CN114032220A - 与紫苏花青素生物合成相关蛋白及其编码基因与应用 - Google Patents
与紫苏花青素生物合成相关蛋白及其编码基因与应用 Download PDFInfo
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Abstract
本发明公布了与紫苏花青素生物合成相关的蛋白及其编码基因与应用。本发明提供一种蛋白,是如下(a)或(b):(a)有序列表中序列2所示的氨基酸序列提供的蛋白质;(b)将序列表中序列2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与植物中花青素含量有关的由序列2衍生的蛋白质。本发明的实验证明,本发明提供的PfANS蛋白及其编码基因,将该基因导入丹参中,得到转基因丹参,研究发现,与对照相比,转基因丹参花青素含量提高,叶片和叶柄呈现出紫红色,说明该蛋白及其编码基因在提高植物花青素方面具有重要的应用价值。本发明将在植物领域中具有广阔的应用空间和市场前景。
Description
技术领域
本发明涉及生物技术领域中与紫苏花青素生物合成相关蛋白及其编码基因与应用。
背景技术
紫苏属植物(Genus Perilla)系唇形科(Lamiaceae)野芝麻亚科 (Lamioideae)塔花族(Sature)紫苏亚族(Perillinae)单种属一年生草本植物,原产于东亚地区,在中国、日本和韩国广泛种植。紫苏(Perilla frutescens (L.)Britt.)是我国重要的药食同源植物之一,具有丰富的药用价值和食用价值。紫苏的叶子可以当中草药、蔬菜和香料使用,种子可以加工成食品和有营养的食用油。目前,由于其重要的经济价值,紫苏被引种到欧洲和北美等地区。我国是紫苏属植物的起源地之一,有丰富的种质资源和悠久的栽培历史,被认为是紫苏属植物多样性变化的中心。目前,我国紫苏栽培的主要区域是河北、四川、陕西、宁夏、甘肃、黑龙江、辽宁、安徽和湖北等省区,其余省区也有零星种植。现代药理学研究表明,紫苏具有保护肝脏、降血脂和降血压、抑制血小板聚集、减少血栓形成、预防癌变、提高记忆能力、抗炎、抗过敏及抗微生物等功效,是卫生部首批颁布的既是食品又是药品的“药食同源”植物之一,可以开发出多种保健食品,在医药、食品工业上具有广泛的用途。
紫苏叶含有多种营养成分,富含胡萝卜素、维生素C、B2、钾和铁等营养元素,有助于增强人体免疫功能。紫苏叶还含有大量的生物活性成分如挥发油、黄酮类、酚酸类和色素类物质,尤其是紫苏含有丰富的花青素,能够对抗氧自由基和超氧阴离子,具有抗衰老的作用。超高液相检测发现紫苏含有20多种花青素,其中锦葵色素、芍药色素和矢车菊色素是紫苏花青素的主要类型。花青素属于黄酮类化合物,在细胞质中合成并在液泡中积累,花青素的生物合成主要经过苯丙氨酸途径和类黄酮途径,最后在花青素合酶(ANS)的作用下催化无色的花青素转化为有色的花青素。ANS 是一种2-酮戊二酸依赖性酶,属于戊二酸依赖型加氧酶家族,是植物花青素生物合成途径中的一个关键酶。ANS基因的表达直接影响植物花青素的积累,研究表明降低ANS的表达会抑制花青素的生物合成,而过表达ANS 可以增加花青素的积累。
发明内容
本发明的一个目的是提供与紫苏花青素生物合成相关的蛋白及其编码基因。
本发明提供的与紫苏花青素物质生物合成相关的蛋白,名称为PfANS,来源于紫苏(Perilla frutescens L.),如下(a)或(b)的蛋白质:
(a)由序列表中序列2所示的氨基酸序列组成的蛋白质;
(b)将序列表中序列2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与植物组织中花青素含量有关的由序列2衍生的蛋白质。
所述序列2由362个氨基酸残基组成。
所述编码上述蛋白的DNA分子也属于本发明的保护范围。
所述DNA分子是如下(1)-(3)中任一种的DNA分子:
(1)编码序列为序列表中序列1所示的DNA分子;
(2)在严格条件下与(1)限定的DNA序列杂交且编码植物花青素含量相关蛋白的DNA分子;
(3)与(1)限定的DNA序列至少具有70%、至少具有75%、至少80%、至少具有85%、至少具有90%、至少具有95%、至少具有96%、至少具有 97%、至少具有98%或至少具有99%同源性且编码植物花青素含量相关蛋白的DNA分子。
所述序列1由1089碱基组成,其开放阅读框(ORF)为自5′末端起第1位 -第1089位碱基,编码氨基酸序列是序列表中序列2所示的蛋白。
上述严格条件可为用6×SSC,0.5%SDS的溶液,在65℃下杂交,然后用2×SSC,0.1%SDS和1×SSC,0.1%SDS各洗膜一次。
含有所述与紫苏花青素生物合成相关蛋白的编码基因的表达盒、重组表达载体、转基因细胞系或重组菌也属于本发明的保护范围。
所述重组表达载体是在载体pCambia1300-GFP的多克隆位点间插入所述编码基因得到的重组表达载体;
所述载体pCambia1300-GFP是通过包括如下步骤的方法得到的:
(1)将pCambia1300载体经过Sal I和Pst I双酶切,回收载体大片段;
(2)将GFP序列用PCR的方法扩增出来,上游引物5′端加Sal I酶切位点,下游引物5′端加Pst I酶切位点。构建到测序载体Zero Background pTOPO-Blunt Cloning Kit,经过Sal I和Pst I双酶切,回收包含GFP基因的片段;
(3)将步骤(1)中回收的载体大片段与步骤(2)中回收的包含GFP 基因的片段连接,得到重组载体pCambia1300-GFP。
所述pCambia1300载体购自CAMBIA公司。
扩增所述与紫苏花青素生物合成相关蛋白的编码基因全长或其任一片段的引物对也属于本发明的保护范围。
本发明的另一个目的是提供一种培育转基因植物的方法。
本发明所提供的培育转基因植物的方法,是将所述与花青素生物合成相关蛋白的编码基因导入目的植物中,得到花青素含量提高的转基因植物。
所述与紫苏花青素生物合成相关蛋白的编码基因是通过所述重组表达载体导入目的植物中。
所述目的植物为双子叶植物或单子叶植物;所述双子叶植物为丹参 (Salviamiltiorrhiza Bge)。
上述花青素含量提高由紫外分光光度计检测结果和叶片、叶柄颜色来体现。
本发明的实验证明,本发明提供了一种PfANS蛋白及其编码基因,将该基因导入丹参中,得到过表达PfANS基因的丹参植株,将转基因丹参植株进行扩繁培养一个月后,观察转PfANS基因丹参的形态变化,与对照相比,转基因丹参的叶片、叶柄颜色有明显的改变,叶片正反面和叶柄颜色由青绿色呈现出紫红色。因此,可以看出,PfANS基因及其所编码的蛋白在植物花青素物质生物合成过程中起着重要的作用。本发明所提供的PfANS蛋白及其编码基因在提高植物花青素含量研究中具有重要的应用价值。本发明将在农业领域具有广阔的应用空间和市场前景。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
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、与花青素生物合成相关的蛋白及其编码基因的获得及功能验证
一、与花青素生物合成相关的蛋白及其编码基因的获得
(一)紫苏(Perilla frutescens L.)PfANS蛋白cDNA的克隆
实验材料:以河北省农林科学院经济作物研究所审定的紫苏品种“冀紫 2号”为实验材料。
1、紫苏总RNA提取
取0.1g紫苏幼嫩叶片在液氮中研磨成粉状,加入2mL离心管,用 TIANGEN的RNAprep pure植物总RNA提取试剂盒(目录号:DP432)提取紫苏总RNA,试剂盒中包括:裂解液RL,去蛋白液RW1,漂洗液RW, RNase-Free ddH2O,RNase-Free吸附柱CR3,RNase-Free过滤柱CS,DNase I,缓冲液RDD,RNase-Free离心管,RNase-Free收集管。取10μL于1.0%琼脂糖凝胶电泳检测其完整性,另取2μL稀释至500μL,用紫外分光光度计检测其质量(OD260)和纯度(OD260/OD280),提取的冀紫2号总RNA,经非变性胶琼脂糖凝胶电泳检测,28S和18S条带清晰,且二者亮度比值为1.5~2 ︰1,表明总RNA没有降解,所得mRNA符合实验要求,可用于紫苏PfANS 蛋白cDNA全长的克隆。
2、PfANS蛋白cDNA的全长克隆
以本实验室构建的冀紫2号转录组数据进行PfANS基因的引物设计,进行PfANS蛋白cDNA的全长克隆。
(1)PfANS蛋白cDNA的全长克隆
根据紫苏转录组数据库获得PfANS基因的unigene序列,设计PfANS基因的5′-端和3′-端引物进行PCR反应。引物序列如下:
引物1:5’ATGGTTACGAGTGCAATGG 3’
引物2:5’CTAGTACGTTGGCTTCTCATCA 3’
PCR得到PfANS基因ORF全长,回收后连接pTOPO-Blunt载体进行TA克隆,以M13F/M13R通用引物进行测序。
以上述提取的总RNA经QuantScript RT Kit(TIANGEN,Beijing)反转录为模板,用高保真的FastPfu酶,进行PCR扩增。琼脂糖凝胶电泳检测PCR 扩增产物,获得1089bp长度的扩增片段。
经过测序,该PCR产物具有序列表中序列1所示的核苷酸,该序列所示的基因命名为PfANS,该基因的编码区为序列表中序列1自5’端第1-1089位核苷酸;序列表中序列1由1089个碱基组成;该基因编码的蛋白命名为PfANS,该蛋白的氨基酸序列为序列表中的序列2;序列表中序列2由362个氨基酸残基组成。
二、紫苏PfANS蛋白在提高植物花青素含量中的应用
1、植物表达载体的构建
根据紫苏PfANS蛋白cDNA的编码序列,设计扩增出完整编码序列的引物序列,正反向引物分别引入Kpn I和BamH I酶切位点,引物序列如下:
引物3:5’GGGGTACCATGGTTACGAGTGCAATGG 3’(下划线部分为 Kpn I酶切位点),
引物4:5’CGGGATCCCTAGTACGTTGGCTTCTCATCA3’(下划线部分为BamH I酶切位点)。
以人工合成的序列表中序列2为模板,PCR扩增后,将产物连接到 pTOPO-Blunt载体(购自北京艾德莱生物科技有限公司,产品目录号为 CV16)上,命名为pTOPO-PfANS载体,进行M13F/M13R的测序,保证紫苏PfANS蛋白cDNA的阅读框及酶切位点的正确。
用Kpn I和BamH I酶切表达载体pCambia1300-GFP,回收载体大片段,同时,用KpnI和BamH I酶切载体pTOPO-PfANS,回收约1.0kb中间片段,将回收的载体大片段与约1.0kb中间片段连接,得到目的质粒。将目的质粒转化大肠杆菌DH5a(购自北京全式金生物技术有限公司,产品目录号为CD201-01),37℃培养20h,进行重组载体的PCR分析和酶切鉴定,并进行测序验证。测序结果表明,在载体pCambia1300-GFP的Kpn I和BamH I酶切位点间插入了序列表中序列2自5′端第1位-第1089位所示的序列,说明重组载体构建正确,将重组载体命名为pC1300-PfANS。
所述pCambia1300-GFP载体是通过包括如下步骤的方法得到的:
(1)将pCambia1300载体(购自CAMBIA公司)经过Sal I和Pst I双酶切,回收载体大片段;
(2)将GFP序列用PCR的方法扩增出来,上游引物5′端加Sal I酶切位点,下游引物5′端加·I酶切位点。构建到测序载体Zero Background pTOPO-Blunt,经过Sal I和Pst I双酶切,回收包含GFP基因的片段;
(3)将步骤(1)中回收的载体大片段与步骤(2)中回收的包含GFP 基因的片段连接,得到重组载体pCambia1300-GFP。
2、植物表达载体转化农杆菌
(1)从-80℃低温冰箱中取出200μL EHA105感受态细胞(购自北京全式金生物技术有限公司),置冰上融化,加入1μg上述步骤1得到的植物表达载体pC1300-PfANS,混匀。
(2)液氮冷冻1min,37℃温育5min。
(3)加入800μL LB液体培养基,28℃培养2-6h。
(4)取100μL菌液至LB固体培养基上(含100μg/mL利福平(Rif)、 50μg/mL卡那霉素(Kan)),涂布均匀,将培养皿封口。倒置培养皿28℃培养2d。
(5)取PCR鉴定呈阳性的单菌落,接种到含有100μg/mL Rif、50μg/mL Kan的LB液体培养基中,28℃培养30h至对数生长期,取适量农杆菌用液体MS培养基稀释50倍备用,即得到导入pC1300-PfANS载体的农杆菌菌液。
3、转PfANS基因的丹参遗传转化及再生
用农杆菌介导的方法将PfANS的cDNA的编码序列导入到丹参中。具体方法如下:
(1)取经过继代培养4-6周的丹参无菌苗叶片,在超净台中切下5×5的丹参叶盘(去掉主叶脉),悬浮于上述步骤2制备好的EHA105/pC1300-PfANS 农杆菌菌液中,10分钟后将侵染后的丹参叶盘接种培养在固体培养基(1.0 mg/L 6-BA、0.1mg/L NAA的MS)上,28°黑暗共培养3天。
(2)将共培养3天后的丹参叶盘用含有500mg/L Car、1.0mg/L 6-BA、0.1 mg/L NAA的MS液体培养基洗涤2次后,将丹参叶盘转至含有1.0mg/L 6-BA、0.1mg/L NAA、100mg/LKam的固体MS培养基上进行选择培养,培养条件为28℃,每天13小时、3000lx的光照。培养4-6周后将丹参不定芽转移到含有1.0mg/L 6-BA、0.1mg/L NAA、100mg/L Kam的1/2MS培养基进行不定根诱导,培养条件为为28℃,每天13小时、3000lx的光照。4-8周后行成完整的再生植株,得到拟转PfANS基因的丹参植株。
(3)用CTAB法提取拟转基因丹参植株和丹参对照植株的基因组DNA。用常规方法进行PCR检测,所使用的PfANS基因引物为:引物1:5’ ATGGTTACGAGTGCAATGG 3’,引物2:5’CTAGTACGTTGGCTTCTCATCA 3’。在0.2mL Eppendorf离心管中加入 10×PCR buffer 2μL、4dNTP(10mol/L)1μL、引物(10μmol/L)均为1μL、模板DNA(50ng/μL)2μL、Taq DNA聚合酶1μL,加H2O至总体积20μL。反应程序为94℃变性4min,58℃复性1min,72℃延伸1min 30s,共35个循环。电泳检测扩增结果见图1(图1中,泳道M为Maker,泳道P为阳性对照(重组质粒pC1300-PfANS);泳道W为阴性对照水;泳道L1-泳道L5为转化PfANS基因的丹参拟转基因植株),从图中可见,泳道L1-L3、L5和阳性对照扩增出1089bp的目标条带,表明PfANS基因已经整合到丹参的基因组中,并证明这些再生植株为转基因植株;对照植株没有扩增出1089bp的目标条带。将经鉴定为转基因的丹参植株扩繁,进行丹参的形态学观察和花青素含量测定。
4、转基因丹参植物形态学观察
将3株过表达PfANS基因的丹参植株和对照丹参进行扩繁培养1个月后 (图2),如图2所示,转PfANS基因的丹参植株叶片正反面和叶柄处都呈现出紫红色,从丹参的形态学上说明过量表达PfANS基因能够改变丹参叶片和叶柄的颜色。
5、转基因丹参花青素含量测定
将上述转基因丹参植株液氮速冻研磨成粉,准确称取100mg,装入10 mL离心管中,加入5mL甲醇(含0.1%盐酸),震荡5次,每次20s,混匀后静置1min,涡旋1min,4℃下,12000r·min-1离心10min。取600μL上清液,加入400μL超纯水和1mL氯仿于10mL离心管中,涡旋1min,4℃下,12000 r·min-1离心2min,取600μL上清,加600μL甲醇(含0.1%盐酸)于2mL离心管中,上下颠倒混匀,紫外分光光度计测其在530nm和657nm的值,△ OD=OD530-0.25×OD657,计算花青素相对含量。如图3所示,转PfANS 基因能够提高丹参的花青素含量。因此,PfANS蛋白及其编码基因可以调控植物花青素的生物合成。
附图说明
图1为转基因植株的PCR检测(部分样品);
图2为转PfANS基因的丹参植株和对照;
图3为转基因丹参植株和对照花青素相对含量测定;
图3中:*表示与对照相比差异显著(P<0.05)
序列表
序列表1
ATGGTTACGAGTGCAATGGGTCCAAGCCCGCGGGTGGAGGAACTGGC CCGAAGAGGACTCAACACGATCCCAAAAGACTACGTGCGGCCCGAA GAAGAGCTGAAAAGTATCATAGGCGACATTTTGGCGGAAGAAAAGAA CAGCGAGGGGCCGCAGTTGCCGACGATCGATCTTGAAGAAATGGATT CAAGAGACGAGGAAAGGCGAAAGAAATGCCATGAGGAACTTAAGAT GGCGGCGACGGATTGGGGGGTGATGCACTTGATCAACCATGGTATACC CGAGGAGCTAATAGATCGTGTAAAGGCTGCGGGAAAGGAGTTTTTCG AGCTGCCCGTGGAGGAGAAGGAGGCATACGCCAACGATCAGGCAGC GGGGAACGTGCAGGGCTACGGCAGCAAGCTCGCTAACAACGCCAGT GGCCAGCTCGAGTGGGAGGACTATTTTTTCCATTGTGTTTACCCGGAA CACAAGACGGACTTGTCTATTTGGCCCACCAAACCACCTGATTATATA CCGGCTACAAGCGAGTATGCAAAGCAGCTAAGAGCCCTAGCAACCAA GATTTTATCAGTCCTATCAATCGGGCTAGGATTGGAAAAAGGTAGGCT AGAAAAAGAAGTTGGTGGTGCGGAGGATCTAATCGTCCAAATGAAGA TAAATTTTTACCCAAAATGCCCTCAGCCGGAGCTCGCCCTCGGCGTGG AAGCTCACACGGATGTAAGCGCCCTCACCTTCATCCTCCACAACATGG TTCCTGGCCTTCAGCTTTTCTATGAGGACAAATGGGTCACTGCAAAAT GTGTCCCCAATTCCATCATCATGCACATCGGAGACACGCTCGAGATTC TGAGTAACGGCAAATACAAGAGCATTCTGCACCGGGGGCTAGTCAAC AAGGAGAAGGTCAGGATTTCTTGGGCGGTTTTTTGTGAGCCGCCCAA GGAGAAAATCGTCCTACAGCCGCTACCTGAGACTGTCTCCGAGGTTG AGCCGCCACGCTTCCCGCCTCGGACGTTTGCTCAGCATCTCAAGCATA AGCTCTTCAGGAAGACTGATGGTGATCTTGATGAGAAGCCAACGTACT AG
序列表2
MVTSAMGPSPRVEELARRGLNTIPKDYVRPEEELKGIIGDILAEEKN SEGPQLPTIDLEEMDSRDEERRKKCHEELKMAATDWGVMHLINHGIPEE LIDRVKAAGKEFFELPMEEKEAYANDQAAGNVQGYGSKLANNASGQLEWEDYFFHCVYPEQKTDLSIWPTKPPDYIPATSEYAKQLRALATKILSVL SIGLGLEKGRLEKEVGGAEDLIVQMKINFYPECPQPELALGVEAHTDVS ALTFILHNMVPGLQLFYEDKWVTAKCVPNSIIMHIGDTLEILSNGKYKSILHRGLVNKEKVRISWAVFCEPPKEKIVLQPLPETVSEVEPPRFPPRTFAQ HLKHKL
Claims (10)
1.一种蛋白,是由序列表中序列2所示的氨基酸序列组成的蛋白质。
2.编码权利要求1所述蛋白的基因。
3.如权利要求2所述的基因,其特征在于:所述基因的编码序列为序列表中序列1所示的DNA分子。
4.含有权利要求2或3所述编码基因的重组载体、表达盒、转基因细胞系或重组菌。
5.如权利要求4所述的重组载体,其特征在于:
载体为将专利要求1所述蛋白的编码基因插入表达载体中,得到表达权利要求1所述蛋白的重组载体。
6.扩增权利要求2或3所述基因全长的引物对。
7.权利要求1所述蛋白、权利要求2或3所述编码基因或权利要求4所述重组载体、表达盒、转基因细胞系或重组菌在调节植物中花青素含量中的应用;
所述植物具体为药用植物。
8.一种培育转基因植物的方法,为将权利要求1所述蛋白的编码基因导入目的植物,获得转基因植物,所述转基因植物组织中花青素含量高于所述目的植物。
9.根据权利要求8所述的方法,其特征在于:所述组织为叶片;
权利要求1所述蛋白的编码基因通过权利要求4或5所述的重组载体导入目的植物。
10.根据权利要求8或9所述的方法,其特征在于:所述目的植物为药用植物。
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