CN111961679A - 博落回八氢番茄红素脱氢酶基因的核苷酸序列及应用 - Google Patents
博落回八氢番茄红素脱氢酶基因的核苷酸序列及应用 Download PDFInfo
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- CN111961679A CN111961679A CN202010876337.4A CN202010876337A CN111961679A CN 111961679 A CN111961679 A CN 111961679A CN 202010876337 A CN202010876337 A CN 202010876337A CN 111961679 A CN111961679 A CN 111961679A
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- macleaya cordata
- nucleotide sequence
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Abstract
本发明提供一种博落回八氢番茄红素脱氢酶基因的核苷酸序列及应用,所述博落回八氢番茄红素脱氢酶的核苷酸序列如SEQ ID NO.1所示,本发明还提供了一种博落回八氢番茄红素脱氢酶的氨基酸序列,所述氨基酸序列如SEQ ID NO.2所示。通过引物设计及PCR扩增的方法获得了博落回八氢番茄红素脱氢酶基因,并将其应用于基于CRISPR/Cas9的博落回基因的构建体系,为首次在博落回植物中实现基因编辑,为博落回中功能基因挖掘、种质创新等提供有力途径。
Description
技术领域
本发明属于基因工程技术领域,具体涉及博落回八氢番茄红素脱氢酶基因的核苷酸序列及应用。
背景技术
博落回(Macleaya cordata(Willd.)R.Br)是一种重要的药用植物,通过研究,目前已成功开发了“博落回提取物”、“博普总碱”两个二类中兽药新药产品,作为饲用抗生素的天然来源替代品广泛用于畜禽水产等养殖业中,随着我国《全国遏制动物源细菌耐药行动计划(2017-2020年)》计划的实施,目前已经全面禁止饲用抗生素的添加,而由博落回开发的系列替抗产品优势逐渐凸显,但是,博落回资源非常有限,若能结合现代分子育种技术对博落回进行定向改良,将为快速获得优质博落回种质提供途径,因此,发展一种针对博落回的CRISPR/Cas体系,并将其应用在博落回基因修饰及种质创新领域显得尤为重要。
对基因进行定点修饰,是生物研究领域重要的方法之一。随着科学的发展,越来越多的沉默技术发展迅速。从经典的MES随机诱变,T-DNA或转座子插入失活到锌指结构(ZFs)与转录激活因子样效应物核酸酶(Transcription activator-like effector nucleases,TALENs)定点突变,这些技术都大大促进了研究基因功能的进程。但由于锌指核糖核酸酶(ZFN)与转录激活因子样效应物核酸酶(TALENs)技术需要针对每一个目的基因设计特定的内切酶,且构建过程繁琐,大大限制了其应用范围。与其他沉默体系相比,CRISPR定点突变技术有其无法比拟的优点,逐渐被广泛地应用与基因定点修饰研究中。
CRISPR/Cas系统(clustered,regularly interspaced,short palindromicrepeats-associated protein systems)最早是在大肠杆菌中发现的,是细菌针对噬菌体等外源DNA的获得性免疫系统,该系统主要依赖于crRNA与Cas蛋白形成的核糖核蛋白复合物识别靶序列上的PAM结构,进而对入侵噬菌体或质粒进行特异性切割。CRISPR系统主要有三种类型,其中II型体系仅需要一个Cas9蛋白、crRNA与tracrRNA就能行使其功能。有研究表明将crRNA与tracrRNA整合成sgRNA并不影响CRISPR/Cas9体系的作用。2013年8月,自然生物技术期刊上首次同时发表了三篇有关CRISPR/Cas9体系成功应用于植物基因修饰的研究。之后,CRISPR/Cas9体系被广泛地应用于拟南芥、烟草、水稻、高粱等模式植物及作物研究上,但CRISPR/Cas9体系在植物药材中的应用还非常少。
PDS基因是八氢番茄红素脱氢酶基因的简称,八氢番茄红素脱氢酶参与催化无色的八氢番茄红素转变成有色类胡萝卜素,是调控叶片白化的关键基因。目前已经使用CRISPR/Cas9技术编辑甘蓝、苹果、香蕉、菠萝等物种中的PDS基因,并成功编辑获得白化植株,但在博落回的研究上则是一片空白。
发明内容
为了克服现有技术中的问题,本申请提供一种博落回八氢番茄红素脱氢酶基因的核苷酸序列及应用,为研究博落回以及博落回八氢番茄红素脱氢酶(PDS)基因提供了基础。
为了实现上述目的,本发明通过以下技术方案实现:
本发明第一方面提供了一种博落回八氢番茄红素脱氢酶基因的核苷酸序列,所述博落回八氢番茄红素脱氢酶的核苷酸序列如SEQ ID NO.1所示。
在一个优选的实施方案中,所述核苷酸序列利用如下所示的引物通过PCR扩增获得,
上游引物McPDS-F:5'—ATGACTTTAACTGGTTCTGTT—3'
下游引物McPDS-R:5'—TTAAGCGATGCTTGCCTCTGT—3'。
在一个优选的实施方案中,所述PCR扩增体系如下:
PCR程序如下:98℃预变性30s;98℃变性10s,58℃退火30s,72℃延伸2min,×30个循环;72℃2min。
本发明第二方面提供了一种编码博落回八氢番茄红素脱氢酶蛋白的氨基酸序列,所述氨基酸序列如SEQ ID NO.2所示。
本发明第三方面提供一种博落回八氢番茄红素脱氢酶基因的核苷酸序列在CRISPR/Cas9体系构建中的应用。
在一个优选的实施方案中,本发明在在博落回八氢番茄红素脱氢酶的基因编码区设计一条靶序列,退火磷酸化反应后,获得靶序列双链,将此靶序列双链插入表达载体中,挑选阳性克隆的质粒进行测序,将测序正确的重组质粒转化农杆菌,利用农杆菌介导侵染植物外植体后进行组织培养,获得突变植株。
在一个优选的实施方案中,所述真核表达载体为pRGEB32。
在一个优选的实施方案中,所述农杆菌为根癌农杆菌GV3101。
本发明通过提取博落回总RNA,然后反转录为cDNA,以博落回cDNA为模板,设计引物利用PDR技术对博落回PDS基因进行扩增,在博落回PDS基因的编码区避开内含子区域设计一个PDS基因靶序列Guide1,结合pRGEB32质粒Bsa I酶切粘性末端设计靶序列引物对,获得靶序列双链后,将pRGEB32质粒用Bsa I酶切成线性化质粒,将上述获得的靶序列双链插入到pRGEB32载体中,将构建正确的重组载体pRGEB32-PDS1转化至GV3101根癌农杆菌,将转化正确的细菌进行扩大培养后侵染博落回外植体,组织培养完成后获得无明显白化表型、有明显白化表型、嵌合表型三种类型的博落回转基因阳性幼苗68株,其中无明显白化表型幼苗29株、嵌合表型幼苗3株、有明显白化表型幼苗36株。测序结果鉴定发现靶位点出现套峰,说明在靶位点存在多种突变类型。68株阳性幼苗进行T-A克隆,转入大肠杆菌DH5α感受态细胞中并挑选约10个阳性克隆进行测序鉴定,总计测序678个单菌落,获得15种突变类型,结果表明,这些突变均发生在Cas9蛋白的剪切位点附近,类型包括碱基缺失、碱基插入、碱基替换等,其主要以碱基缺失突变为主,最长的碱基缺失达29bp。
与现有技术相比,本发明的有益效果如下:
本发明公布了一种博落回八氢番茄红素脱氢酶(PDS)基因序列,通过引物设计及PCR扩增的方法得到了博落回八氢番茄红素脱氢酶基因,并将其应用于基于CRISPR/Cas9的博落回基因的构建体系,为首次在博落回植物中实现基因编辑,为博落回中功能基因挖掘、种质创新等提供有力途径。
附图说明
图1为本发明实施例2中博落回PDS基因的靶点序列设计示意图;
图2为本发明实施例2中pRGEB32质粒图谱;
图3为本发明实施例2中博落回PDS基因编辑转基因阳性幼苗,其中A为无明显白化表型幼苗,B为嵌合表型幼苗,C为有明显白化表型幼苗;
图4为本发明实施例2中博落回PDS-Guide1靶位点测序结果图;其中方框中为PDS-Guide1靶位点,箭头为套峰出现的起始位点;
图5为本发明实施例2中博落回PDS-guide1靶位点T-A克隆测序突变类型及对应的峰图,其中方框中为突变的位点及具体的突变类型,阴影部分为靶位点。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明中生物材料的来源
pRGEB32质粒由湖南省中医药研究院张水寒实验室馈赠,其他常规试剂、药品及耗材均为现有技术中常用物质。
本发明第一方面提供了一种博落回八氢番茄红素脱氢酶基因的核苷酸序列,所述博落回八氢番茄红素脱氢酶基因的核苷酸序列如SEQ ID NO.1所示。
本发明第二方面提供了一种编码博落回八氢番茄红素脱氢蛋白的氨基酸序列,所述氨基酸序列如SEQ ID NO.2所示。
本发明第三方面提供一种博落回八氢番茄红素脱氢酶的核苷酸序列在CRISPR/Cas9体系构建中的应用。
实施例一博落回PDS基因的合成
1.博落回总RNA的提取
使用植物RNA提取试剂盒提取博落回叶片中的总RNA,方法如下:
(1)匀浆处理:100mg博落回新鲜叶片组织磨粉,加入450μL RL并震荡混匀。
(2)转移至过滤柱CS上,12,000rpm离心2-5min,取上清至RNase-Free管中。
(3)加入0.5倍体积的无水乙醇颠倒数次,再转到CR3中,12,000rpm离心30-60s,弃液,将CR3放回管中。
(4)向CR3加入350μL去蛋白液RW1,12,000rpm离心60s,弃废液。
(5)配DNase I的配制:(10μL DNase I加70μLRDD溶液)。
(6)向CR3中央加入80μL的DNase I,静置15min。
(7)加入350μL去蛋白液RW1,12,000rpm离心60s,弃废液。
(8)向CR3中加入500μL RW,室温静置2min,12,000rpm离心30-60s,弃废液,将CR3放回管中。
(9)重复上步骤。
(10)12,000rpm离心2min,倒掉废液,开盖室温放置2min。
(11)将CR3放入RNase-Free离心管中,加30-100μL RNase-Free ddH2O,室温放置2min,12,000rpm离心2min,得到RNA溶液。
2.RNA反转录为cDNA
使用PrimeScriptTM1st Strand cDNA Synthesis Kit试剂盒将上一步提取到的总RNA反转录成双链cDNA。具体操作步骤如下:
(1)反应体系一:
在PCR管中配制下列反应混合液,如表1所示:
表1
试剂 | 使用量 |
Oligo dT Primer | 1μL |
dNTP Mixture | 1μL |
模板RNA | 1μg |
RNase free ddH<sub>2</sub>O | 补足10μL |
Total | 10μL |
65℃热激5min后,放于冰上迅速冷却。
(2)加入反应体系二,如表2所示:
表2
试剂 | 使用量 |
上述变性后反应液 | 10μL |
5×PrimeScript Buffer | 4μL |
RNase Inhibitor | 0.5μL |
PrimeScript RTase | 1μL |
RNase free ddH<sub>2</sub>O | 4.5μL |
Total | 20μL |
缓慢混匀后短暂离心,放于PCR仪上进行反转录反应,30℃10min,42℃30~60min,95℃5min,4℃保持。
3.目的基因扩增
以博落回cDNA为模板,使用表3所示的引物在PCR反应体系下扩增目的基因,引物合成委托北京擎科生物科技有限公司完成。
表3
引物名称 | 序列(5’-3’) |
McPDS-F | ATGACTTTAACTGGTTCTGTT(序列如SEQ ID NO.3所示) |
McPDS-R | TTAAGCGATGCTTGCCTCTGT(序列如SEQ ID NO.4所示) |
反应体系如表4所示:
表4
成分 | 50μL反应体系 |
Q5 Hot Start High-Fidelity 2X Master Mix | 25μL |
McPDS-F | 1.25μL |
McPDS-R | 1.25μL |
博落回cDNA | 1μL |
ddH<sub>2</sub>O | 21.5μL |
轻轻混匀,短暂离心5s。扩增条件:98℃预变性30s,(98℃变性10s,58℃退火30s,72℃延伸2min)×30个循环,72℃2min,4℃保持,即得博落回PDS基因。
实施例2
1.博落回PDS基因靶序列设计及序列合成
(1)使用植物基因组编辑靶位点设计软件CRISPR-P 2.0(http://crispr.hzau.edu.cn/CRISPR2/),在博落回PDS基因的编码区避开内含子区域设计一个PDS基因靶序列Guide1,如图1中阴影部分序列所示,核苷酸序列为GAAACAATGAAATGCTTACA,如SEQ ID NO.5所示。
其中靶序列的设计原则有:a、靶序列主要包括20个碱基,并且这20个碱基3’端是NGG(N为任意碱基)3个碱基的PAM区(Protospacer adjacent motif,PAM),如图1中方框部分序列所示;b、靶位点尽量选择在基因编码区的前端。
(2)利用(1)中基因靶序列Guide1(20bp),结合图2所示的pRGEB32质粒Bsa I酶切粘性末端设计靶序列引物对pRGEB32-PDS-Guide 1-Oligo 1和pRGEB32-PDS-Guide 1-Oligo 2,具体序列见表5,下划线部分为与pRGEB32质粒Bsa I酶切粘性末端相互补的碱基片段,引物合成委托北京擎科生物科技有限公司完成。
表5
2.博落回PDS基因敲除载体构建
(1)将上述表5合成的靶序列引物对进行退火磷酸化反应,反应条件为37℃,30min,转95℃,5min,再以5℃/min降温速度降至25℃,获得靶序列双链。
(2)将pRGEB32质粒(湖南省中医药研究院张水寒实验室馈赠)用Bsa I酶切成线性化质粒,反应条件为37℃,15min,并进行1%的琼脂糖凝胶电泳,电泳条件为120V,20min,切胶回收目标片段,用凝胶回收试剂盒纯化目的DNA,片段大小约15868bp。
(3)将(1)中制备的靶序列双链通过连接酶与(2)中制备的pRGEB32线性化载体相连接,连接条件为室温放置10min,取5μL的连接产物至50μL的DH5α感受态细胞细胞中,并混合(刚在冰上解冻时加入),冰上孵育30min,42℃热激90s,然后立即放入冰水中静置3min,向离心管中加入500μL LB液体培养基,37℃,180r条件下震荡培养45min,涂布于含卡那霉素的LB固体筛选培养基上,37℃培养12h。
(4)使用Primer Premier 6.0引物设计软件在pRGEB32载体靶位点连接位点的上下游各约300-400bp左右分别设计PCR扩增的上下游引物,引物序列见表6,重组质粒扩增目标条带616bp,主要用于载体构建结果的鉴定,引物合成委托北京擎科生物科技有限公司完成。
表6
(5)利用M13R和pRGEB32-R引物对菌落进行PCR鉴定,筛选阳性克隆,鉴定正确重组载体命名为:pRGEB32-PDS1。
3.根癌农杆菌介导的博落回遗传转化
(1)取5μL重组载体pRGEB32-PDS1至50μL的GV3101根癌农杆菌感受态细胞中,并混合(刚在冰上解冻时加入),液氮速冻1min,37℃水浴5min,向离心管中加入1ml LB液体培养基,28℃,120r条件下震荡培养4h,涂布于含卡那霉素和利福平的LB固体筛选培养基上,28℃培养48h,待长出根癌农杆菌单菌落。
(2)挑选根癌农杆菌单克隆提取质粒,PCR鉴定(M13R/pRGEB32-R引物对)筛选阳性克隆,并提取质粒委托北京擎科生物科技有限公司进行测序,测序正确阳性克隆进行扩大培养,培养至OD600为0.6-0.8时进行博落回外植体(茎段及叶盘)的侵染。
(3)完成侵染的博落回外植体转移至含有乙酰丁香酮的MS培养基中共培养三天,后转移至含潮霉素和特美汀的MS筛选培养基进行筛选培养,完成整个组培过程,并获得无明显白化表型、有明显白化表型、嵌合表型三种类型的博落回转基因阳性幼苗。
实施例3基因编辑结果鉴定
使用Primer Premier 6.0引物设计软件在PDS基因靶位点上下游各约300-400bp左右分别设计PCR扩增的上下游引物,引物序列见表7,扩增目标条带约795bp,主要用于靶位点突变鉴定,引物合成委托北京擎科生物科技有限公司完成。
表7
引物名称 | 序列(5’-3’) |
PDS-F | GCAGTGGAAGGAACACTCTATGATT(序列如SEQ ID NO.10所示) |
PDS-R | ATCTTTAACGGTAAAGCCATCTTGA(序列如SEQ ID NO.11所示) |
农杆菌介导的pRGEB32-PDS1质粒首次获得转基因阳性幼苗68株,其中无明显白化表型幼苗29株、嵌合表型幼苗3株、有明显白化表型幼苗36株。分别提取68株阳性博落回育苗的DNA,利用表7所示的PDS-F和PDS-R引物对目的片段进行PCR扩增,测序鉴定,发现靶位点出现套峰,结果如图4所示,其中方框中为PDS-Guide1靶位点,箭头为套峰出现的起始位点,图4说明在靶位点存在多种突变类型。
目的片段PCR产物(PDS-F/PDS-R)的测序结果表明在68株转基因博落回中有59株出现突变现象,这表明博落回中PDS基因的靶位点PDS-guide1的打靶效率达86.76%,为非常高效的博落回基因编辑体系。
为进一步确定转基因博落回中的基因编辑突变形式,针对上述的68株阳性幼苗进行T-A克隆,转入大肠杆菌DH5α感受态细胞中并挑选约10个阳性克隆进行测序鉴定,总计测序678个单菌落,获得15种突变类型,结果如图5所示,这些突变均发生在Cas9蛋白的剪切位点附近,类型包括碱基缺失、碱基插入、碱基替换等,其主要以碱基缺失突变为主,最长的碱基缺失达29bp。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明,不能认定本发明的具体实施只限于这些说明。对于本发明所属领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干简单推演或替换,都应当视为属于本发明的保护范围。
序列表
<110> 湖南美可达生物资源股份有限公司
<120> 博落回八氢番茄红素脱氢酶基因的核苷酸序列及应用
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1752
<212> DNA
<213> Macleaya cordata
<400> 1
atgactttaa ctggttctgt ttctgttgta aatttgagcg ggcaaggtac tacaatcaac 60
atttggaact caaattccag cagaagatgt tgtttctcta tcaattctgg agataacaat 120
ctattagcat ttggagggag tgattctatg gggcagcccc tgaaatcacg aaaggcacat 180
gctgttgcaa ttcggccaag gaggaatatt ggccctttgc aggttgtttg catggactac 240
ccaagacctg aactcgaaaa tactgttaat ttcatagagg ctgcttactt atcttcatcc 300
ttccgtactt ctgcccgtcc aaataaacca ttacaagttg taattgctgg tgcaggcttg 360
gctggtctat ctacggcaaa atatctggca gatgcaggtc acaaacccat attgctggaa 420
gcaagagatg ttctgggtgg aaaggtggct gcatggaagg atgatgatgg agactggtac 480
gagaccggcc tacacatatt ttttggagct tatccgaacg tgcagaattt gtttggagaa 540
cttggtatta atgatcggct gcagtggaag gaacactcta tgatttttgc catgccaagc 600
aagccaggag agttcagccg atttgatttt cccgatgttc ttcctgcacc cttaaatggg 660
atctgggcta tcttaagaaa caatgaaatg cttacatggc cggagaaagt acggtttgcc 720
attggactct tgcctgcaat ggttggtgga caggcttatg tggaggctca agatggcttt 780
accgttaaag attggatgag aaagcagggt gttccagatc gagtaactga tgaggtgttt 840
atcgccatgt caaaggcact aaacttcata aacccagatg agctttcgat gcaatgcatt 900
ttgattgcct tgaatcgatt tcttcaggaa aagcatggtt ccaagatggc ctttttagat 960
ggtaaccctc ctgagagact ttgcatgcca atcgttgagc atatccagtc actaggtggc 1020
caagttcaac tcaattcacg aattcaaaag atcgagctga aaaatgatgg aactgtgaag 1080
cgtcttatac tcactaatgg caatgcaata gaaggagatg cctatgtaat tgcaacacca 1140
gttgatatcc tgaagcttct tctacctgaa gactggaaag agattccata cttcaagaga 1200
ttggacaaac tagttggtgt tcctgtgatt aacgttcata tatggtttga caggaaactg 1260
aagaacacat atgatcatct actcttcagc agaagtcccc tcttgagtgt atatgcagac 1320
atgtctgtaa catgtaagga atattacgat ccaaaccgct ccatgcttga gttggttttt 1380
gcacctgctg aagactggat ctcatgcagt gacacggaaa ttattgaagc taccctgaag 1440
gagcttgcga aactctttcc tgatgaaatt gctgcagatc agagcaaagc aaaaatattg 1500
aagtaccaca ttgttaaaac accacggtct gtatataaaa ctgttcctga ttgtgaacca 1560
tgccgtccat tgcaaagatc tccagtagag ggattctatt tggctggtga ctacacaaag 1620
caaaagtatt tggcttcaat ggaaggtgca gttttgtctg ggaagctctg cgcgcaggct 1680
attgtgcagg actatgagtt actcgctgct cggggagaaa aaaccaggac aacagaggca 1740
agcatcgctt aa 1752
<210> 2
<211> 583
<212> PRT
<213> Macleaya cordata
<400> 2
Met Thr Leu Thr Gly Ser Val Ser Val Val Asn Leu Ser Gly Gln Gly
1 5 10 15
Thr Thr Ile Asn Ile Trp Asn Ser Asn Ser Ser Arg Arg Cys Cys Phe
20 25 30
Ser Ile Asn Ser Gly Asp Asn Asn Leu Leu Ala Phe Gly Gly Ser Asp
35 40 45
Ser Met Gly Gln Pro Leu Lys Ser Arg Lys Ala His Ala Val Ala Ile
50 55 60
Arg Pro Arg Arg Asn Ile Gly Pro Leu Gln Val Val Cys Met Asp Tyr
65 70 75 80
Pro Arg Pro Glu Leu Glu Asn Thr Val Asn Phe Ile Glu Ala Ala Tyr
85 90 95
Leu Ser Ser Ser Phe Arg Thr Ser Ala Arg Pro Asn Lys Pro Leu Gln
100 105 110
Val Val Ile Ala Gly Ala Gly Leu Ala Gly Leu Ser Thr Ala Lys Tyr
115 120 125
Leu Ala Asp Ala Gly His Lys Pro Ile Leu Leu Glu Ala Arg Asp Val
130 135 140
Leu Gly Gly Lys Val Ala Ala Trp Lys Asp Asp Asp Gly Asp Trp Tyr
145 150 155 160
Glu Thr Gly Leu His Ile Phe Phe Gly Ala Tyr Pro Asn Val Gln Asn
165 170 175
Leu Phe Gly Glu Leu Gly Ile Asn Asp Arg Leu Gln Trp Lys Glu His
180 185 190
Ser Met Ile Phe Ala Met Pro Ser Lys Pro Gly Glu Phe Ser Arg Phe
195 200 205
Asp Phe Pro Asp Val Leu Pro Ala Pro Leu Asn Gly Ile Trp Ala Ile
210 215 220
Leu Arg Asn Asn Glu Met Leu Thr Trp Pro Glu Lys Val Arg Phe Ala
225 230 235 240
Ile Gly Leu Leu Pro Ala Met Val Gly Gly Gln Ala Tyr Val Glu Ala
245 250 255
Gln Asp Gly Phe Thr Val Lys Asp Trp Met Arg Lys Gln Gly Val Pro
260 265 270
Asp Arg Val Thr Asp Glu Val Phe Ile Ala Met Ser Lys Ala Leu Asn
275 280 285
Phe Ile Asn Pro Asp Glu Leu Ser Met Gln Cys Ile Leu Ile Ala Leu
290 295 300
Asn Arg Phe Leu Gln Glu Lys His Gly Ser Lys Met Ala Phe Leu Asp
305 310 315 320
Gly Asn Pro Pro Glu Arg Leu Cys Met Pro Ile Val Glu His Ile Gln
325 330 335
Ser Leu Gly Gly Gln Val Gln Leu Asn Ser Arg Ile Gln Lys Ile Glu
340 345 350
Leu Lys Asn Asp Gly Thr Val Lys Arg Leu Ile Leu Thr Asn Gly Asn
355 360 365
Ala Ile Glu Gly Asp Ala Tyr Val Ile Ala Thr Pro Val Asp Ile Leu
370 375 380
Lys Leu Leu Leu Pro Glu Asp Trp Lys Glu Ile Pro Tyr Phe Lys Arg
385 390 395 400
Leu Asp Lys Leu Val Gly Val Pro Val Ile Asn Val His Ile Trp Phe
405 410 415
Asp Arg Lys Leu Lys Asn Thr Tyr Asp His Leu Leu Phe Ser Arg Ser
420 425 430
Pro Leu Leu Ser Val Tyr Ala Asp Met Ser Val Thr Cys Lys Glu Tyr
435 440 445
Tyr Asp Pro Asn Arg Ser Met Leu Glu Leu Val Phe Ala Pro Ala Glu
450 455 460
Asp Trp Ile Ser Cys Ser Asp Thr Glu Ile Ile Glu Ala Thr Leu Lys
465 470 475 480
Glu Leu Ala Lys Leu Phe Pro Asp Glu Ile Ala Ala Asp Gln Ser Lys
485 490 495
Ala Lys Ile Leu Lys Tyr His Ile Val Lys Thr Pro Arg Ser Val Tyr
500 505 510
Lys Thr Val Pro Asp Cys Glu Pro Cys Arg Pro Leu Gln Arg Ser Pro
515 520 525
Val Glu Gly Phe Tyr Leu Ala Gly Asp Tyr Thr Lys Gln Lys Tyr Leu
530 535 540
Ala Ser Met Glu Gly Ala Val Leu Ser Gly Lys Leu Cys Ala Gln Ala
545 550 555 560
Ile Val Gln Asp Tyr Glu Leu Leu Ala Ala Arg Gly Glu Lys Thr Arg
565 570 575
Thr Thr Glu Ala Ser Ile Ala
580
<210> 3
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgactttaa ctggttctgt t 21
<210> 4
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ttaagcgatg cttgcctctg t 21
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gaaacaatga aatgcttaca 20
<210> 6
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ggcagaaaca atgaaatgct taca 24
<210> 7
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
aaactgtaag catttcattg tttc 24
<210> 8
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gagcggataa caatttcaca cagg 24
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gacccgaatt tgtggacctg 20
<210> 10
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gcagtggaag gaacactcta tgatt 25
<210> 11
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
atctttaacg gtaaagccat cttga 25
Claims (10)
1.一种博落回八氢番茄红素脱氢酶基因的核苷酸序列,其特征在于,所述博落回八氢番茄红素脱氢酶的核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的博落回八氢番茄红素脱氢酶基因的核苷酸序列,其特征在于,所述核苷酸序列利用如下所示的引物通过PCR扩增获得,
上游引物McPDS-F:5'—ATGACTTTAACTGGTTCTGTT—3'
下游引物McPDS-R:5'—TTAAGCGATGCTTGCCTCTGT—3'。
4.含有权利要求1所述的核苷酸序列的载体。
5.含有权利要求1所述的核苷酸序列的工程菌。
6.一种编码博落回八氢番茄红素脱氢酶蛋白的氨基酸序列,其特征在于,所述氨基酸序列如SEQ ID NO.2所示。
7.一种如权利要求1所述的博落回八氢番茄红素脱氢酶基因的核苷酸序列在CRISPR/Cas9体系构建中的应用。
8.根据权利要求7所述的博落回八氢番茄红素脱氢酶基因的核苷酸序列在CRISPR/Cas9体系构建中的应用,其特征在于,包括如下步骤,
在博落回八氢番茄红素脱氢酶的基因编码区设计一条靶序列,退火磷酸化反应后,获得靶序列双链,将此靶序列双链插入表达载体中,挑选阳性克隆的质粒进行测序,将测序正确的重组质粒转化农杆菌,利用农杆菌介导侵染植物外植体后进行组织培养,获得突变植株。
9.根据权利要求7所述的博落回八氢番茄红素脱氢酶基因的核苷酸序列在CRISPR/Cas9体系构建中的应用,其特征在于,所述表达载体为pRGEB32。
10.根据权利要求7所述的博落回八氢番茄红素脱氢酶基因的核苷酸序列在CRISPR/Cas9体系构建中的应用,其特征在于,所述农杆菌为根癌农杆菌GV3101。
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