CN111254150A - 一种银杏GbFLSa基因及其表达蛋白和应用 - Google Patents
一种银杏GbFLSa基因及其表达蛋白和应用 Download PDFInfo
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- CN111254150A CN111254150A CN202010092212.2A CN202010092212A CN111254150A CN 111254150 A CN111254150 A CN 111254150A CN 202010092212 A CN202010092212 A CN 202010092212A CN 111254150 A CN111254150 A CN 111254150A
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Abstract
本发明公开了一种银杏GbFLSa基因及其表达蛋白和应用,属于植物基因工程技术领域。银杏GbFLSa基因的核苷酸序列如SEQ ID No.1所示,其表达蛋白的氨基酸序列如SEQ ID No.3所示,通过将银杏的GbFLSa基因转基因到山新杨中,发现转基因杨树中儿茶素、表儿茶素、表没食子儿茶素、没食子儿茶素等原花青素含量显著降低,此外,DFR、ANS、LAR酶基因的表达水平也显著低于对照。表明GbFLSa编码的是一种功能蛋白,对原花青素的生物合成起负调控作用。有助于揭示GbFLSa在植物代谢中的作用,更好地了解类黄酮生物合成的潜在分子机制。
Description
技术领域
本发明属于植物基因工程技术领域,更具体地说,涉及一种银杏GbFLSa基因及其表达蛋白和应用。
背景技术
黄酮广泛分布于植物界,其根据C环氧化和饱和程度的不同,可以分为不同的亚族,是具有代表性的重要次生产物之一。种类丰富的黄酮类化合物都是由15个碳原子构成,排列成C6-C3-C6的构型,即两个芳环中间与一个成环的C3联结起来。众所周知,植物黄酮类化合物的生物合成是一个复杂的过程,涉及多种重要酶类。其合成以香豆酰CoA和丙二酰CoA为前体,在查尔酮合酶(chalcone synthase,CHS)的作用下形成黄色的查尔酮,这一步是类黄酮合成途径的第一个限速步骤。然后由查尔酮异构酶(chalconeisomerase,CHI)催化查尔酮生成无色的柚皮素,柚皮素经黄烷酮3-羟化酶(flavanone 3-hydroxylase,F3H)催化,生成二氢山奈酚类(DHK),DHK随后分形成二氢槲皮素(DHQ)和二氢杨梅素(DHM)。上述3个二氢黄酮醇类化合物可在黄酮醇合成酶(flavonol synthase,FLS)作用下进入黄酮醇合成途径,产生山奈酚、槲皮素和杨梅素;也可由二氢黄酮醇还原酶(DFR)催化进入花青素合成途径。由于黄酮醇合酶(flavonol synthase,FLS)和DFR催化反应的底物相同,因此存在竞争关系。其中FLS是合成下游途径中的一个关键酶,它属于依赖铁离子和2-酮戊二酸的双加氧酶(2-oxoglutarate-dependentdioxygenase,2-ODD)。FLS作为类黄酮合成途径和儿茶素合成途径连接中间桥梁,在植物中的保守度很高。目前已经在矮牵牛、烟草、拟南芥和水稻等植物中研究。
银杏是多年生的落叶乔木,被公认为“活化石”,是现存种子植物中最古老的孑遗植物。银杏也是一种典型的雌雄异株裸子植物,具有很高的药用、经济和生态价值。银杏叶片中含有的黄酮类化合物,对治疗老年性痴呆症,高血压和心血管疾病等有很好的疗效。且黄酮含量是衡量银杏叶提取物制剂质量的关键因素之一,如何提高银杏叶黄酮的含量已经成为研究的热点之一。目前,针对银杏类黄酮的研究主要是集中在提取工艺和药理作用等方面,且提高银杏类黄酮产量的方法以激素调控和培养条件优化等为主。通过针对次生代谢途径的遗传调控来增加类黄酮的产量是提高黄酮类化合物产量的潜在策略。主要由于银杏缺乏组培和遗传转化体系,基因组较大,尚不能关联到染色体层面,且功能基因组学研究不深入。这使得银杏黄酮生物合成中分子机制和具体的次生代谢途径研究尚不清晰。
目前,FLS已在模式植物中被证实与生物体内类黄酮的积累有关,然而,在银杏中仅对GbFLS进行了原核表达和体外活性检测研究,还没有针对银杏FLS的关键酶基因进行植物体内功能的研究。
发明内容
针对现有技术存在的上述问题,本发明所要解决的技术问题在于提供一种银杏GbFLSa基因;本发明所要解决的另一技术问题是提供银杏GbFLSa基因的表达蛋白;本发明所要解决的再一技术问题是提供银杏GbFLSa基因的应用。
为了解决上述技术问题,本发明所采用的技术方案如下:
一种银杏GbFLSa基因,其核苷酸序列如SEQID No.1所示。
SEQID No.1所示的核苷酸序列经过一个或几个核苷酸取代和/或缺失和/或添加且与原花青素合成相关的基因也属于本发明的保护范围。
银杏GbFLSa基因的表达蛋白,其氨基酸序列如SEQ ID No.3所示。
含有银杏GbFLSa基因的载体、重组菌或宿主细胞。
银杏GbFLSa基因在调控原青花素的生物合成中的应用。
一种制备原花青素含量降低的转基因植物的方法,包括以下步骤:将银杏GbFLSa基因导入目的植物,培养筛选得到原花青素含量降低的转基因植物。
进一步的,银杏GbFLSa基因通过重组表达载体导入目的植物。
进一步的,重组表达载体为Pro35S::GbFLSa。
相比于现有技术,本发明的有益效果为:本申请利用RACE技术从银杏中分离了GbFLSa基因。GbFLSa的全长cDNA序列为1314bp,包含的1092bp ORF编码了363个氨基酸,通过PCR扩增GbFLSa cDNA的ORF,利用Gateway技术(Invitrogen,CA,Carlsbad)将位于CaMV35S启动子下游的ccdB基因克隆到PBI121(含有HA标签)载体上。将含有Pro35S::GbFLSa的载体导入农杆菌菌株EHA105中进行转化。利用山新杨稳定和高效的遗传转化体系,将银杏的GbFLSa基因转基因到山新杨中。发现转基因杨树中儿茶素、表儿茶素、表没食子儿茶素和没食子儿茶素的含量显著低于对照。此外,DFR、ANS和LAR这3个酶基因的表达量也均显著低于对照。过量表达GbFLSa对原花青素(儿茶素、表儿茶素、表没食子儿茶素和没食子儿茶素)的生物合成具有负调控作用。
附图说明
图1是GbFLSa蛋白结构域分析及系统发育分析图;图中,A为银杏GbFLSa蛋白结构域的NCBI保守分析图;B为GbFLSa和其他FLSs蛋白的系统发育分析图;其中,GenBank的登录号如下:Populus euphratica(XP_011001283.1),Populus trichocarpa(XP_002325697.1),Populus alba(TKS05742.1),Fagopyrum dibotrys(AHN19765.1),Fagopyrum esculentum(AEC33115.1),Fagopyrum tataricum(AEC33116.1),Allium cepa(AQR58516.1),Malus domestica(XP_028949987.1),Prunus avium(XP_021810338.1),Picea sitchensis(ABK26270.1),Pinus tabuliformis(AHW42460.1);
图2是GbFLSa的qRT-PCR表达分析图;图中,A为GbFLSa在不同组织中的相对表达量图;B为GbFLSa基因在4-10月银杏叶片中的相对表达量图;
图3是GbFLSa原核表达分析图;SDS-PAGE凝胶(A)和western blot(B)分析pET-30a(+)中克隆的GbFLSa,并在BL21(DE3)菌株中表达;Lane M1:蛋白质分子量标准marker;LaneM2:免疫印迹marker;Lane PC1:BSA(1μg);Lane PC2:BSA(2μg);Lane NC:细胞未诱导裂解;Lane 1:15℃诱导细胞裂解16h;Lane 2:37℃诱导细胞裂解4h;Lane NC1:未诱导的细胞裂解液上清;Lane3:细胞裂解液的上清液,15℃诱导16h;Lane 4:细胞裂解液上清,37℃诱导4h;Lane NC2:未诱导的细胞裂解物碎片;Lane 5:细胞裂解物的碎片在15℃下诱导16h;Lane 6:细胞裂解物的碎片在37℃下诱导4h;Western blot的主要抗体是anti-Hisantibody;箭头指向为GbFLSa;
图4是GbFLSa-GFP融合蛋白与35S:GFP对照在杨树叶肉原生质体中的瞬时表达图;比例尺=10μm;GFP:为GFP荧光;Auto为叶绿素自发荧光;Bright为明场;Merged 1为GFP和Auto的合并;Merged 2=Merged 1+明场;
图5是非转基因杨树(WT)和转基因杨树的比较图;图中,A为非转基因杨树(WT)和8个不同的GbFLSa转基因杨树株系转录水平的相对定量分析图;B为30日龄WT与转基因杨树表型的比较图;C为非转基因杨树(WT)和3个独立转基因株系的不定根数的比较图;D为非转基因杨树(WT)和3个独立转基因株系的最大不定根长的比较图;E为非转基因杨树(WT)和3个独立转基因株系的株高的比较图;
图6是非转基因与转基因杨树组间原花青素相关代谢产物含量(A)和酶基因相对表达量(B)图;图中,DFR:二氢黄酮醇还原酶;ANS:花青素合成酶;LAR:无色花青素还原酶。
具体实施方式
下面结合具体实施例进一步说明本发明,但这些实施例并不用来限制本发明。
以下实施例所采用的植物材料及其生长条件为:以25年生银杏(叶、茎、根、叶柄、种仁、芽)为材料,进行GbFLSa基因的组织表达研究。以1年生银杏幼苗为材料,在不同的叶片发育阶段,从4月到10月每月采集一次叶片,研究该基因的表达模式。收集后,将植物材料快速冷冻在液氮中,置于-80℃的超低温冰箱中。
此外,无性系山新杨(Populus davidiana Dode×Populus bolleana Lauchecv.,clone“Shanxin Yang”)组培苗在16h光照和8h暗光周期下和25℃(白天)和18℃(夜晚)条件下进行生长。
实施例1
1、通过RACE技术克隆GbFLSa基因
采用植物基因组DNA试剂盒Plant Genomic DNA Kit(cetyltrimethylammoniumbromide)(Zoman,Beijing,China)进行银杏DNA的提取。采用RNAprep Pure Plant kit(Polysaccharides&Polyphenolics-rich,TIANGEN,Beijing,China)从银杏叶中提取总RNA。基于银杏转录组数据(NCBI Short Reads Archive database under access numberSRP137637)设计GbFLSa基因的特异性引物,利用快速扩增cDNA末端(RACE)技术克隆GbFLSa全长cDNA序列。嵌套引物设计用于扩增全长cDNA采用SMATer RACE 5′/3′Kit(Clontech,Palo Alto,CA,USA)试剂盒。所有引物均采用Oligo 6.0软件设计(表1)。通过PCR扩增,切胶回收纯化,经pMD19-T载体转入大肠杆菌感受态细胞。采用PCR检测菌落,选择阳性菌落进行Sanger测序。通过拼接5′和3′-RACE序列得到GbFLSa的全长,并利用NCBI ORF Finder预测开放阅读框。之后,GbFLSa ORF在以下程序中PCR扩增:95℃反应3min;35循环下的95℃30s,55℃ 40s,72℃ 90s:最后在72℃下延长10min。PCR产物经1%琼脂糖凝胶电泳检测,目的片段经琼脂糖凝胶DNA纯化试剂盒回收(TaKaRa,Dalian,China),然后将目标片段连接到pMD19-T载体上,转化到大肠杆菌TOP 10中。取单菌落进行培养。筛选阳性克隆送Sanger测序。
采用BioEdit进行DNA和蛋白序列分析。用ExPASy ProtParam(http://web.expasy.org/protparam/)对其理化性能进行了预测。利用在线SOPMA(https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html)对GbFLSa基因的二级结构进行预测。利用在线BLAST(https://blast.ncbi.nlm.nih.gov/)工具获得了同源比对的相似序列。利用DNAMAN软件对推导出的GbFLSa氨基酸序列和FLSs同源蛋白序列进行多重比对。此外,采用极大似然法,分析FLSs氨基酸序列,并利用MEGA7.0软件进行1000次自举复制构建系统发育树。
通过RACE技术分离了GbFLSa基因。经测定GbFLSa的全长cDNA序列为1314bp,包含的1092bp ORF编码了363个氨基酸。GbFLSa蛋白预测分子量为40.93kDa,理论pI为6.14,失稳指数为34.33,这将蛋白质归类为稳定的。脂肪族指数为92.12,总平均亲水性为-0.261。
NCBI保守域数据库检索发现,GbFLSa属于2OG-Fe(II)加氧酶超家族(图1A)。该家族包括2-氧戊二酸盐(2OG)和铁(II)依赖性双加氧酶超家族成员。将推导出的GbFLSa氨基酸序列与其他FLS蛋白序列进行比较,发现GbFLSa具有保守性。多重序列比对表明,所有这些FLS氨基酸序列都有2-氧戊二酸结合残基、铁结合残基和推测的DHQ结合残基,它们也有负责FLS多肽适当折叠的残基。系统发育分析表明,GbFLSa与裸子植物的北美云杉和油松同源(图1B)。此外,同一被子植物科的所有成员都归在一起。这些结果表明,系统发育分析与物种间的遗传关系吻合良好。
2、实时荧光定量PCR分析
用RNAprep植物试剂盒(TIANGEN,Beijing,China)提取总RNA,然后用PrimeScriptTMRT Master Mix(TAKAEA,Dalian,China)转录总RNA。将cDNA作为模板稀释3倍。用于qRT-PCR扩增的GbFLSa引物和内参比基因引物见表1。在ABI ViiA 7Real-time PCRplatform使用FastStart Universal SYBR Green Master with ROX for RT-PCR Kit(Roche,Indianapolis,IN,USA)进行qRT-PCR分析。10μL反应体系和PCR反应程序如下:95℃反应2min;40个循环下反应95℃ 15s和95℃ 1min。
表1 GbLSa基因克隆,qRT-PCR和载体构建的引物
利用qRT-PCR检测了GbFLSa在不同时间和不同部位的表达谱(图2)。如图2所示,除了根外,GbFLSa在茎、叶、种仁、芽和叶柄中均有表达(图2A)。GbFLSa在茎和叶中的表达水平显著高于其他组织。此外,在4-10月的叶片发育过程中GbFLSa基因均有表达(图2B)。相对表达水平在4月达到顶峰,并在4月至8月期间开始下降,九月达到次峰值。
3、质粒的构建、转化、原核表达和亚细胞定位
原核表达:通过克隆得到基因的全长序列,将基因的ORF序列进行优化基因合成并将其复制到大肠杆菌表达的目标载体pET-30a(+)上,亚克隆方案:Ndel-ATG-His tag-FLS基因-Stop codon--Hindlll。然后从超低温冰箱取出BL21(DE3)感受态细胞,于冰上融化后加入100ng目标基因质粒,轻轻吹打混匀后,冰浴30min,42℃热击90s,冰上放置3min后,再加入100μL液体LB,37℃ 200rpm培养60min,4000rpm离心10min,留100μL菌液和菌体吹打混匀后,涂至Kan抗性平板倒置37℃过夜培养。分别挑选3个单克隆,接种于4mL 50μg/mL kan的LB液体中,摇床37℃ 200rpm培养至OD600=0.7,向2个试管中分别加0.5mM IPTG,15℃培养16h和37℃培养4h,最后一支试管为阴性参照。采用SDS-PAGE检测蛋白质的表达和溶解度。
全菌样品:取200μL培养基离心后的沉淀,重悬于5×loading buffer(30%glycerol,10%SDS,300mM Tris,250mM DTT),100℃加热10min,7000rpm离心5min。
上清和包涵体样品:取300μL培养基离心后的沉淀,用100μLBug Buster ProteinExtration Reagent试剂,室温孵育10min。15000rpm离心10min,分别取上清和沉淀。混合25μL 5×loading buffer到100μL上清,作为上清样品。用50μL 5×loading buffer重悬沉淀,作为包涵体样品。100℃加热10min后,15000rpm离心5min。
亚细胞定位的方法:将不含终止密码子的GbFLSa编码区域克隆到载体pCRTM8/GW/TOPOTM(Invitrogen,Carlsbad,CA,USA)中,进行简单的TOPO克隆反应。对标记蛋白的亚细胞定位,使用LR克隆酶将插入物GbFLSa片段从入门载体转移到目的载体(p2GWF7),并在插入物的C端放置绿色荧光蛋白GFP标记。生成的GFP融合载体(35S::GbFLSa-GFP)为双35S花椰菜花叶病毒(CaMV)启动子驱动的高拷贝载体,以氨苄青霉素为细菌选择筛选标记。原生质体分离和PEG介导转染如参照Tan et al.(2013)所述。所有荧光实验均独立重复三次。
经IPTG诱导的pET-30a(+)GbFLSa重组质粒在E.coli BL21中成功表达(图3),分子质量约为41kDa,与预期的目标蛋白大小一致(40.93kDa)。菌体经超声波破碎后,分别收集上清和沉淀进行SDS-PAGE电泳分析,发现沉淀中有明显的蛋白特异条带,上清中的特异条带较弱,表明该重组蛋白主要以包涵体形式存在。SDS-PAGE结果显示(图3),在E.coli BL21(DE3)中成功诱导表达了GbFLSa蛋白,与预期蛋白分子量大小一致,最佳表达条件是15℃,诱导16h,为进一步研究GbFLSa的功能提供实验依据。纯化的重组GbFLSa蛋白的westernblot(图3)结果证实了其对His抗体的特异性免疫反应。
将对照35S:GFP和GbFLSa-GFP转化为山新杨叶肉原生质体。通过共聚焦显微镜,我们发现GbFLSa-GFP融合蛋白主要定位于细胞质内(图4),因此GbFLSa是一种细胞质蛋白。
4、GbFLSa在杨树中的异源过量表达
通过PCR扩增GbFLSa cDNA的ORF,利用Gateway技术(Invitrogen,CA,Carlsbad)将位于CaMV 35S启动子下游的ccdB基因克隆到PBI121(含有HA标签)载体上。将含有Pro35S::GbFLSa的载体导入农杆菌菌株EHA105中进行转化。利用山新杨稳定和高效的遗传转化体系,将银杏的GbFLSa基因转基因到山新杨中。采用卡那霉素(Kan)抗性筛选后,随机选择3个非转基因杨树和8个不同的转基因杨树株系叶片进行qRT-PCR测定并观察同一时期转基因苗和非转基因苗的生长状况。
通过qRT-PCR对转基因植株和未转基因植株中GbFLSa的表达量进行检测,如图5A,GbFLSa在未转基因杨树中并没有检测到其表达,GbFLSa相对于内参基因的表达量在0.00036~0.02129范围内,其中表达量最高的为L5株系,其次为L7和L2。确定GbFLSa成功整合到受体植物基因组中,并成功表达。对转基因植株与未转基因植株进行表型观测,如图5B所示,生长1个月左右的GbFLSa转基因山新杨与未转基因山新杨的表型存在一定的差异,整体而言,转基因植株比未转基因杨树的长势好,尤其是L5株系(L5的株高显著高于对照)。通过统计分析得出转基因植株L2,L5和L7株系的最大不定根长均极显著大于非转基因苗(F=11.931,P=0.003)(图5D)。
以上说明对本发明而言只是说明性的,而非限制性的,本领域普通技术人员理解,在不脱离所附权利要求所限定的精神和范围的情况下,可做出许多修改、变化或等效,但都将落入本发明的保护范围。
序列表
<110> 南京林业大学
<120> 一种银杏GbFLSa基因及其表达蛋白和应用
<130> 1
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1314
<212> DNA
<213> GbFLSa的全长cDNA(银杏)
<400> 1
cattggcatt ggcatgggtt cgaaacacat ggtaagagtg caaacacttg cagagagtgg 60
aatccaaact attccacgaa agtatgctag ccgtcttgac attgacaaag cagaagctaa 120
ggcgacaaag gttgaagaag attcaaccgc agatcttccc ataatagaca tatctgattt 180
gaacaataca aatacagttg cccaaattgt caaagcagcc aaagagtggg gtttcttcca 240
gattatcaac catgctattc cagagccact tattgatgga gttcaagcgg ttagcaaaag 300
attttttgat cttccggtgg agcagaagga ggtttatgcc aataagcccg gcgccatcta 360
tggctaccac actaaattgg tggagtccca agatgtggga ttagattggg cagatcacta 420
ttttaatctg gtgtggcctc ctgccaggag agacatgacc tcatggccta cacagcctgc 480
atctttcatc gaaacaatgg atgaatacag cagggaagcg ctgaaattgt tcgagagcct 540
tctgcaggca ctatctcttg gtctgggggt gcgggaagag tccctgaacg agggagtggg 600
cggcaataag aaagaaatat ttgttgcaat aaattactac ccgccatgtc ctcagccgga 660
gcttgcattt ggactggcac ctcattctga caatcaggcc ctaacaatac ttattcaaga 720
ccaggtgccc ggtctgcaaa tttgcaagga cggcaaatgg cttgatgtca aatgcatacc 780
gggcgctctc gtcgtcaatg ttggcgatca gttagagata ctgagcaacg gcaagtacaa 840
gagcgttgag cacagggcta tggtgaataa ggagaaggtg aggatgtcat gggcaatgtt 900
tttagctccc ccacgtgaag gtttgtcgtt ggtcatctct cctcttgtag aactcgtaga 960
cgcagagaat cctcccttat acagagcagt ttgttataag gattatgttt tggaattcag 1020
gaaacagagg gtgtttggaa aaaggtttat tgatagattc aagcaacttt ctttcttaaa 1080
ttctaagaca cagtataaag ggtaagttgt tgcccattgg ctgtggtaaa caatttacca 1140
ggtggtatgg tgtaggtatg agattttgct agtgatcgag tgttactgga attaacttga 1200
tgatgattga atggctatag atttttttac taatttatgc ttacagcccg taagctcaat 1260
gggaataaaa ttttatttct ctcttaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 1314
<210> 2
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<212> DNA
<213> GbFLSa ORF(银杏)
<400> 2
atgggttcga aacacatggt aagagtgcaa acacttgcag agagtggaat ccaaactatt 60
ccacgaaagt atgctagccg tcttgacatt gacaaagcag aagctaaggc gacaaaggtt 120
gaagaagatt caaccgcaga tcttcccata atagacatat ctgatttgaa caatacaaat 180
acagttgccc aaattgtcaa agcagccaaa gagtggggtt tcttccagat tatcaaccat 240
gctattccag agccacttat tgatggagtt caagcggtta gcaaaagatt ttttgatctt 300
ccggtggagc agaaggaggt ttatgccaat aagcccggcg ccatctatgg ctaccacact 360
aaattggtgg agtcccaaga tgtgggatta gattgggcag atcactattt taatctggtg 420
tggcctcctg ccaggagaga catgacctca tggcctacac agcctgcatc tttcatcgaa 480
acaatggatg aatacagcag ggaagcgctg aaattgttcg agagccttct gcaggcacta 540
tctcttggtc tgggggtgcg ggaagagtcc ctgaacgagg gagtgggcgg caataagaaa 600
gaaatatttg ttgcaataaa ttactacccg ccatgtcctc agccggagct tgcatttgga 660
ctggcacctc attctgacaa tcaggcccta acaatactta ttcaagacca ggtgcccggt 720
ctgcaaattt gcaaggacgg caaatggctt gatgtcaaat gcataccggg cgctctcgtc 780
gtcaatgttg gcgatcagtt agagatactg agcaacggca agtacaagag cgttgagcac 840
agggctatgg tgaataagga gaaggtgagg atgtcatggg caatgttttt agctccccca 900
cgtgaaggtt tgtcgttggt catctctcct cttgtagaac tcgtagacgc agagaatcct 960
cccttataca gagcagtttg ttataaggat tatgttttgg aattcaggaa acagagggtg 1020
tttggaaaaa ggtttattga tagattcaag caactttctt tcttaaattc taagacacag 1080
tataaagggt aa 1092
<210> 3
<211> 363
<212> PRT
<213> GbFLSa(银杏)
<400> 3
Met Gly Ser Lys His Met Val Arg Val Gln Thr Leu Ala Glu Ser Gly
1 5 10 15
Ile Gln Thr Ile Pro Arg Lys Tyr Ala Ser Arg Leu Asp Ile Asp Lys
20 25 30
Ala Glu Ala Lys Ala Thr Lys Val Glu Glu Asp Ser Thr Ala Asp Leu
35 40 45
Pro Ile Ile Asp Ile Ser Asp Leu Asn Asn Thr Asn Thr Val Ala Gln
50 55 60
Ile Val Lys Ala Ala Lys Glu Trp Gly Phe Phe Gln Ile Ile Asn His
65 70 75 80
Ala Ile Pro Glu Pro Leu Ile Asp Gly Val Gln Ala Val Ser Lys Arg
85 90 95
Phe Phe Asp Leu Pro Val Glu Gln Lys Glu Val Tyr Ala Asn Lys Pro
100 105 110
Gly Ala Ile Tyr Gly Tyr His Thr Lys Leu Val Glu Ser Gln Asp Val
115 120 125
Gly Leu Asp Trp Ala Asp His Tyr Phe Asn Leu Val Trp Pro Pro Ala
130 135 140
Arg Arg Asp Met Thr Ser Trp Pro Thr Gln Pro Ala Ser Phe Ile Glu
145 150 155 160
Thr Met Asp Glu Tyr Ser Arg Glu Ala Leu Lys Leu Phe Glu Ser Leu
165 170 175
Leu Gln Ala Leu Ser Leu Gly Leu Gly Val Arg Glu Glu Ser Leu Asn
180 185 190
Glu Gly Val Gly Gly Asn Lys Lys Glu Ile Phe Val Ala Ile Asn Tyr
195 200 205
Tyr Pro Pro Cys Pro Gln Pro Glu Leu Ala Phe Gly Leu Ala Pro His
210 215 220
Ser Asp Asn Gln Ala Leu Thr Ile Leu Ile Gln Asp Gln Val Pro Gly
225 230 235 240
Leu Gln Ile Cys Lys Asp Gly Lys Trp Leu Asp Val Lys Cys Ile Pro
245 250 255
Gly Ala Leu Val Val Asn Val Gly Asp Gln Leu Glu Ile Leu Ser Asn
260 265 270
Gly Lys Tyr Lys Ser Val Glu His Arg Ala Met Val Asn Lys Glu Lys
275 280 285
Val Arg Met Ser Trp Ala Met Phe Leu Ala Pro Pro Arg Glu Gly Leu
290 295 300
Ser Leu Val Ile Ser Pro Leu Val Glu Leu Val Asp Ala Glu Asn Pro
305 310 315 320
Pro Leu Tyr Arg Ala Val Cys Tyr Lys Asp Tyr Val Leu Glu Phe Arg
325 330 335
Lys Gln Arg Val Phe Gly Lys Arg Phe Ile Asp Arg Phe Lys Gln Leu
340 345 350
Ser Phe Leu Asn Ser Lys Thr Gln Tyr Lys Gly
355 360
<210> 4
<211> 45
<212> DNA
<213> GbFLSa_5'OUTER 正向PCR引物(artificial)
<400> 4
ctaatacgac tcactatagg gcaagcagtg gtatcaacgc agagt 45
<210> 5
<211> 23
<212> DNA
<213> GbFLSa_5'OUTER 反向PCR引物(artificial)
<400> 5
ttccccttgt ggttgttttc ttc 23
<210> 6
<211> 22
<212> DNA
<213> GbFLSa_5'INNER 正向PCR引物(artificial)
<400> 6
ctaatacgac tcactatagg gc 22
<210> 7
<211> 22
<212> DNA
<213> GbFLSa_5'INNER 反向PCR引物(artificial)
<400> 7
aaatctcata cctacaccat ac 22
<210> 8
<211> 23
<212> DNA
<213> GbFLSa_3'OUTER 正向PCR引物(artificial)
<400> 8
ggagttcaag cggttagcaa aag 23
<210> 9
<211> 45
<212> DNA
<213> GbFLSa_3'OUTER 反向PCR引物(artificial)
<400> 9
actctgcgtt gataccactg cttgccctat agtgagtcgt attag 45
<210> 10
<211> 21
<212> DNA
<213> GbFLSa_3'INNER 正向PCR引物(artificial)
<400> 10
gttggtcatc tctcctcttg t 21
<210> 11
<211> 22
<212> DNA
<213> GbFLSa_3'INNER 反向PCR引物(artificial)
<400> 11
gccctatagt gagtcgtatt ag 22
<210> 12
<211> 19
<212> DNA
<213> GbFLSa_ORF 正向PCR引物(artificial)
<400> 12
atgggttcga aacacatgg 19
<210> 13
<211> 20
<212> DNA
<213> GbFLSa_ORF 反向PCR引物(artificial)
<400> 13
ccctttatac tgtgtcttag 20
<210> 14
<211> 21
<212> DNA
<213> GbFLSa_qPCR 正向引物(artificial)
<400> 14
tttggactgg cacctcattc t 21
<210> 15
<211> 20
<212> DNA
<213> GbFLSa_qPCR 反向引物(artificial)
<400> 15
tcttgtactt gccgttgctc 20
Claims (7)
1.一种银杏GbFLSa基因,其核苷酸序列如SEQ ID No.1所示。
2.权利要求1所述的银杏GbFLSa基因的表达蛋白,其氨基酸序列如SEQ ID No.3所示。
3.含有权利要求1所述的银杏GbFLSa基因的载体、重组菌或宿主细胞。
4.权利要求1所述的银杏GbFLSa基因在调控原青花素的生物合成中的应用。
5.一种制备原花青素含量降低的转基因植物的方法,其特征在于,包括以下步骤:将权利要求1所述的银杏GbFLSa基因导入目的植物,培养筛选得到原花青素含量降低的转基因植物。
6.根据权利要求5所述的方法,其特征在于,所述银杏GbFLSa基因通过重组表达载体导入目的植物。
7.根据权利要求6所述的方法,其特征在于,所述重组表达载体为Pro35S∷GbFLSa。
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Cited By (6)
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CN112079911A (zh) * | 2020-09-04 | 2020-12-15 | 扬州大学 | 一种促进银杏类黄酮合成的关键基因GbMYB6及其表达的蛋白、载体和应用 |
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CN113846026A (zh) * | 2021-09-22 | 2021-12-28 | 江南大学 | 合成阿福豆素和儿茶素的酿酒酵母菌株及其应用 |
CN113846026B (zh) * | 2021-09-22 | 2023-10-27 | 江南大学 | 合成阿福豆素和儿茶素的酿酒酵母菌株及其应用 |
CN117264967A (zh) * | 2023-09-15 | 2023-12-22 | 南京林业大学 | 一种银杏GbWOX3A基因及其在植物组织培养中的应用 |
CN117264967B (zh) * | 2023-09-15 | 2024-04-26 | 南京林业大学 | 一种银杏GbWOX3A基因及其在植物组织培养中的应用 |
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