CN113322262A - 一种控制火龙果甜菜色素合成的基因HuDOPA - Google Patents
一种控制火龙果甜菜色素合成的基因HuDOPA Download PDFInfo
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Abstract
本发明公开了一种控制火龙果甜菜色素合成的基因HuDOPA,该基因的核苷酸序列如序列表中SEQ ID NO.1所示,氨基酸序列如序列表中SEQ ID NO.2所示;本发明HuDOPA基因可直接应用于火龙果果肉颜色方面生物育种,还可作为火龙果重要农业形状的关键基因进行品种资源精细鉴定,可通过过量表达HuDOPA基因,或者基因沉默,以及基因编辑,RNAi干扰等方式,以定量/定性形式改变作物,水果、蔬菜、花卉中的甜菜色素的成分比例,从而可以获得不同颜色的特殊品种、品质的生物育种新策略。
Description
技术领域
本发明涉及调控甜菜色素合成领域,特别涉及一种控制火龙果甜菜色素合成的基因HuDOPA。
背景技术
火龙果(Hylocereus undatus)属仙人掌科(Cactaceae)量天尺属(Hylocereus)植物,独特的外形,丰富的营养价值广受消费者的喜爱。火龙果不仅营养价值丰富,还有很好的药用价值和观赏价值。市场上常有的品种是红皮红肉和红皮白肉两个种类。
火龙果的果皮和果肉中含有大量的甜菜色素,甜菜色素是一种天然可食用色素,常用于食品的着色与护色,还具有抗氧化活性、抗癌、溶血作用、降血脂、促进酶活性和治疗糖尿病等特性,在医疗、保健、日用化工等方面均具有较高的应用价值。但目前,与其他色素的研究相比,甜菜色素的代谢合成机制还有很多的不完善。
2020年10月的《分子植物育种》中,由郭攀阳等人撰写的《红皮红肉和红皮白肉火龙果果肉呈色相关基因差异表达分析》,公开了在红皮红肉火龙果发育过程中伴随着大量甜菜色素的合成,酪氨酸作为前体在其中起关键作用,且该色素的合成需要大量氨基酸、酶、及转录因子的参与。也有其他研究表明甜菜色素合成途径中有几个关键酶,都是自发反应构成的,其中最主要的酪氨酸酶、多巴双加氧酶和葡糖基转移酶,但目前对于甜菜色素合成途径尚未明确。
发明内容
针对上述问题,本发明提出一种控制火龙果甜菜色素合成的基因HuDOPA。
本发明所述一种控制火龙果甜菜色素合成的基因HuDOPA,该基因的核苷酸序列如序列表中SEQ ID NO.1第1位至第816位所示。
进一步的,该基因编码的蛋白的氨基酸序列如序列表中SEQ ID NO.2第1位至第271位所示。
进一步的,所述基因通过特异性引物从火龙果品种中筛选而得。
进一步的,所述基因作为重组载体或宿主细胞中的一部分。
进一步的,所述宿主细胞是指除人或动物的生殖细胞或胚胎干细胞以外的细胞。
进一步的,所述基因HuDOPA在控制植物甜菜色素合成和/或火龙果品种资源精细鉴定中的应用。
进一步的,所述控制植物甜菜色素合成为提高植物甜菜色素累积。
进一步的,所述控制植物甜菜色素合成的应用方法,包括以下步骤:构建HuDOPA表达载体,并利用该表达载体转化植物细胞,进而培育成转基因植株。
进一步的,所述植物为水果、蔬菜和/或花卉。
进一步的,所述植物为火龙果。
与现有技术相比,本发明的有益效果是:
(1)本发明发现一种调控火龙果甜菜色素的新基因HuDOPA,可直接应用于火龙果果肉颜色方面生物育种,还可作为火龙果重要农业形状的关键基因进行品种资源精细鉴定。
(2)本发明基因HuDOPA可通过过量表达,或者基因沉默,以及基因编辑,RNAi干扰等方式,以定量/定性形式改变作物,水果、蔬菜、花卉中的甜菜色素的成分比例,从而可以获得不同颜色的特殊品种、品质的生物育种新策略。
(3)本发明基因HuDOPA还可作为果实特异表达的基因和颜色变化育种的标记基因,可以直观观察到颜色变化。
附图说明
图1为甜菜色素的生物合成途径,注:Ⅰ:酪氨酸酶;Ⅱ:4,5多巴雌二醇双加氧酶;Ⅲ:细胞色素P450(CYP76AD1);Ⅳ:葡糖基环状多巴;Ⅴ:自发反应;Ⅵ:葡糖基转移酶;
图2为HuDOPA基因RT-PCR扩增的电泳图;
图3为HuDOPA基因编码蛋白序列中各氨基酸的含量;
图4为HuDOPA基因编码蛋白的疏水区分布;
图5为实施例3中火龙果果实发育不同时期的甜菜碱含量图;
图6为实施例3中HuDOPA基因在果实组织特异表达的结果示意图;
图7为实施例3中火龙果果实发育不同时期的HuDOPA基因表达情况,注:p0:小花蕾;p1:中花蕾;p2:大花苞;P3:果实膨大期;p4:绿熟期;p5破色期;p6:着色期;p7:果肉成熟果皮未完全成熟;p8:成熟期;
图8为不同发育时期阶段的红心火龙果果实,注:图中第1排为红心火龙果果实,第2排为与第1排红心火龙果果实对应的果实总剖图;第1-2排从左到右的发育时期依次为p0,p1,p2,p3,p4,p5,p6,p7,p8;
图9为实施例4中HuDOPA基因在烟草叶片中的亚细胞定位的明视野图;
图10为实施例4中HuDOPA基因在烟草叶片中的亚细胞定位的荧光图;
图11为实施例4中HuDOPA基因在烟草叶片中的亚细胞定位的叠加图;
图12为实施例5中PNC-cam1304-35SHuDOPA过量表达Micro-Tom番茄表型,注:图中第1排的番茄为对照组,第2排的番茄为实验组;
具体实施方式
为了更好理解本发明技术内容,下面提供具体实施例,对本发明做进一步的说明。
本发明研究使用的火龙果的品种为“美红一号”;
实施例1 HuDOPA基因的分离
HuDOPA基因的分离,包括以下步骤:
S1.利用全长cDNA测序法获得全长序列;
S2.根据生物学信息设计以下引物
正向引物(SEQ ID NO.3):ATGGGTGTTGGCAAAGAAGTGTCG
反向引物(SEQ ID NO.4):GATGGAAGTGAACTTGTAGGAGGC
S3.PCR扩增:基于火龙果果实发育阶段的组装数据进行RT-PCR克隆,利用上述引物,以火龙果cDNA为模板,PrimeSTAR Max DNA聚合酶扩增,PCR反应条件见下表:
反应体系:
反应程序:
S4.PCR反应产物回收:PCR产物先用1%琼脂糖凝胶电泳做定性分析,再用GelExtracti on Kit(OMEGA)回收纯化目的片段;
S5.菌液PCR及DNA测序:将回收纯化的目的片段连接到PNC-cam1304-subC/N和PNC-cam1304-35S载体上再转入感受态细胞中,在LB-Kan平板上37℃培养16h,随机挑取圆润的单克隆,摇菌(LB+Kan),震荡培养16h,菌液PCR检测,为阳性的菌液,送至上海生工生物公司测序。
实施例2基因序列的生物信息学比对及其编码蛋白分子特征分析
生物信息学比对:用BLAST程序在NCBI数据库中进行HuDOPA基因及其编码蛋白的同源性检索,将序列SEQ ID NO.1片段在DNA水平上进行比对分析,将序列SEQ ID NO.2片段在氨基酸水平上进行比对分析;结果显示,在DNA水平上,序列SEQ ID NO.1片段与巨柱仙人掌(Carnegiea gigantea,NCBI登录号MN136222.1)DODAa1的mRNA序列的同源性为97.06%;在氨基酸水平上,序列SEQ ID NO.2片段与巨柱仙人掌(Carnegiea gigantea,NCBI登录号QED21473.1)DODAa1的氨基酸序列的同源性为97.05%,与马齿苋(Portulacagrandiflora,NCBI登录号Q7XA48.1)4,5-DOPA dioxygenase extradiol的氨基酸序列的同源性为87.82%。
蛋白分子特征分析:通过生物信息分分析该蛋白分子特征。利用生物信息bioedit软件对火龙果HuDOPA蛋白中的氨基酸含量进行分析吗,结果发现:丙氨酸、丝氨酸和亮氨酸在蛋白序列中含量较高,分别占总含量的9.2%、8.9%和8.1%(见图3)。
在WoLFPSORT在线软件(http://www.genscript.com/wolf-psort.html)预测HuDOPA蛋白定位于细胞质内。
采用ExPASy(ProtScale)在线软件对蛋白亲/疏水性进行分析,HuDOPA蛋白平均疏水性(GRAVY):-0.156,推断其为亲水性蛋白。
通过DNAMAN软件预测火龙果HuDOPA蛋白的二级结构,我们发现火龙果HuDOPA蛋白的二级结构多数为不规则的卷曲占42.07%和α螺旋结构占32.47%,延伸链占19.19%,β转角6.27%。
在Swissmodel在线软件(http://swissmodel.expasy.org/)上预测了蛋白的三级结构可能的构型。用NCBI的Conserved domains(http://www.ncbi.nlm.nih.gov)进行保守区查找,获得序列的保守结构域,发现HuDOPA蛋白含有4,5-DOPA dioxygenase、LigB、PRK10628等区域。
实施例3 HuDOPA基因在火龙果中的表达分析
1.火龙果不同发育时期的甜菜碱含量变化情况;
分别在火龙果的果实膨大期(P3),绿熟期(P4),破色期(P5),着色期(P6),果肉成熟果皮未完全成熟(P7),成熟期(P8)取样,采用紫外分光光度法测定甜菜碱含量,结果表明,随着火龙果的发育,甜菜碱含量逐渐增加。
2.HuDOPA基因在果实组织特异表达
表达量的测定方法,包括以下步骤:
(1)果实总RNA的提取和cDNA合成:选择多糖多酚RNA提取试剂盒(TIANGEN,天根生化科技(北京)有限公司),实验方法步骤参见试剂盒说明书。RNA提取完成后,用1.2%琼脂糖凝胶120V电泳15min检测其完整性。cDNA合成参见RevertAid First Strand cDNASynthesis Kit(ThermoFisher Scientific)。
(2)通过实时荧光定量PCR对基因表达进行分析,采用TBPremix Ex TaqTM(TliRNaseH Plus)(Takara)试剂盒,加样完成后,颠倒混匀,瞬离数秒,置于实时荧光定量PCR仪中完成反应,所用引物同实施例1,选用SYBR Green PCR试剂盒(TaKaRa)进行qRT-PCR验证,反应条件如下:
反应体系:
反应程序:
通过研究发现在茎、根、花、果皮、果肉组织中均有表达,在果肉中表达量最高,花中表达量最少,果肉的表达量大约为花的28倍(见图6)。果肉中的表达量大约为茎的6.5倍。花和根中HuDOPA基因表达量相近。
3.红肉火龙果不同发育时期的基因表达量
采用qRT-PCR对不同基因在火龙果各个发育时期的表达量进行分析,表达量测定方法同上;结果显示,在不同发育时期223840、28540、HuPPH1、HuPPH2、HuDOPA、HubHLH表现明显上升趋势,在第4个时期果实基因表达量有明显变化(见图7),因此果肉含量在不同时期的表达变话存在明显差异。
实施例4瞬时转化表达试验
1.重组质粒的构建:将HuDOPA基因克隆至载体,得到重组质粒PNC-cam1304-subC-HuDOPA-GFP。
2.转化:重组质粒转化农杆菌GV3101后,于28℃培养2-3d,挑取圆润的单菌落接种到LB-kan+rif液体培养基中摇培至0.6<OD600<0.8,室温5000rpm离心10min收集菌体,弃掉培养基,用MS+AS液体培养基重悬后,放于28℃培养箱孵育培养3h后,用1mL的注射器吸取菌液,自烟草叶片背面远离叶脉处将菌液注入烟草。
3.荧光共聚焦显微镜的观察:3d后剪取叶片制成植物玻片于荧光共聚焦显微镜下观察GFP,发现在荧光场,PNC-cam1304-subC-HuDOPA-GFP融合蛋白的绿色荧光特异性出现在细胞质中,说明HuDOPA定位在细胞质中,与预测结果一致。
实施例5瞬时转化验证HuDOPA基因功能试验
以PNC-cam1304-35S作为对照,PNC-cam1304-35SHuDOPA作为实验组,在同一时间段分别注射侵染番茄,待番茄转色成熟时同时采收;结果表明:PNC-cam1304-35SHuDOPA实验组番茄与对照组相比,实验组的番茄果颜色有明显变化,在幼果却表现出“早熟”的特征(见图12),说明本申请的HuDOPA基因能够控制色素的合成。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 中国热带农业科学院三亚研究院
<120> 一种控制火龙果甜菜色素合成的基因HuDOPA
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 816
<212> DNA/RNA
<213> 火龙果(Hylocereus undatus 'Foo-Lon')
<400> 1
atgggtgttg gcaaagaagt gtcgttcaag gagactttct atgtgtctca tgggaatccg 60
gccatgttgg ccgatgtgtc gttcatagca cggaacttcc tgctggggtg gaagaagaat 120
gtcttcccca tcaaacccaa gtcgatcctg gttgtctctg ctcactggga gactgatgtg 180
ccttctgtat ctgccggtga acatcctgat gtcatttacg atttcagcga tgttcctgac 240
tgcatgttcc agatgaagta cccagctcta gggtcaccaa aactggccaa aagggtgcag 300
gagctactga tagcaggagg gttcaagaca gcgagcctag acgagagtcg tgggttcgac 360
cacagctcat gggtgcccct gagcctcatg taccctgagg ctgacatccc ggtgtgccag 420
ctctcagtcc agcctcacct aagcgcgagc caccacttcg acatagggag ggctttggct 480
cctctcaagg aggaaggggt cctgttcatt gggtctgggg gtgcagtgca cccgtctgat 540
gacaccccac actggtctga tggggttgcc ccttgggctg ctgagtttga tcaatggctt 600
gaggatgctc tcattaatgg aaggtacgat gatgtgaataa ttatcaaaca aaagcacct 660
tctgggtgga aaatagcaca tccaattcca gaacactttt taccattgca tgtagccatg 720
ggtgcagctg gtgaaaaatc aaaggcagag ctcatttatc gtacgtggga tcatggtact 780
cttggctatg cctcctacaa gttcacttcc atctga 816
<210> 2
<211> 271
<212> PRT
<213> 火龙果(Hylocereus undatus 'Foo-Lon')
<400> 2
Met Gly Val Gly Lys Glu Val Ser Phe Lys Glu Thr Phe Tyr Val Ser
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His Gly Asn Pro Ala Met Leu Ala Asp Val Ser Phe Ile Ala Arg Asn
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Phe Leu Leu Gly Trp Lys Lys Asn Val Phe Pro Ile Lys Pro Lys Ser
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Ile Leu Val Val Ser Ala His Trp Glu Thr Asp Val Pro Ser Val Ser
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Ala Gly Glu His Pro Asp Val Ile Tyr Asp Phe Ser Asp Val Pro Asp
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Cys Met Phe Gln Met Lys Tyr Pro Ala Leu Gly Ser Pro Lys Leu Ala
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Lys Arg Val Gln Glu Leu Leu Ile Ala Gly Gly Phe Lys Thr Ala Ser
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Leu Asp Glu Ser Arg Gly Phe Asp His Ser Ser Trp Val Pro Leu Ser
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Leu Met Tyr Pro Glu Ala Asp Ile Pro Val Cys Gln Leu Ser Val Gln
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Pro His Leu Ser Ala Ser His His Phe Asp Ile Gly Arg Ala Leu Ala
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Pro Leu Lys Glu Glu Gly Val Leu Phe Ile Gly Ser Gly Gly Ala Val
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His Pro Ser Asp Asp Thr Pro His Trp Ser Asp Gly Val Ala Pro Trp
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Ala Ala Glu Phe Asp Gln Trp Leu Glu Asp Ala Leu Ile Asn Gly Arg
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Tyr Asp Asp Val Asn Asn Tyr Gln Thr Lys Ala Pro Ser Gly Trp Lys
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Ile Ala His Pro Ile Pro Glu His Phe Leu Pro Leu His Val Ala Met
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Gly Ala Ala Gly Glu Lys Ser Lys Ala Glu Leu Ile Tyr Arg Thr Trp
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Asp His Gly Thr Leu Gly Tyr Ala Ser Tyr Lys Phe Thr Ser Ile
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<210> 3
<211> 24
<212> DNA/RNA
<213> 火龙果(Hylocereus undatus 'Foo-Lon')
<400> 3
atgggtgttg gcaaagaagt gtcg 24
<210> 4
<211> 24
<212> DNA/RNA
<213> 火龙果(Hylocereus undatus 'Foo-Lon')
gatggaagtg aacttgtagg aggc 24
Claims (9)
1.一种控制火龙果甜菜色素合成的基因HuDOPA,其特征在于,该基因的核苷酸序列如序列表中SEQ ID NO.1第1位至第816位所示。
2.如权利要求1所述的一种控制火龙果甜菜色素合成的基因HuDOPA,其特征在于,该基因编码的蛋白的氨基酸序列如序列表中SEQ ID NO.2第1位至第271位所示。
3.如权利要求1所述的一种控制火龙果甜菜色素合成的基因HuDOPA,其特征在于,所述基因通过特异性引物从火龙果品种中筛选而得。
4.如权利要求1所述的一种控制火龙果甜菜色素合成的基因HuDOPA,其特征在于,所述基因作为重组载体或宿主细胞中的一部分。
5.如权利要求1-4任一项所述的一种控制火龙果甜菜色素合成的基因HuDOPA的应用,其特征在于,所述基因HuDOPA在控制植物甜菜色素合成和/或火龙果品种资源精细鉴定中的应用。
6.如权利要求5所述的应用,其特征在于,所述控制植物甜菜色素合成为提高植物甜菜色素累积。
7.如权利要求5所述的应用,其特征在于,所述控制植物甜菜色素合成的应用方法,包括以下步骤:构建HuDOPA表达载体,并利用该表达载体转化植物细胞,进而培育成转基因植株。
8.如权利要求6或7所述的应用,其特征在于,所述植物为水果、蔬菜和/或花卉。
9.如权利要求8所述的应用,其特征在于,所述水果为火龙果。
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