CN113512556A - 一种与β-紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因及其编码蛋白和应用 - Google Patents
一种与β-紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因及其编码蛋白和应用 Download PDFInfo
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Abstract
本发明公开了一种与β‑紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因及其编码蛋白和应用，该基因的核苷酸序列如序列表Seq ID No:1所示，该编码蛋白的氨基酸序列如序列表Seq ID No:2所示，本发明的与β‑紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因通过基因工程技术裂解β‑胡萝卜素形成β‑紫罗兰酮，提高辣椒β‑紫罗兰酮的含量进而改善辣椒的风味，提高辣椒的经济价值。
Description
技术领域
本发明属于基因工程技术领域，具体涉及辣椒(遵辣)类胡萝卜素双加氧裂解酶基因CaCCD1A及其编码蛋白在辣椒类胡萝卜素代谢以及挥发物代谢中的应用。
背景技术
辣椒(Capsicum annuum L.)是一种药食同源的蔬菜，起源于中美洲和南美洲的热带地区，现在在世界范围内已广泛种植(Ziino et al.，2009)。我国是辣椒种植大国，国内知名的辣椒品种多样，主要包括朝天椒、秦椒、羊角椒、小米椒等。根据联合国粮食和农业组织的数据，在1996-2016年间，中国的鲜食辣椒总产量翻了三番以上，干辣椒的总产量也渐近番了两番，由此可见辣椒在我国饮食文化上的地位。
随着生活水平的提高，人们评判辣椒的品质不仅仅局限在它的色泽以及辣度，同时对它的风味品质也提出了更高的要求。辣椒果实的香气是由许多挥发性化合物综合构成。现如今，在辣椒果实中已经鉴定到上百种挥发性化合物，主要包括脂肪酸和芳香族氨基酸的衍生物，单萜和倍半萜，以及类胡萝卜素衍生物(Kocsis et al.，2002；Auldridge etal.，2006a；Ziino et al.，2009；Borovsky et al.，2013)。β-紫罗兰酮，因其阈值较低，对辣椒的整体风味影响很大，然而关于它在辣椒中的合成途径，目前并没有相关的研究报道。
类胡萝卜素裂解双加氧酶可以作用于类胡萝卜素的双键位置，对类胡萝卜素进行氧化裂解，形成一系列的类胡萝卜素衍生物。第一个被发现的类胡萝卜素裂解双加氧酶是VP14，随后对它的同源基因在其它物种中开始了广泛研究(Schwartz et al.，1997)。高等植物中类胡萝卜素裂解双加氧酶根据系统发育和基因功能被分为5个家族，CCD1，CCD4，CCD7，CCD8以及NCED。
CCD1直系同源物非常相似，并且是类胡萝卜素裂解双加氧酶家族中唯一胞质定位的成员。CCD1酶的特点是作用底物广泛，裂解方式多样。如番茄中的CCD1对不同的类胡萝卜素底物在不同的双键位置切割，形成β-紫罗兰酮，假紫罗兰酮和香叶基丙酮香味物质(Simkin et al.，2004)。此外，在拟南芥、桃子、玫瑰、桂花、葡萄、水稻等多种物种的研究均表明，CCD1与风味物质的形成有关(Auldridge et al.，2006b；Huang et al.，2009；Ilg etal.，2009；Baldermann et al.，2010；Brandi et al.，2011)。除CCD1外，CCD4是另一个参与植物脱辅机类胡萝卜素香气物质形成的家族，此外，CCD4还可以参与植物呈色物质的合成，它可以在C9-C10双键上氧化裂解β-胡萝卜素或者β-脱辅基8’胡萝卜醛裂解为β-紫罗兰酮(Rubio et al.，2008；Adami et al.，2013)，同时，它也可以在C7-C8双键位置处将β-胡萝卜素、β-隐黄质和玉米黄素，产生新的有色物质β-脱辅机8’胡萝卜醛和β-柠乌素(Mein etal.，2011)。CCD7和CCD8的顺序切割可以形成控制侧根和侧芽萌发的植物激素：独脚金内酯(Walter and Strack，2011)。NCED与上述4个亚家族成员的同源性较低，它可以裂解新黄质或紫黄质产生黄质醛，是脱落酸(ABA)生物合成途径的关键酶。
研究表明，β-紫罗兰酮是辣椒果实中的重要的香气物质，并且随着辣椒果实的成熟，它的含量亦有增加的趋势。然而，尚未有β-紫罗兰酮在辣椒中的合成途径的报道。
发明内容
发明目的：为了克服现有技术中存在的不足，本发明提供一种与β-紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因及其编码蛋白和应用，该基因转录编码类胡萝卜素裂解双加氧酶CCD1A，可以裂解β-胡萝卜素形成β-紫罗兰酮。对与β-紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因(CCD1A)的发现和功能鉴定有利于利用基因工程技术提高辣椒中β-紫罗兰酮的含量进而改善辣椒的风味物质，提高辣椒的经济价值。
技术方案：为实现上述目的，本发明采用的技术方案为：
一种与β-紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因，该基因的核苷酸序列如序列表Seq ID No:1所示。
一种与β-紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因的编码蛋白，该编码蛋白的氨基酸序列如序列表Seq ID No:2所示。
本发明的类胡萝卜素裂解双加氧酶基因来源于遵辣(Capsicum annuum L.)，名称为CaCCD1A，以下该基因简称为CaCCD1A。
所述的类胡萝卜素裂解双加氧酶来源遵辣(Capsicum annuum L.)。
一种重组表达载体，包括与β-紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因。
一种转化子，包括重组表达载体的宿主细胞。
一种转化子，它的宿主为微生物、植物或转基因细胞系。
一种与β-紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因的应用，应用于辣椒类胡萝卜素代谢基因工程中风味品质提高的用途。
一种与β-紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因(CaCCD1A)的获取方法，包括以下步骤：
步骤1，总RNA的提取
选取遵辣(Capsicum annuum L.)，应用RNAiso试剂盒(Takara)提取得到遵辣果实的总RNA，经紫外分光光度计检测确认RNA的完整性以及浓度。
步骤2，遵辣类胡萝卜素裂解双加氧酶基因(CaCCD1A)的克隆及表达载体的构建
应用1μg步骤1获得的总RNA作为反转录模板，按照PrimeScript 1st Strand cDNASynthesis Kit(Takara)的说明书操作，经反转录合成cDNA第一链后，应用发转录PCR程序进行PCR扩增，以获得与β-紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因的ORF全长(带有pMAL-c5x同源臂)，其中：
F端引物如下：
gcgatatcgtcgacggatccATGGGGAGAAAAGAAGAAGGAGATGATGG
R端引物如下：
taattacctgcagggaattcTCACAGTTTGGCTTGTTCCAGAATT
其中，小写字母代表同源臂序列，大写字母代表引物的基因序列；
步骤3，将带有pMAL-c5x同源臂的与β-紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因片段根据ClonExpress MultiS One Step Cloning Kit(Vazyme)试剂盒的操作说明进行同源重组，测序后经拼接获得具有完整编码区的辣椒类与β-紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因(CaCCD1A)，CaCCD1A的ORF序列Seq ID No:1。
步骤4，酶活鉴定
利用大肠杆菌色素系统进行辣椒CaCCD1A类胡萝卜素裂解双加氧酶基因的酶活鉴定，该色素系统因为含有类胡萝卜素合成途径的基因可使大肠杆菌产生β-胡萝卜素(pAC-BETA)，它可以使大肠杆菌菌落或菌液呈现亮黄色。在检测辣椒CaCCD1A类胡萝卜素裂解双加氧酶基因的酶活时，需要将其表达载体pMAL-c5x-CaCCD1A与产生β-胡萝卜素的大肠杆菌共转。进一步通过HPLC分析其色素的变化，同时采用SPME萃取辣椒CaCCD1A类胡萝卜素裂解双加氧酶基因对底物的裂解产物，对萃取得到的产物进行质谱分析。空载体pMAL-c5x与底物共转作为负对照。
本发明的CaCCD1A类胡萝卜素裂解双加氧酶基因来源于辣椒，其基因工程受体植物适用于水稻等植物。
本发明相比现有技术，具有以下有益效果：
1.利用本法发明的辣椒CaCCD1A类胡萝卜素裂解双加氧酶基因可以作为目的基因构建植物表达载体，可以利用花椰菜花叶病毒CAM35S启动子、乙醇诱导启动子等，必要时可以包括增强子。为了简化转化植株的鉴定，可以使用选择性标记(如抗生素酶)。所用的表达载体可使用Ti质粒、Ri质粒以及植物病毒载体等。转化方法可用农杆菌介导法或其他方法转化植物。
2.本发明首次发现辣椒中参与β-紫罗兰酮释放的一个关键酶辣椒CaCCD1A类胡萝卜素裂解双加氧酶，并证实该酶参与辣椒中β-紫罗兰酮的释放。
3.本发明获得编码该酶的基因序列，为利用基因工程技术改进辣椒中β-紫罗兰酮的释放，提高辣椒风味品质提供了理论基础。
4.通过本发明提供的辣椒CaCCD1A类胡萝卜素裂解双加氧酶基因和蛋白，对辣椒进行基因工程改进，其产业价值着重两个方面，通过提高β-紫罗兰酮的含量提高辣椒果实的风味品质，从而提高辣椒的经济价值。
附图说明
图1为CaCCD1A核苷酸和推定氨基酸序列；其中，“*”代表终止密码子。
图2为CaCCD1A与β-胡萝卜素共孵育的酶活鉴定结果，包括HPLC以及GC-MS分析。其中pMAL-c5x空载作为阴性对照。
图3为不同物种与β-紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因的多序列比对，标记区域为保守的组氨酸以及谷氨酸或天冬氨酸残基。下划线标注的为预测的转运肽。
图4为不同物种中与β-紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因的系统发育树分析。
具体实施方式
下面结合附图和具体实施例，进一步阐明本发明，应理解这些实例仅用于说明本发明而不用于限制本发明的范围，在阅读了本发明之后，本领域技术人员对本发明的各种等价形式的修改均落于本申请所附权利要求所限定的范围。
本实施例的与β-紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因(CaCCD1A)的基因获得过程如下：
1)遵辣CaCCD1A转录本鉴定和编码区克隆
根据拟南芥编码CCD1基因的开放阅读框(ORF)，在遵辣基因组数据库(http://peppersequence.genomics.cn)搜索遵辣中的同源序列，搜索方法为tblastX，找到了辣椒中的一个转录本NM_001324566。根据该转录本基因序列设计扩增带有pMAL-c5x同源臂的ORF引物。选用RNAiso试剂(Takara)提取辣椒果实的总RNA，经紫外分光光度计检测其纯度及浓度。用1μg总RNA作为反转录的模板，用PrimeScript 1st Strand cDNA Synthesis Kit(Takara)试剂盒经发转录合成cDNA第一链之后，用高保真酶PrimeSTAR DNA polymerase(Takara)进行PCR扩增，PCR反应程序如下：98℃预变性2min，98℃变性30s，58℃退火30s，72℃延伸2min，35个循环后，72℃延伸5min。反应结束后，根据ClonExpress MultiS One StepCloning Kit(Vazyme)试剂盒的操作说明进与线性的pMAL-c5x进行同源重组。连接产物转化大肠杆菌(Top10)，筛选获得阳性克隆，得到携带CaCCD1A的重组载体，命名为pMAL-CaCCD1A，如图1所示，该基因全1644bp，共编码含有547个氨基酸残基的蛋白序列和一个终止密码子，经分析，该蛋白的N端没有叶绿体转运肽，与现有报道的CCD1的定位情况一致。将所推测的蛋白序列与其他物种的同源CCD1以及CCD4多序列对比(图3所示)，说明所获得的CaCCD1A与其余物种的CCD1同源。
CaCCD1A的异源表达和功能鉴定
将pMAL-CaCCD1A载体转化进入合成β-胡萝卜素的TOP10大肠杆菌，在含有羧苄霉素(100μg mL-1)和氯霉素(34μg mL-1)的LB固体培养基上过夜筛选。挑单菌转入含有相同类型和浓度的LB液体培养基培养过夜(5mL LB液体培养基)。第二天，按照1:50的比例进行转接，当菌液OD600达到0.6-0.8范围时，加入0.5mM isopropylβ-D-thiogalactopyranoside(IPTG)放于28℃摇床4h诱导蛋白表达。随后插入SPME萃取大肠杆菌释放的挥发性产物。在萃取8个小时后，将SPME吸附的化合物进行GC-MS分析鉴定。所用的GC-MS为Agilent7890AGC(Agilent，Santa Clara，CA)，同时配有5977A MS检测器(Agilent)。使用HP-5MS(Agilent，30m×0.25mm；film thickness，0.25μm)柱子分离混合物。在萃取过程中使用氦气作为载气，并保持流速为1mL min–1。炉温程序为：40℃保持2min，随后以每分钟上升5℃的速率上升至150℃。最后在260℃中保持10min。在整个实验过程中，均要加入空载pMAL-c5x作为对照。我们在GC-MS的分析中我们发现，当用产生β-胡萝卜素的大肠杆菌与CaCCD1A共孵育时，可以检测到β-紫罗兰酮的产生，说明CaCCD1A可以作用于β-胡萝卜素的9-10/9’-10’位置处，对其进行氧化裂解(图2所示)。
色素提取及HPLC分析
将含有表达β-胡萝卜素色素的菌株中转入表达载体pMAL-CaCCD1A，pMAL-c5x空载体作为阴性对照。从菌液的颜色来看，当转入pMAL-CaCCD1A后，菌液的颜色由黄色几乎变为白色。为了从色素分析角度解释CaCCD1A的裂解活性，我们进一步对上述菌液进行HPLC分析。收集5mL转入表达载体pMAL-CaCCD1A以及空载的色素表达菌株的菌体，离心富集后，加入400μL的80％的丙酮，剧烈振荡充分混匀，以彻底提取材料中的色素，随后依次加入250μL的乙酸乙酯和250μL的水，转速13400g，4℃离心20min。吸取上清，并将提取物用0.22μm有机系的滤膜进行过滤。所提取的色素用反向高效液相色谱方法，在Spherisorb ODS2 column(5μm，4.6×250mm)(Waters)柱子上分离，流动相在乙腈：水：三已胺(9:1:0.01)中线性展开乙酸乙酯(0-100％)。运行时间为37min，流速为1ml min-1，柱温保持30℃。检测器的检测波长为440nm。类胡萝卜素的鉴定根据每个物质的特征吸收光谱来确定。所有的化学试剂均为色谱纯。分析结果表明，当表达色素的菌株与空载pMAL-c5x共转时，大肠杆菌会产生大量的β-胡萝卜素。但是当加入CaCCD1A时，β-胡萝卜素几乎检测不到。这些结果进一步从色素角度证实了CaCCD1A可以对底物β-胡萝卜素进行裂解(图2所示)。
系统学分析
为了确定所克隆的CaCCD1A所属的类型，我们检索了其他物种CCD家族成员，对它们进行聚类分析。所有序列采用Clustal W进行序列比对。分析结果表明，CaCCD1A确实属于CCD1家族的成员(图4所示)。
以上所述仅是本发明的优选实施方式，应当指出：本发明内容不仅限于上述各实施例的内容，含有本发明基因的重组表达载体、转基因细胞系及宿主菌均属本发明的保护范围。对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 南京大学
<120> 一种与β-紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因及其编码蛋白和应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1644
<212> DNA
<213> 辣椒(Capsicum annuum)
<400> 1
atggggagaa aagaagaagg agatgatgga gtggagagaa gtgaaggagg agttgtgtta 60
gtgaacccga agccacgaaa gggatttagt gccaaggcat tagacttatt ggaaaagggg 120
attgttaagt tagtgcacga ttcatccaag ccacttcact atcttcaagg caattttgca 180
cctactgatg aaactcctcc tcttaatgac ctccttgtac atggccatct tccggagtgc 240
cttaatggtg aatttgttag agttggtcca aatccaaagt ttgctccggt tgctggatat 300
cactggtttg atggggatgg catgattcat ggcttacgca ttaaagatgg aaaagcaacg 360
tatgtctcac gttatgtgag aacttcacgt ctaaagcaag aagagttctt tggtggagct 420
aagtttatga aggttggaga tcttaaaggg ctgtttggtc tgttcacagt ttatatgcaa 480
atgctcagag caaagctgaa agtattggac atctcctatg gaaatggcac agctaataca 540
gctttagcat accaccatgg gaagcttttg gcactttcag aagctgataa accttatgca 600
cttaaagttc tagaggatgg agatcttcaa acacttggca tgctggatta tgacaaaaga 660
ttgacacatt ccttcacggc ccatccaaaa gttgatcctg taactggtga gatgtttacc 720
tttggctact ctcacgatcc accttacatc acttataggg ttatatcgaa ggatggaatt 780
atgcaggacc cggttccaat aacaatacca gagcctatca tgatgcatga ttttgctatt 840
actgaaaatt atgcaatcat gatggatctt cctttgtgtt ttcgtccgaa ggaaatggtg 900
aaaaacaata agctggcatt tacctttgat gctactaaaa aggctcgatt tggagtcctt 960
gcacgatatg caaacaatga ggccctaatc aagtggtttg agcttcctaa ttgcttcata 1020
ttccataatg ccaacgcttg ggaggaggga gatgaagttg tgctgatcac ctgccgcctt 1080
atgaatccag acctagactt ggtcaatgga gccgttaaag aaaagcttga gaatttctgc 1140
aatgagctat atgaaatgag atttaacatg aagagtggtg cagcttcaca gaagaaacta 1200
tcggagtctg ctgttgattt tccaaggatc aatgagaact acactggcag gaagcagcgt 1260
tatgtatatg gaaccacttt gaacagcatt gctaaggtaa ctggaatcat caaatttgat 1320
ttgcatgctg aaccaaagac tggaaaatca cagcttgaag ttggaggaaa tgttcaaggc 1380
atatttgatc ttggacctgg cagatttggt tcggaggctg tatttgttcc tagtcagcct 1440
gatacagaat gtgaagagga cgatggcttc ttgatattct ttgtacatga tgagaacact 1500
ggaaagtcag ctgtgaatgt aattgatgca aaaacaatgt ccgcggagcc tgtggcagta 1560
gttgaattgc caaaaagagt tccttatgga ttccatgctt tctttgtcac agaggaacaa 1620
attctggaac aagccaaact gtga 1644
<210> 2
<211> 547
<212> PRT
<213> 辣椒(Capsicum annuum)
<400> 2
Met Gly Arg Lys Glu Glu Gly Asp Asp Gly Val Glu Arg Ser Glu Gly
1 5 10 15
Gly Val Val Leu Val Asn Pro Lys Pro Arg Lys Gly Phe Ser Ala Lys
20 25 30
Ala Leu Asp Leu Leu Glu Lys Gly Ile Val Lys Leu Val His Asp Ser
35 40 45
Ser Lys Pro Leu His Tyr Leu Gln Gly Asn Phe Ala Pro Thr Asp Glu
50 55 60
Thr Pro Pro Leu Asn Asp Leu Leu Val His Gly His Leu Pro Glu Cys
65 70 75 80
Leu Asn Gly Glu Phe Val Arg Val Gly Pro Asn Pro Lys Phe Ala Pro
85 90 95
Val Ala Gly Tyr His Trp Phe Asp Gly Asp Gly Met Ile His Gly Leu
100 105 110
Arg Ile Lys Asp Gly Lys Ala Thr Tyr Val Ser Arg Tyr Val Arg Thr
115 120 125
Ser Arg Leu Lys Gln Glu Glu Phe Phe Gly Gly Ala Lys Phe Met Lys
130 135 140
Val Gly Asp Leu Lys Gly Leu Phe Gly Leu Phe Thr Val Tyr Met Gln
145 150 155 160
Met Leu Arg Ala Lys Leu Lys Val Leu Asp Ile Ser Tyr Gly Asn Gly
165 170 175
Thr Ala Asn Thr Ala Leu Ala Tyr His His Gly Lys Leu Leu Ala Leu
180 185 190
Ser Glu Ala Asp Lys Pro Tyr Ala Leu Lys Val Leu Glu Asp Gly Asp
195 200 205
Leu Gln Thr Leu Gly Met Leu Asp Tyr Asp Lys Arg Leu Thr His Ser
210 215 220
Phe Thr Ala His Pro Lys Val Asp Pro Val Thr Gly Glu Met Phe Thr
225 230 235 240
Phe Gly Tyr Ser His Asp Pro Pro Tyr Ile Thr Tyr Arg Val Ile Ser
245 250 255
Lys Asp Gly Ile Met Gln Asp Pro Val Pro Ile Thr Ile Pro Glu Pro
260 265 270
Ile Met Met His Asp Phe Ala Ile Thr Glu Asn Tyr Ala Ile Met Met
275 280 285
Asp Leu Pro Leu Cys Phe Arg Pro Lys Glu Met Val Lys Asn Asn Lys
290 295 300
Leu Ala Phe Thr Phe Asp Ala Thr Lys Lys Ala Arg Phe Gly Val Leu
305 310 315 320
Ala Arg Tyr Ala Asn Asn Glu Ala Leu Ile Lys Trp Phe Glu Leu Pro
325 330 335
Asn Cys Phe Ile Phe His Asn Ala Asn Ala Trp Glu Glu Gly Asp Glu
340 345 350
Val Val Leu Ile Thr Cys Arg Leu Met Asn Pro Asp Leu Asp Leu Val
355 360 365
Asn Gly Ala Val Lys Glu Lys Leu Glu Asn Phe Cys Asn Glu Leu Tyr
370 375 380
Glu Met Arg Phe Asn Met Lys Ser Gly Ala Ala Ser Gln Lys Lys Leu
385 390 395 400
Ser Glu Ser Ala Val Asp Phe Pro Arg Ile Asn Glu Asn Tyr Thr Gly
405 410 415
Arg Lys Gln Arg Tyr Val Tyr Gly Thr Thr Leu Asn Ser Ile Ala Lys
420 425 430
Val Thr Gly Ile Ile Lys Phe Asp Leu His Ala Glu Pro Lys Thr Gly
435 440 445
Lys Ser Gln Leu Glu Val Gly Gly Asn Val Gln Gly Ile Phe Asp Leu
450 455 460
Gly Pro Gly Arg Phe Gly Ser Glu Ala Val Phe Val Pro Ser Gln Pro
465 470 475 480
Asp Thr Glu Cys Glu Glu Asp Asp Gly Phe Leu Ile Phe Phe Val His
485 490 495
Asp Glu Asn Thr Gly Lys Ser Ala Val Asn Val Ile Asp Ala Lys Thr
500 505 510
Met Ser Ala Glu Pro Val Ala Val Val Glu Leu Pro Lys Arg Val Pro
515 520 525
Tyr Gly Phe His Ala Phe Phe Val Thr Glu Glu Gln Ile Leu Glu Gln
530 535 540
Ala Lys Leu
545
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gcgatatcgt cgacggatcc 20
<210> 4
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggggagaa aagaagaagg agatgatgg 29
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
taattacctg cagggaattc 20
<210> 6
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tcacagtttg gcttgttcca gaatt 25
Claims (8)
1.一种与β-紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因，其特征在于：该基因的核苷酸序列如序列表Seq ID No:1所示。
2.一种与β-紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因的编码蛋白，其特征在于：该编码蛋白的氨基酸序列如序列表Seq ID No:2所示。
3.根据权利要求1或2所述的胡萝卜素裂解双加氧酶基因的编码蛋白，其特征在于：类胡萝卜素裂解酶来源于辣椒。
4.一种重组表达载体，其特征在于：包括权利要求1或2所述的基因。
5.一种转化子，其特征在于：包括权利要求4所述的重组表达载体的宿主细胞。
6.根据权利要求5所述的一种转化子，其特征在于：它的宿主为微生物、植物或转基因细胞系。
7.一种与β-紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因的获取方法，其特征在于：包括以下步骤：
步骤1，选取遵辣，应用试剂盒提取得到遵辣果实的总RNA；
步骤2，应用步骤1获得的总RNA作为反转录模板，经反转录合成cDNA第一链后，应用发转录PCR程序进行PCR扩增，以获得与β-紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因的ORF全长，此时的与β-紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因带有pMAL-c5x同源臂，其中：
F端引物如下：
gcgatatcgtcgacggatccATGGGGAGAAAAGAAGAAGGAGATGATGG
R端引物如下：
taattacctgcagggaattcTCACAGTTTGGCTTGTTCCAGAATT
其中，小写字母代表同源臂序列，大写字母代表引物的基因序列；
步骤3，将带有pMAL-c5x同源臂的与β-紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因片段进行同源重组，测序后经拼接获得具有完整编码区的辣椒类与β-紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因。
8.一种与β-紫罗兰酮合成相关的类胡萝卜素裂解双加氧酶基因的应用，其特征在于：用于辣椒类胡萝卜素代谢基因工程中β-紫罗兰酮含量的提高。
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| CN115161298A (zh) * | 2022-06-26 | 2022-10-11 | 上海龙殷生物科技有限公司 | 一种双加氧酶氨基酸序列、基因及其生物生香应用 |
| CN116042663A (zh) * | 2022-07-06 | 2023-05-02 | 中国科学院昆明植物研究所 | 一种铁皮石斛β-紫罗兰酮合成关键酶基因DoCCD1及其克隆方法和应用 |
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Application publication date: 20211019 |