；
（4）加一抗混合液（20 ml 1×PBT + 5 ul PybHLH特异性一抗+ 1 ml Blocking SolutionⅠ），室温摇床2 h；
（5）回收一抗混合液，加1×PBT室温摇床10-20 min；
（6）弃1×PBT，加入二抗混合液(20 ml 1×PBT + 1μl 商业化羊抗兔二抗，室温摇床1 h；
（7）弃二抗混合液；加入20 ml 1×PBT，摇床10 min，重复洗3次。
3、 结果观察
弃1×PBT，加1 ml工作液(500 μl稳定剂 + 500 μl发光底物，混匀)，浸润整个PVDF膜，转入成像系统Chemidoc XRS(BIO-RAD)，将成像系统上方调至0档，选择应用程序中的Chemi Hi Sensitivity选项进行成像，Western Blot检测结果如图6，PybHLH菌体总蛋白在大约99 kDa出现条带，与预期的结果一致，说明本发明的PybHLH特异性抗体能用于准确的检测PybHLH蛋白。
B、35S::PybHLH转基因烟草的检测
蛋白样品制备：蛋白样品和1/5体积5×蛋白上样缓冲液（Tris-HCl（pH6.8）250 mM，SDS 10%，BPB 0.5%，甘油 50%，β-巯基乙醇：5%），95℃加热5 min后置于冰上5 min，常温13 000 rpm、离心1min。
1、SDS-PAGE检测
（1）配制SDS-PAGE凝胶；
（2）电泳：需要恒流：浓缩胶30 mA，分离胶40 mA。
2、Western操作
（1）转膜：设定电流为2.0 mA/cm2，转膜40 min；
（2）封闭（5%的脱脂牛奶）；
（3）添加一抗（PybHLH抗体）；
（4）添加二抗（羊抗兔的商业化抗体）；
（5）结果观察
弃二抗，洗涤后，加1 ml工作液，浸润整个PVDF膜，使用成像系统Chemidoc XRS(BIO-RAD)进行成像观察，结果如图7，泳道1、2、3、4为转基因烟草总蛋白，5为野生型烟草的总蛋白，在转基因烟草中检测到PybHLH蛋白的表达，而在野生型对照中未检测到PybHLH蛋白，这表明本发明制备的多克隆抗体专一性强，适合进行PybHLH蛋白的表达检测。
实施例3：免疫共沉淀和Far Western blotting 分析PyMYB与PybHLH的互作
1、利用云红梨1号果皮可溶性蛋白（500ug）提取物用来进行免疫共沉淀分析，往果皮总蛋白溶液中加入5ug特异性抗体anti-PyMYB（PyMYB特异性抗体是本实验室完成，具体步骤如下：克隆获得云红梨1号PyMYB基因，构建原核表达载体，获得PyMYB重组蛋白；对获得PyMYB重组蛋白进行纯化，收集纯化的蛋白，收集纯化的PyMYB蛋白送抗体制备公司制备多克隆抗体）和anti-GST（GST抗体购于康为世纪），室温震荡孵育2h；然后往蛋白液中加入20ul蛋白A/G琼脂糖并且4℃震荡孵育过夜；孵育后的蛋白混合液4℃，3500 g离心5min；收集的蛋白沉淀物用遇冷的1×PBS洗三次，蛋白沉淀物最后溶解于40ul 1×电泳上样缓冲液中，取20ul在12%的聚丙烯酰胺凝胶上进行SDS-PAGE，分离后的蛋白通过半干转膜仪转移到PVDF膜上，膜首先用PybHLH特异性抗体处理并且与含有辣根过氧化物酶的羊抗兔和羊抗鼠lgG抗体结合反应，用凝胶成像系统Chemidoc XRS(BIO-RAD)进行观察，结果表明（见图8），泳道1、2、3为用anti-PyMYB免疫共沉淀后的总蛋白，泳道4为果皮为用anti-GST进行免疫沉淀的负对照，这表明本发明制备的多克隆抗体专一性强，适合运用在免疫共沉淀中进行PybHLH蛋白互作分析。
2、取50ug GST-MYB融合蛋白进行SDS-PAGE，然后在转膜缓冲液中电转移到硝酸纤维素（PVDF）膜上，转移到膜上的蛋白分别在浓度为6M、3M、1M、 0M盐酸胍AC buffer(10%Glycerol，0.1M NaCl，20mM Tris-HCl at pH 7.5，1mM EDTA，0.1%Tween-20，2%Milk powder，1mM DTT)中进行变性/复性；膜分别在含有6M、3M、1M盐酸胍的AC缓冲液中室温孵育2h，然后在0M盐酸胍的AC 缓冲液中4℃震荡孵育过夜；然后在含有5%脱脂奶粉的封闭液(50 mM Tris-HCl，pH 8.0，0.15M NaCl，0.02% Tween 20)中室温封闭2h；然后，PVDF膜放入分别含有30ul GST-PybHLH和GST探针的蛋白结合缓冲液中，4℃震荡孵育过夜；用anti-PybHHL多克隆抗体进行Western blotting分析；结果表明（见图9），泳道1和3为GST-PybHLH探针处理，2为GST探针处理，这表明本发明制备的多克隆抗体专一性强，适合运用在Far Western blotting中进行PybHLH与PyMYB互作分析。
SEQUENCE LISTING
<110> 昆明理工大学
<120> 云南红梨PybHLH基因及其原核表达载体和应用
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 30
<212> DNA
<213> 人工序列
<400> 1
gtcgacatgg ctcagaatca tgagagggtg 30
<210> 2
<211> 31
<212> DNA
<213> 人工序列
<400> 2
ctcgaggcac ttaccagcaa ttttccaaag c 31
<210> 3
<211> 1947
<212> DNA
<213> Pyrus Pyrifolia
<400> 3
atggctcaga atcatgagag ggtgccgggg aatctgagaa aacagtttgc tgttgctgtg 60
aggagtatta agtggagcta tgcaattttc tggtcattat caactgcaca acaaggggtg 120
ctggaatggg gtgaggggta ctacaatgga gacatcaaaa cccgaaaaac agttgaaggt 180
gtggaactta aaaccgataa aatgggttta cagaggaatg tgcaactcag agagctgtat 240
aagtctcttc tagaaggtga gactgagaca gagcggcaag ctaaagcgcc ttctggtgta 300
ttgtgcccgg aagatctcac agatgccgag tggtattact tgctttgcat gtccttcata 360
ttcaatcctg gcgaaggttt gcctggaaga gcattggcaa gtggccaaac catttggttg 420
tgcaatgctc aacatgcgga tagtaaagtt ttctcgcgct ctttgccggc gaagagtgca 480
tctgttcaga ctgtagtctg ctttccctac ctggggggtg ttgttgagct aggtgtgact 540
gagctggtat cggaggacct taatctcatt caacacatca aggcttcctt actagatttt 600
tcaaagcctg attgctgtga gaaatcttcc tctgcccctc acaaagcaga cgatgattca 660
gagcaaatag ttgccaaggt tgaccatgac gtagttgata cattgccttt agagaaccta 720
tattcccctt cggaagaaat caaatttgat cagaggggaa ttaacggttt acacggaatc 780
catgaagagg tcaacatgga ctcttctgat gaatgttcta atggttgtga tcacaatcat 840
cagacagaag actccatgat gcttgaaggt accaatgctg tggcttctca ggttcagagt 900
tggcatttca tggatgaaga cttcagcagc gggcgtcaag attccatgaa ttctagtgac 960
tccatatctg aagcttttgt taatcaagga aaggctcact cttttgctga acgtgagaat 1020
gtgaaccata tccatttaaa ggaacttcaa aacttcaatg acacaaaact aagttctttg 1080
tatcttggat ctgttgatga acatgtacac tacaaaagaa ctctttctac tcttctagga 1140
agctcaatga ggctgattga aaatccatgt ttttgcgacg gagagagcaa atccagtttt 1200
gtgaaatgga agaaagaagt tgttcgtagt tgtaggtcaa cagtacatca gaagacatta 1260
aagaagattt tgttcacagt tcctttaatg tatggtgttc gctctcgtat ggcaaccggt 1320
aaagaaaata cgggaaaaga tttgctccca aatttgcaag gtgacgatat taacagggaa 1380
catgaaaaaa ggagagagaa cgaaaaattg ttggtcctca ggtcgatggt tccttctatc 1440
actgaggttg atatccttga tgatacgatc aagtacttga aagagcttga ggcacgagca 1500
gaagagatgg aatcctgcat ggacaccgtg gaagctattt ctagagggaa attcctgaac 1560
agggtagagc agacatcaga taactatgat aaaacaaaga tgaacaatgt gaaaaagtct 1620
ttagtaaaga agagaaaggc ctgtgacatt gacaaaactg acccctatcc caatatgctt 1680
gtttccggag aaagcttgcc actagatgtg aaagtgtgcg taaacgagca agaggttctg 1740
atagagatga gatgccctta ccgggaatat atcttgcttg atataatgga tgccattaac 1800
aatctgtact tagatgcaca ctcggtccaa tcatccatcc ttgacggtgt tctcatgttg 1860
agccttaaat caaagtttcg aggagcggcg atttcaccag tggggatgat taagcaggcg 1920
ctttggaaaa ttgctggtaa gtgctga 1947
<210> 4
<211> 650
<212> PRT
<213> Pyrus Pyrifolia
<400> 4
Val Asp MET Ala Gln Asn His Glu Arg Val Pro Gly Asn Leu Arg Lys Gln Phe Ala Val
1 5 10 15 20
Ala Val Arg Ser Ile Lys Trp Ser Tyr Ala Ile Phe Trp Ser Leu Ser Thr Ala Gln Gln
25 30 35 40
Gly Val Leu Glu Trp Gly Glu Gly Tyr Tyr Asn Gly Asp Ile Lys Thr Arg Lys Thr Val
45 50 55 60
Glu Gly Val Glu Leu Lys Thr Asp Lys MET Gly Leu Gln Arg Asn Val Gln Leu Arg Glu
65 70 75 80
Leu Tyr Lys Ser Leu Leu Glu Gly Glu Thr Glu Thr Glu Arg Gln Ala Lys Ala Pro Ser
85 90 95 100
Gly Val Leu Cys Pro Glu Asp Leu Thr Asp Ala Glu Trp Tyr Tyr Leu Leu Cys MET Ser
105 110 115 120
Phe Ile Phe Asn Pro Gly Glu Gly Leu Pro Gly Arg Ala Leu Ala Ser Gly Gln Thr Ile
125 130 135 140
Trp Leu Cys Asn Ala Gln His Ala Asp Ser Lys Val Phe Ser Arg Ser Leu Pro Ala Lys
145 150 155 160
Ser Ala Ser Val Gln Thr Val Val Cys Phe Pro Tyr Leu Gly Gly Val Val Glu Leu Gly
165 170 175 180
Val Thr Glu Leu Val Ser Glu Asp Leu Asn Leu Ile Gln His Ile Lys Ala Ser Leu Leu
185 190 195 200
Asp Phe Ser Lys Pro Asp Cys Cys Glu Lys Ser Ser Ser Ala Pro His Lys Ala Asp Asp
205 210 215 220
Asp Ser Glu Gln Ile Val Ala Lys Val Asp His Asp Val Val Asp Thr Leu Pro Leu Glu
225 230 235 240
Asn Leu Tyr Ser Pro Ser Glu Glu Ile Lys Phe Asp Gln Arg Gly Ile Asn Gly Leu His
245 250 255 260
Gly Ile His Glu Glu Val Asn MET Asp Ser Ser Asp Glu Cys Ser Asn Gly Cys Asp His
265 270 275 280
Asn His Gln Thr Glu Asp Ser MET MET Leu Glu Gly Thr Asn Ala Val Ala Ser Gln Val
285 290 295 300
Gln Ser Trp His Phe MET Asp Glu Asp Phe Ser Ser Gly Arg Gln Asp Ser MET Asn Ser
305 310 315 320
Ser Asp Ser Ile Ser Glu Ala Phe Val Asn Gln Gly Lys Ala His Ser Phe Ala Glu Arg
325 330 335 340
Glu Asn Val Asn His Ile His Leu Lys Glu Leu Gln Asn Phe Asn Asp Thr Lys Leu Ser
345 350 355 360
Ser Leu Tyr Leu Gly Ser Val Asp Glu His Val His Tyr Lys Arg Thr Leu Ser Thr Leu
365 370 375 380
Leu Gly Ser Ser MET Arg Leu Ile Glu Asn Pro Cys Phe Cys Asp Gly Glu Ser Lys Ser
385 390 395 400
Ser Phe Val Lys Trp Lys Lys Glu Val Val Arg Ser Cys Arg Ser Thr Val His Gln Lys
405 410 415 420
Thr Leu Lys Lys Ile Leu Phe Thr Val Pro Leu MET Tyr Gly Val Arg Ser Arg MET Ala
425 430 435 440
Thr Gly Lys Glu Asn Thr Gly Lys Asp Leu Leu Pro Asn Leu Gln Gly Asp Asp Ile Asn
445 450 455 460
Arg Glu His Glu Lys Arg Arg Glu Asn Glu Lys Leu Leu Val Leu Arg Ser MET Val Pro
465 470 475 480
Ser Ile Thr Glu Val Asp Ile Leu Asp Asp Thr Ile Lys Tyr Leu Lys Glu Leu Glu Ala
485 490 495 500
Arg Ala Glu Glu MET Glu Ser Cys MET Asp Thr Val Glu Ala Ile Ser Arg Gly Lys Phe
505 510 515 520
Leu Asn Arg Val Glu Gln Thr Ser Asp Asn Tyr Asp Lys Thr Lys MET Asn Asn Val Lys
525 530 535 540
Lys Ser Leu Val Lys Lys Arg Lys Ala Cys Asp Ile Asp Lys Thr Asp Pro Tyr Pro Asn
545 550 555 560
MET Leu Val Ser Gly Glu Ser Leu Pro Leu Asp Val Lys Val Cys Val Asn Glu Gln Glu
565 570 575 580
Val Leu Ile Glu MET Arg Cys Pro Tyr Arg Glu Tyr Ile Leu Leu Asp Ile MET Asp Ala
585 590 595 600
Ile Asn Asn Leu Tyr Leu Asp Ala His Ser Val Gln Ser Ser Ile Leu Asp Gly Val Leu
605 610 615 620
MET Leu Ser Leu Lys Ser Lys Phe Arg Gly Ala Ala Ile Ser Pro Val Gly MET Ile Lys
625 630 635 640
Gln Ala Leu Trp Lys Ile Ala Gly Lys Cys
645 650
Claims (4)
1.云南红梨PybHLH基因，其特征在于：PybHLH基因的核苷酸序列如SEQ ID NO：3所示或编码如SEQ ID NO：4所示的氨基酸序列的蛋白质。
2.权利要求1所述云南红梨PybHLH基因的原核表达载体pGEX-4T-PybHLH，其特征在于：该载体含有PybHLH基因，上游有Ptac启动子和细菌核糖体结合位点RBS，Ptac启动子的下游有可被IPTG诱导的操作子序列，PybHLH基因的N端含有GST标签。
3.权利要求2的所述云南红梨PybHLH基因的原核表达载体在制备云南红梨色素合成特异性调控蛋白PybHLH中的应用。
4.权利要求2的所述云南红梨PybHLH基因的原核表达载体在制备PybHLH特异性抗体中的应用。
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| CN108642073A (zh) * | 2018-05-18 | 2018-10-12 | 南京农业大学 | 一种梨PbrRALF2蛋白质的体外表达及其多克隆抗体的制备方法 |
| CN109182292A (zh) * | 2018-09-25 | 2019-01-11 | 安徽农业大学 | 一种草莓谷胱甘肽转移酶FaGST基因及其表达蛋白和应用 |
| CN111499708A (zh) * | 2020-05-11 | 2020-08-07 | 中国科学院武汉植物园 | 葡萄VabHLH036基因在提高植物抗寒能力中的应用 |
| CN111733166A (zh) * | 2020-06-18 | 2020-10-02 | 中国农业科学院郑州果树研究所 | 刺葡萄花色苷合成基因VdbHLH037及其应用 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108165539A (zh) * | 2017-12-13 | 2018-06-15 | 南京农业大学 | 一种梨S7-RNase蛋白的体外表达方法及其多克隆抗体的制备方法 |
| CN108642073A (zh) * | 2018-05-18 | 2018-10-12 | 南京农业大学 | 一种梨PbrRALF2蛋白质的体外表达及其多克隆抗体的制备方法 |
| CN109182292A (zh) * | 2018-09-25 | 2019-01-11 | 安徽农业大学 | 一种草莓谷胱甘肽转移酶FaGST基因及其表达蛋白和应用 |
| CN109182292B (zh) * | 2018-09-25 | 2022-02-08 | 安徽农业大学 | 一种草莓谷胱甘肽转移酶FaGST基因及其表达蛋白和应用 |
| CN111499708A (zh) * | 2020-05-11 | 2020-08-07 | 中国科学院武汉植物园 | 葡萄VabHLH036基因在提高植物抗寒能力中的应用 |
| CN111499708B (zh) * | 2020-05-11 | 2022-04-05 | 中国科学院武汉植物园 | 葡萄VabHLH036基因在提高植物抗寒能力中的应用 |
| CN111733166A (zh) * | 2020-06-18 | 2020-10-02 | 中国农业科学院郑州果树研究所 | 刺葡萄花色苷合成基因VdbHLH037及其应用 |
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