CN106636146A - 一种降低尼古丁转化率的cyp82e5基因突变体及其应用 - Google Patents
一种降低尼古丁转化率的cyp82e5基因突变体及其应用 Download PDFInfo
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- CN106636146A CN106636146A CN201710082843.4A CN201710082843A CN106636146A CN 106636146 A CN106636146 A CN 106636146A CN 201710082843 A CN201710082843 A CN 201710082843A CN 106636146 A CN106636146 A CN 106636146A
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Abstract
本发明公开了一种降低尼古丁转化率的CYP82E5基因突变体及其应用。所述的降低尼古丁转化率的CYP82E5基因突变体，相对于核苷酸序列为SEQ ID NO.1所示的CYP82E5基因，所述的降低尼古丁转化率的CYP82E5基因突变体含有的CYP82E5基因序列中392位的G被A取代发生了点突变，所述的降低尼古丁转化率的CYP82E5基因突变体的核苷酸序列如SEQ ID NO.3所示。应用为所述的降低尼古丁转化率的CYP82E5基因突变体在获得尼古丁低转化率的烟草植株中的应用。本发明所述的降低尼古丁转化率的CYP82E5基因突变体获得的云烟87材料中，叶片尼古丁转化率比对照下降约20%，显著降低了尼古丁的转化率。
Description
技术领域
本发明属于遗传工程技术领域，具体涉及一种降低尼古丁转化率的CYP82E5基因突变体及其应用。
背景技术
尼古丁（nicotine）、去甲基尼古丁（nornicotine）、假木贼碱（anabasine）、和新烟碱（anatabine）是烟草（Nicotiana tobacum）中主要的生物碱，其中尼古丁是首要的生物碱，占生物碱总含量的90-95%，去甲基尼古丁含量通常低于总生物碱的3.5%，是尼古丁通过去甲基化反应转化生成。去甲基尼古丁能够对人体健康产生危害，主要表现在它是卷烟烟气中潜在致癌物质亚硝基去甲基尼古丁（nitrosonornicotine, NNN）的合成前体，可能导致食管癌、口腔癌的发生。去甲基尼古丁也能直接诱导吸烟者血浆中蛋白的异常糖基化，也有研究表明它能与常用类固醇类药物发生共价反应，影响药效和毒性。因此，减少尼古丁转化生成去甲基尼古丁，即降低尼古丁转化率具有重大意义。
国际上有两种类型的烟草在生产中广泛应用，白肋烟和烤烟，其中白肋烟生产主要在西方发达国家及南美洲，我国主要种植烤烟。白肋烟CYP82E2亚家族的CYP82E4、CYP82E5、CYP82E10基因能够编码具有活性的尼古丁去甲基酶，是尼古丁转化的关键酶。Gavilano等通过RNAi技术抑制白肋烟强“转化株”CYP82E4及其同源基因的表达，去甲基尼古丁合成受到显著抑制，转基因植株的尼古丁转化率最低只有0.8%，甚至低于白肋烟“非转化株”普遍大约3-5%的转化率。Julio等人在1132个EMS诱变突变株中筛选得到10株在CYP82E4基因座发生点突变的烟草，其中无义突变及错义突变株中尼古丁转化率降至极其微量水平。Lewis等人用EMS诱变的方法分别获得了CYP82E4、CYP82E5、CYP82E10发生突变的白肋烟材料，发现CYP82E5、CYP82E10基因突变不影响尼古丁转化率，三个基因同时突变的突变株中尼古丁转化率远远低于对照株。上述研究表明白肋烟中CYP82E4是决定尼古丁转化率的关键基因，CYP82E5、CYP82E10基因无明显影响。白肋烟和烤烟中尼古丁转化机制不同，但目前尚无烤烟尼古丁去甲基酶基因的功能研究发表，降低其尼古丁转化率的方法仍需探索。
发明内容
本发明的第一目的在于提供一种降低尼古丁转化率的CYP82E5基因突变体；第二目的在于提供所述的降低尼古丁转化率的CYP82E5基因突变体的应用。
本发明的第一目的是这样实现的，相对于核苷酸序列为SEQ ID NO.1所示的CYP82E5基因，所述的降低尼古丁转化率的CYP82E5基因突变体含有的CYP82E5基因序列中392位的G被A取代发生了点突变，所述的降低尼古丁转化率的CYP82E5基因突变体的核苷酸序列如SEQ ID NO.3所示。
本发明的第二目的是这样实现的，所述的降低尼古丁转化率的CYP82E5基因突变体在获得尼古丁低转化率的烟草植株中的应用。
本发明所述的降低尼古丁转化率的CYP82E5基因突变体获得的云烟87材料中，叶片尼古丁转化率比对照下降约20%，显著降低了尼古丁的转化率。
附图说明
图1为CYP82E5基因突变毛细管电泳检测；
图2为突变体材料测序结果比对；
图3为突变体株系572号的测序峰图；
图4为突变体材料尼古丁转化率分析。
具体实施方式
下面结合实施例和附图对本发明作进一步的说明，但不以任何方式对本发明加以限制，基于本发明教导所作的任何变换或替换，均属于本发明的保护范围。
本发明所述的降低尼古丁转化率的CYP82E5基因突变体，相对于核苷酸序列为SEQID NO.1所示的CYP82E5基因，所述的降低尼古丁转化率的CYP82E5基因突变体含有的CYP82E5基因序列中392位的G被A取代发生了点突变，所述的降低尼古丁转化率的CYP82E5基因突变体的核苷酸序列如SEQ ID NO.3所示。
所述的降低尼古丁转化率的CYP82E5基因突变体编码的氨基酸序列如SEQ IDNO.4所示。
本发明所述的降低尼古丁转化率的CYP82E5基因突变体应用为所述的降低尼古丁转化率的CYP82E5基因突变体在获得尼古丁低转化率的烟草植株中的应用。
下面以具体实施案例对本发明做进一步说明：
实施例1
烤烟突变体库创制
一、烤烟种子EMS处理
烤烟（品种：云烟87）种子用50%的市售商品漂白液浸泡12分钟后，用去离子水快速清洗，并在去离子水中浸泡12小时。弃去离子水，加入等体积0.5% EMS（甲基磺酸乙酯）处理12小时。弃处理液，加入去离子水清洗6-8次，每次约1分钟。种子收集于布氏漏斗，抽干待用。
二、M1植株田间种植
EMS处理完毕种子播种于漂浮盘，每穴一粒种子，出苗后移栽至田间，正常农艺措施管理。现蕾后单株套袋编号收种获得M2种子。
三、突变体基因组DNA提取及混样
利用试剂盒提取基因组DNA，步骤如下：
称取0.1g鲜样，液氮研磨、细碎后，转入2.0 ml样品管中，立即加入600μL AP1缓冲液和4μL RNaseA贮存液（100 mg/ml）。65℃中水浴处理10分钟，期间颠倒EP管2-3次。加入190μLAP2缓冲液，混匀置于冰上5分钟， 14000 rpm室温离心5分钟。吸取上清液至QIAshredderMini柱，14000 rpm室温离心2分钟。取450—650μL过滤液至2.0ml离心管中，再加入675-900μL的缓冲液AP3/E。取650μL混合物至2 ml的DNeasy柱内，室温以≥8000rpm离心1-2分钟。将上述的DNeasy柱放入新的2 ml收集管中，加入500 μL AW缓冲液，以≥8000rpm离心2分钟，丢弃流出液，再用AW缓冲液重复清洗一次。将DNeasy柱放在1.5 ml离心管中，加入100μL AE缓冲液，室温放置5分钟，离心所得滤液即为基因组DNA。
大田种植约2200份M2代EMS突变体植株，采集叶片并利用上述方法提取基因组DNA，将所有样品DNA浓度稀释至40 ng/μl，最终建立M2代云烟87突变体的DNA库，每8份DNA混样，构成8倍混合池，保存于96孔板中。
实施例2
CYP82E5基因终止突变体筛选
一、Tilling引物
CYP82E5基因有两个外显子，利用Tilling技术筛选第一个外显子区域的突变体。根据目地基因的基因组序列，
正向引物为CYP82E5_F:5’-GGTAATTTTGTATTTATTATATTATGCG-3’；
反向引物为CYP82E5_R：5’-TCATCCTTAGTATTTAGATAATCTAATT-3’。
二、PCR扩增条件
PCR 反应体系如下：总体积为10 μL，其中20 ng/μL DNA 样品1.0 μL、10×PCR buffer1.0 μL， dNTPs 0.8 μL，引物各0.16 μL， Taq DNA酶0.1 μL，ddH2O 6.78 μL。
PCR 反应程序如下： 95℃预变性3 分钟；94℃变性30 秒，62℃退火30 秒（每个循环下降1℃），72℃延伸90 秒，运行7 个循环；94℃变性30 秒，58℃退火30 秒，72℃延伸90秒，运行40 个循环；最后72℃延伸5 分钟。PCR 扩增产物可以在4℃保存。
三、PCR产物酶切及电泳
利用CEL I酶特异性切割异源双链的特性，对PCR产物进行酶切，酶切体系如下：PCR 产物4 μL， 10×buffer 1μL，CEL I酶 0.5μL，补充H2O至总体积为10μL。酶切体系利用自动毛细管电泳系统进行分离，分离条件如下：样品上样电压为9KV，上样30 sec，Marker的上样电压为7.5KV，上样5sec，预电泳电压9KV，分离电泳电压9 kv，运行时间80 min，电泳结果用Prosize 2.0软件分析。Tilling筛选共获得9个CYP82E5基因突变体，其中编号572单株，有一个密码子从TGG突变为TAG，为CYP82E5基因终止突变，结果见表1和图1。
表1 云烟87突变体库中CYP82E5基因突变体分析
实施例3
CYP82E5基因终止突变验证
一、M3代突变体基因组DNA提取及PCR扩增
根据M2代植株Tilling筛选结果，选择CYP82E5基因目标区域发生突变单株的种子（M3代，编号572），在育苗盘中播种。采用试剂盒法提取幼苗叶片基因组DNA。以CYP82E5_F和CYP82E5_R引物，以基因组DNA为模板扩增CYP82E5基因第一个外显子区域。PCR 反应体系如下：总体积为25 μL，其中20 ng/μL DNA 样品1.0 μL、10×PCR buffer 2.5 μL， dNTPs 2 μL，引物各0.5 μL， Taq DNA酶0.3 μL，ddH2O 18.2 μL。PCR 反应程序如下： 95℃预变性3分钟；94℃变性30 秒，55℃退火30 秒（每个循环下降1℃），72℃延伸90 秒，运行30 个循环； 72℃延伸5 分钟。PCR 扩增产物可以在4℃保存。
二、PCR产物TOPO克隆及测序
PCR产物回收后连接pTOPO载体（Invitrogen），体系如下：PCR产物4μL，pCR-BluntII-TOPO质粒1μL，salt Solution 1μL。反应组分25℃孵育30min，转E.coli.感受态细胞中混匀，冰浴30min，42℃热休克90sec后立即放入冰浴中2min，加入0.35mL的LB液体培养基，37℃ 210rpm振荡培养1h。离心1min（7500rpm），弃上清至约100μl混匀，均匀涂布于Km抗性培养基上，37℃过夜培养。选择阳性克隆提取质粒后进行Sanger测序，结果见图2和图3。
实施例4
CYP82E5基因终止突变体去甲基尼古丁含量分析
一、突变体材料样品制备
烟草生长至成熟期，取整株烟叶样品60 ℃烘干，粉碎过60目筛待测。准确称取烟样0.5g于50 mL离心管中，加入5 mL 10%的NaOH溶液，摇匀，浸泡15 min，加入20 mL含内标的萃取液，超声60 min，在离心机上5000 r/min离心5 min，取2 mL下层二氯甲烷清液过装有2 g无水硫酸钠的微孔过滤器后，用气相色谱-串联质谱仪分析。
二、叶片去甲基尼古丁含量分析
采用GC-MS-MS法分析叶片中去甲基尼古丁含量。传输线温度：230℃，离子源温度：210℃；电离模式：电子轰击电离（EI）；轰击能量：70 eV；灯丝电流：50 μA，电子倍增器电压1200V；碰撞气：氩气（纯度≥99.999％）；碰撞池压力：0.3Pa）；溶剂延迟时间：4 min；数据采集模式：MRM。保留时间（min）：10.48；定量离子对：119﹥92；碰撞能(eV) ：15；定性离子对：119﹥65；碰撞能(eV)： 25； 驻留时间(s)： 0.15。CYP82E5基因纯合终止突变体叶片尼古丁转化率为1.34，对照云烟87为1.69，突变体下降约20%。尼古丁转化率＝[去甲基尼古丁/去甲基尼古丁＋尼古丁]*100%，具体结果见图4。
SEQUENCE LISTING
<110> 云南省烟草农业科学研究院
<120> 一种降低尼古丁转化率的CYP82E5基因突变体及其应用
<130> 2017
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 1554
<212> DNA
<213> CYP82E5基因核苷酸
<400> 1
atggtttctc ccgtagaagc cattgtagga ctagtaaccc ttacacttct cttctacttc 60
ctatggccca aaaaatttca aataccttca aaaccattac caccgaaaat tcccggaggg 120
tggccggtaa tcggccatct tttctacttc gatgatgacg gcgacgaccg tccattagct 180
cgaaaactcg gagacttagc tgacaaatac ggcccggttt tcactttccg gctaggcctt 240
ccgcttgtgt tagttgtaag cagttacgaa gctgtaaaag actgcttctc tacaaatgac 300
gccattttct ccaatcgtcc agcttttctt tacggtgaat accttggcta caataatgcc 360
atgctatttt tgacaaaata cggaccttat tggcgaaaaa atagaaaatt agtcattcag 420
gaagttctct ctgctagtcg tctcgaaaaa ttgaagcacg tgagatttgg taaaattcaa 480
acgagcatta agagtttata cactcgaatt gatggaaatt cgagtacgat aaatctaact 540
gattggttag aagaattgaa ttttggtctg atcgtgaaaa tgatcgctgg gaaaaattat 600
gaatccggta aaggagatga acaagtggag agatttagga aagcgtttaa ggattttata 660
attttatcaa tggagtttgt gttatgggat gcttttccaa ttccattgtt caaatgggtg 720
gattttcaag gccatgttaa ggccatgaaa aggacattta aggatataga ttctgttttt 780
cagaattggt tagaggaaca tgtcaagaaa agagaaaaaa tggaggttaa tgcacaaggg 840
aatgaacaag atttcattga tgtggtgctt tcaaaaatga gtaatgaata tcttgatgaa 900
ggttactctc gtgatactgt cataaaagca acagtgttta gtttggtctt ggatgctgcg 960
gacacagttg ctcttcacat gaattgggga atggcattac tgataaacaa tcaacatgcc 1020
ttgaagaaag cacaagaaga gatcgataaa aaagttggta aggaaagatg ggtagaagag 1080
agtgatatta aggatttggt ctacctccaa gctattgtta aagaagtgtt acgattatat 1140
ccaccaggac ctttattagt acctcatgaa aatgtagagg attgtgttgt tagtggatat 1200
cacattccta aagggactag actattcgcg aacgttatga aattgcagcg cgatcctaaa 1260
ctctggtcaa atcctgataa gtttgatcca gagagattct tcgctgatga tattgactac 1320
cgtggtcagc actatgagtt tatcccattt ggttctggaa gacgatcttg tccggggatg 1380
acttatgcat tacaagtgga acacctaaca atagcacatt tgatccaggg tttcaattac 1440
aaaactccaa atgacgagcc cttggatatg aaggaaggtg caggattaac tatacgtaaa 1500
gtaaatcctg tagaagtgac aattacggct cgcctggcac ctgagcttta ttaa 1554
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Asn Gln His Ala Leu Lys Lys Ala Gln Glu Glu Ile Asp Lys Lys Val
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Gly Lys Glu Arg Trp Val Glu Glu Ser Asp Ile Lys Asp Leu Val Tyr
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ctatggccca aaaaatttca aataccttca aaaccattac caccgaaaat tcccggaggg 120
tggccggtaa tcggccatct tttctacttc gatgatgacg gcgacgaccg tccattagct 180
cgaaaactcg gagacttagc tgacaaatac ggcccggttt tcactttccg gctaggcctt 240
ccgcttgtgt tagttgtaag cagttacgaa gctgtaaaag actgcttctc tacaaatgac 300
gccattttct ccaatcgtcc agcttttctt tacggtgaat accttggcta caataatgcc 360
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attttatcaa tggagtttgt gttatgggat gcttttccaa ttccattgtt caaatgggtg 720
gattttcaag gccatgttaa ggccatgaaa aggacattta aggatataga ttctgttttt 780
cagaattggt tagaggaaca tgtcaagaaa agagaaaaaa tggaggttaa tgcacaaggg 840
aatgaacaag atttcattga tgtggtgctt tcaaaaatga gtaatgaata tcttgatgaa 900
ggttactctc gtgatactgt cataaaagca acagtgttta gtttggtctt ggatgctgcg 960
gacacagttg ctcttcacat gaattgggga atggcattac tgataaacaa tcaacatgcc 1020
ttgaagaaag cacaagaaga gatcgataaa aaagttggta aggaaagatg ggtagaagag 1080
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ccaccaggac ctttattagt acctcatgaa aatgtagagg attgtgttgt tagtggatat 1200
cacattccta aagggactag actattcgcg aacgttatga aattgcagcg cgatcctaaa 1260
ctctggtcaa atcctgataa gtttgatcca gagagattct tcgctgatga tattgactac 1320
cgtggtcagc actatgagtt tatcccattt ggttctggaa gacgatcttg tccggggatg 1380
acttatgcat tacaagtgga acacctaaca atagcacatt tgatccaggg tttcaattac 1440
aaaactccaa atgacgagcc cttggatatg aaggaaggtg caggattaac tatacgtaaa 1500
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ggtaattttg tatttattat attatgcg 28
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Claims (3)
1.一种降低尼古丁转化率的CYP82E5基因突变体，其特征在于在于相对于核苷酸序列为SEQ ID NO.1所示的CYP82E5基因，所述的降低尼古丁转化率的CYP82E5基因突变体含有的CYP82E5基因序列中392位的G被A取代发生了点突变，所述的降低尼古丁转化率的CYP82E5基因突变体的核苷酸序列如SEQ ID NO.3所示。
2.根据权利要求1所述的降低尼古丁转化率的CYP82E5基因突变体，其特征在于所述的降低尼古丁转化率的CYP82E5基因突变体编码的氨基酸序列如SEQ ID NO.4所示。
3.一种权利要求1或2所述的降低尼古丁转化率的CYP82E5基因突变体应用，其特征在于所述的降低尼古丁转化率的CYP82E5基因突变体在获得尼古丁低转化率的烟草植株中的应用。
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| CN115404250A (zh) * | 2021-05-29 | 2022-11-29 | 重庆博腾制药科技股份有限公司 | 一种利用还原方式制备(s)-尼古丁的方法 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108424922A (zh) * | 2018-04-20 | 2018-08-21 | 云南省烟草农业科学研究院 | 一种降低尼古丁转化率的cyp82e10基因错义突变体m271及其应用 |
| CN108424922B (zh) * | 2018-04-20 | 2021-04-16 | 云南省烟草农业科学研究院 | 一种降低尼古丁转化率的cyp82e10基因错义突变体m271及其应用 |
| CN115404250A (zh) * | 2021-05-29 | 2022-11-29 | 重庆博腾制药科技股份有限公司 | 一种利用还原方式制备(s)-尼古丁的方法 |
| CN115404250B (zh) * | 2021-05-29 | 2024-08-06 | 重庆博腾制药科技股份有限公司 | 一种利用还原方式制备(s)-尼古丁的方法 |
| US12319947B2 (en) | 2021-05-29 | 2025-06-03 | Porton Pharma Solutions Ltd. | Method for preparing (S)-nicotine by reduction |
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