CN113151317A - 一种烟草δ(24)-固醇还原酶基因及其应用 - Google Patents
一种烟草δ(24)-固醇还原酶基因及其应用 Download PDFInfo
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- CN113151317A CN113151317A CN202110580248.XA CN202110580248A CN113151317A CN 113151317 A CN113151317 A CN 113151317A CN 202110580248 A CN202110580248 A CN 202110580248A CN 113151317 A CN113151317 A CN 113151317A
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Abstract
本发明涉及一种烟草δ(24)‑固醇还原酶基因及其应用,δ(24)‑固醇还原酶基因的核苷酸序列如SEQ ID NO.1所示。本申请中,通过对特定δ(24)‑固醇还原酶基因的初步研究,发现其与烟草叶片中豆甾醇含量高度相关,在本氏烟草中将该基因沉默,烟草叶片中豆甾醇含量发生了明显降低,降低了27.9%,而甾醇总量变化不明显。基于这一特性,可利用基因工程手段,为烟叶品质调控、烟草新品种培育提供一定的应用基础和参考借鉴。
Description
技术领域
本发明属于烟草基因工程领域,具体涉及一种烟草δ(24)-固醇还原酶基因及其应用。
背景技术
植物甾醇是生物膜系统的重要组成成分,可以调控植物生长发育,响应多种生物和非生物胁迫。甾醇类物质约占烟叶质量的0.1%~0.3%,烟草中的甾醇类化合物主要有胆甾醇(cholesterol)、豆甾醇(stigmasterol)、菜油甾醇(campasterol)、和β-谷甾醇((β-sitosterol)等。已有文献报道其中的豆甾醇是卷烟烟气苯并芘的重要来源。因此,降低烟草成熟叶片中豆甾醇含量可有效降低卷烟烟气苯并芘含量。
目前植物中甾醇的合成代谢已有研究,但栽培烟草中调控豆甾醇合成的基因却鲜有报道。烟草中影响豆甾醇含量的基因功能研究将为烟叶安全性改善、烟草品种遗传改良提供理论支持,对提高我国烟草产品安全性具有重要的意义。
发明内容
本发明的目的在于提供一种烟草δ(24)-固醇还原酶基因及其应用,以改善烟草中豆甾醇含量,从而为烟叶品质调控、烟草新品种培育奠定一定基础。
为实现上述发明目的,本申请采用如下技术方案:
一种烟草δ(24)-固醇还原酶基因,核苷酸序列如SEQ ID NO.1所示,含有1695个碱基,命名为NtDHCR24。
进一步的,烟草δ(24)-固醇还原酶基因的编码蛋白,氨基酸序列如SEQ ID NO.2所示,由564个氨基酸残基组成。
进一步的,烟草δ(24)-固醇还原酶基的PCR扩增制备方法,包括如下步骤:
(1)提取基因组,并反转录为cDNA备用;
(2)设计PCR扩增用引物,并进行PCR扩增,具体引物序列设计如下:
NtDHCR24-F:5’-AAGTTGGAGATTGTCTTGAGTG-3’,
NtDHCR24-R:5’-CAGTTGGCGGCATTTCTC-3’。
进一步的,在步骤(1)中提取基因组时,以烟草品种红花大金元叶片为样品。
上述任一项的烟草δ(24)-固醇还原酶基因的应用,利用基因沉默技术,通过调节烟草甾醇δ(24)-固醇还原酶的表达量,来调节控制烟叶中豆甾醇含量。
进一步的,通过转基因技术、瞬时表达技术或基因组编辑技术,构建含有烟草δ(24)-固醇还原酶基因的病毒诱导沉默载体、RNAi干涉载体、超表达载体,转化烟草,筛选获得豆甾醇含量变化的烟草新品种。
具体例如:利用病毒诱导的基因沉默(VIGS)的技术,干扰烟草δ(24)-固醇还原酶基因的表达使其沉默,烟草δ(24)-固醇还原酶基因沉默植株中豆甾醇含量显著下降,进而获得豆甾醇含量下降的植物新品种。
本发明的有益效果是:
基于甾醇对植物生长发育和对烟叶安全性的重要作用,对烟草甾醇调控基因进行深入研究,利用基因工程构建新的烟草品种,为改善烟草品种奠定良好应用基础。本申请中,通过对特定烟草δ(24)-固醇还原酶基因NtDHCR24的初步研究,发现其与烟草叶片中豆甾醇含量高度相关,在将该基因沉默后,烟草叶片中豆甾醇含量发生了降低。基于这一特性,可为烟叶品质调控、烟草新品种培育提供一定的应用基础和参考借鉴。
附图说明
图1为与对照植株相比,NtDHCR24基因沉默植株中该基因的相对表达量;
图2为病毒诱导基因沉默的烟叶及对照烟叶中的甾醇含量比较。
具体实施方式
以下通过具体实施例对本发明的技术方案进行详细的说明,以下的实施例仅是示例性的,仅能用来解释和说明本发明的技术方案,而不能解释为是对本发明技术方案的限制。
在本申请的各实施例中,没有注明具体技术或条件者,按照本领域内现有技术或条件进行,所使用的材料或设备未注明生产厂商者,均为可以通过购买获得的常规产品。
本发明除非另有说明,否则百分号为体积百分数,比例为体积比。
生物材料:
本氏烟草,一种现常用烟草材料,育苗钵中育苗,待发芽后两周进行分苗,种于塑料钵(10cm×10cm)中,22℃、16h光/8h暗条件下进行日常肥水管理等栽培管理。
下述实施例中所采用的VIGS载体是一种来自烟草脆裂病毒的病毒载体(tobaccorattle virus,TRV),所具体利用的TRV2(一种常用载体)带有卡那霉素筛选标记和35S启动子,同时TRV2带有EcoR I和BamH I等多克隆位点,可以用来携带和转化外源基因。
实验试剂:
LB液体培养基,1L含量中含有:10g细菌蛋白胨(bacteriological peptone);10g氯化钠(NaCl);5g酵母抽提物(yeast extract),高温高压灭菌。
YEB液体培养基,1L含量中含有:5g牛肉浸膏(beef extract);5g细菌蛋白胨(bacteriological peptone);5g蔗糖(sucrose);1g酵母抽提物(yeast extract);2mL 1M硫酸镁(MgSO4),高温高压灭菌。
1M 2-(N-吗啉)乙磺酸(MES)储备液:ddH2O溶解,过滤灭菌,-20℃储存备用。
200mM乙酰丁香酮(Acetosyringone,As)储备液:二甲基亚砜(DSMO)溶解,-20℃储存备用。
实施例1
本实施例就烟草NtDHCR24基因克隆及沉默载体的构建过程简要介绍如下。
(1)烟草NtDHCR24基因克隆
根据前期对于烟草基因组及相关NtDHCR24基因研究,选择特异编码序列为目标片段,设计PCR扩增用引物序列如下:
NtDHCR24-F:5’-AAGTTGGAGATTGTCTTGAGTG-3’,
NtDHCR24-R:5’-CAGTTGGCGGCATTTCTC-3’。
以烟草红花大金元叶片(先提取基因组,再反转录为cDNA)的cDNA为模板,进行PCR扩增获得NtDHCR24基因;
PCR扩增程序为:95℃预变性3min;95℃变性15s,55℃退火15s,72℃延伸2min,34个循环后,72℃彻底延伸5min;
对PCR扩增产物进行琼脂糖凝胶电泳检测,并回收电泳产物备用。
(2)构建重组TRV2-NtDHCR24载体
将步骤(1)中的PCR扩增产物进行EcoRI、BamHI双酶切,同时对空载体TRV2进行EcoRI、BamHI双酶切,分别回收酶切产物,利用T4DNA连接酶进行连接。
将连接产物转化大肠杆菌感受态DH5α,转化操作结束后将转化产物涂布在含50mg/L Kan的LB固体培养基上,37℃过培养夜。
挑选阳性单菌落扩增后进一步进行PCR鉴定,并结合测序验证,确保获得构建正确的重组载体TRV2-NtDHCR24。
实施例2
在实施例1基础上,利用农杆菌介导的VIGS技术,进一步将所构建的重组TRV2-NtDHCR24载体转化了烟草植株,并就相关植物表型变化情况做了验证分析,具体实验过程简介如下。
(1)转化农杆菌
需要说明的是,参考实施例1操作及现有技术,制备了TRV2-GFP重组载体作为对照,具体转化过程为:
将TRV2-GFP(载体对照)及TRV2-NtDHCR24的阳性克隆质粒,分别通过电击转化方式转化进入农杆菌GV3101感受态细胞中,利用含50mg/L Kan和50mg/L Rif的YEB平板进行培养筛选,在28℃倒置培养2d后,利用菌落PCR筛选带有目的基因的农杆菌。
(2)制备转染用菌液
将步骤(1)中筛选所得阳性农杆菌克隆在5mL的YEB液体培养基(含50mg/L Kan和50mg/L Rif)中,28℃、250rpm条件下培养过夜;
取50uL过夜培养物接种至50mL的YEB液体培养基(含50mg/L Kan)中,培养至OD600=1.0-1.5左右,然后4000g离心5min,收集菌体,再用MMA重悬,调节OD600=1.0左右;
最后室温放置3h左右后,作为转染用菌液。
(3)瞬时转化
以3-4w(周)苗龄的本氏烟草叶片为实验材料,利用1mL规格注射器,将步骤(2)中所制备转染用菌液注射至烟草叶片中,注射后的烟草继续在人工培养箱内培养,观察表型变化。
进一步通过qRT-PCR对NtDHCR24基因表达情况进行了检测,结果如图1所示,可以看出,TRV2-NtDHCR24的侵染植株中,NtDHCR24的表达量显著降低,qRT-PCR引物如下:
NtDHCR24-F:5’-CCAAGAGGAAGAAGCAGGTG-3’,
NtDHCR24-R:5’-ACGCTGCTCGTATGATTTCC-3’。
进一步地,对实验组(TRV2-NtDHCR24浸染植株)和对照组(TRV2-GFP浸染植株)中的叶片甾醇含量情况进行了检测(检测方法参照《基于气质和液质联用技术的烟草鲜烟叶代谢组学分析流程》(郑庆霞等,烟草科技,2019)),结果如图2所示。
从图2结果可以看出,实验组中豆甾醇含量与对照组相比均有显著下降,降低27.9%,而其他几种甾醇含量和总甾醇含量无显著改变。这进一步表明,通过沉默NtDHCR24基因,可对烟草叶片中豆甾醇的含量进行调控,进而可为烟叶品质调控、烟草新品种培育奠定一定技术基础。
通过转基因技术、瞬时表达技术或基因组编辑技术,构建含有NtDHCR24基因的病毒诱导沉默载体、RNAi干涉载体、超表达载体或基因组编辑载体,转化烟草,筛选获得烟叶中豆甾醇含量变化的烟草新品种。
以上显示和描述了本发明的基本原理和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
序列表
<110> 云南中烟工业有限责任公司
<120> 一种烟草δ(24)-固醇还原酶基因及其应用
<130> WPC211446
<141> 2021-05-25
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1695
<212> DNA
<213> 人工序列(NtDHCR24)
<400> 1
atgtcggatg ctaaggcccc ccttcgtccc aagaggaaga tgcagttggt ggactttctt 60
atccaattca gatggatcct tgttatcttc tttgtccttc ctttctcgtt cctgtattac 120
ttctccatat atctagggga tgttaaatct gagaggaaat cttacgaaca gcgtcagaag 180
gaacacgatg aaaatgttaa agaggtcgtg aagcgccttg gggagaggga tgcatcaaag 240
gatggtcttg tctgcacagg caggcctccg tgggtggttg ttggaatgag aaatgttgac 300
tataagcgtg ctcgtcattt tgaagttgat ctttctaagt tcagaaatat acttgaaatt 360
gacaaggaaa gaatgattgc cagagttgag cctctggtca atatgggcca aatatctaga 420
gttactgttc caatgaatct ttcccttgca gttcttgctg agcttgatga tctgacagtt 480
ggtggtctga tcaatggctt cgggattgaa ggaagctctc acatctttgg attgttctct 540
gacactgttg tgtcacttga ggtagttcta gcagacggac gggtggttag agctacaaag 600
gacaatgagt attctgatct tttctatgct atcccgtggt ctcaggggac attgggtctt 660
cttgtttcag ctgagatcaa gcttataccg gttaaggagt acgtgagact tacttacaaa 720
cctgtaactg gtaatcttaa agagcttgcg caggcttatg cggatacttt tgcacctaga 780
gatggggacc aggacaatcc ttctaaagtt ccagagatgg tagaaggcat gatttatagt 840
cccacggaag gtgttatgat gaccggtaga tatgcttcga aacaggaagc caagcaaagg 900
ggtaatgtaa tcaacaatta tggttggtgg ttcaaaccat ggttttacca gcatgctcaa 960
actgcactga aaagagggga atttgtggag tacattccaa ctagggacta ctaccacagg 1020
cacacaagat ccttgtattg ggaagggaaa ctaattcttc catttggtga tcagttctgg 1080
tttaggtttc tcctaggatg gctcatgcca cccaagattg ctctgctcaa ggccactcaa 1140
agtgaggcta tcaggaacta ttaccatgac catcatgtta ttcaggatct gcttgttcct 1200
ctttacaagg ttggagattg tcttgagtgg gtccaccgcg aaatggaggt atatcccatc 1260
tggctctgcc cacacagaat ttacaagctg cctgtgaaac ctatgatcta tcctgaacca 1320
ggatttgagg cacaccgcag gcagggtgac actgaatatg ctcaaatgta tactgatatc 1380
ggcgtctact atgttcctgg agcagtcctg agaggtgagc cctttgatgg tgcagagaaa 1440
tgccgccaac tggagctttg gttgatcgaa aaccatgggt tccaggctca atacgcggtc 1500
actgaactga cagagaagaa cttctggagg atgtttgata ataccctcta cgagcagtgc 1560
agaaaaaaat ataaagccat cggaaccttc atgagtgcgt actataaatc caaaaaagga 1620
aggaagacgg agaaggaggt gcaggaagcc gagcaagaga aagctgaaca agagactccc 1680
gaagtcgatg agtaa 1695
<210> 2
<211> 564
<212> PRT
<213> 人工序列(NtDHCR24)
<400> 2
Met Ser Asp Ala Lys Ala Pro Leu Arg Pro Lys Arg Lys Met Gln Leu
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Val Asp Phe Leu Ile Gln Phe Arg Trp Ile Leu Val Ile Phe Phe Val
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Leu Pro Phe Ser Phe Leu Tyr Tyr Phe Ser Ile Tyr Leu Gly Asp Val
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Lys Ser Glu Arg Lys Ser Tyr Glu Gln Arg Gln Lys Glu His Asp Glu
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Asn Val Lys Glu Val Val Lys Arg Leu Gly Glu Arg Asp Ala Ser Lys
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Asp Gly Leu Val Cys Thr Gly Arg Pro Pro Trp Val Val Val Gly Met
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Arg Asn Val Asp Tyr Lys Arg Ala Arg His Phe Glu Val Asp Leu Ser
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Val Glu Pro Leu Val Asn Met Gly Gln Ile Ser Arg Val Thr Val Pro
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Met Asn Leu Ser Leu Ala Val Leu Ala Glu Leu Asp Asp Leu Thr Val
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Gly Gly Leu Ile Asn Gly Phe Gly Ile Glu Gly Ser Ser His Ile Phe
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Gly Leu Phe Ser Asp Thr Val Val Ser Leu Glu Val Val Leu Ala Asp
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Glu Ile Lys Leu Ile Pro Val Lys Glu Tyr Val Arg Leu Thr Tyr Lys
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Pro Val Thr Gly Asn Leu Lys Glu Leu Ala Gln Ala Tyr Ala Asp Thr
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Phe Ala Pro Arg Asp Gly Asp Gln Asp Asn Pro Ser Lys Val Pro Glu
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Met Val Glu Gly Met Ile Tyr Ser Pro Thr Glu Gly Val Met Met Thr
275 280 285
Gly Arg Tyr Ala Ser Lys Gln Glu Ala Lys Gln Arg Gly Asn Val Ile
290 295 300
Asn Asn Tyr Gly Trp Trp Phe Lys Pro Trp Phe Tyr Gln His Ala Gln
305 310 315 320
Thr Ala Leu Lys Arg Gly Glu Phe Val Glu Tyr Ile Pro Thr Arg Asp
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Tyr Tyr His Arg His Thr Arg Ser Leu Tyr Trp Glu Gly Lys Leu Ile
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Leu Pro Phe Gly Asp Gln Phe Trp Phe Arg Phe Leu Leu Gly Trp Leu
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Met Pro Pro Lys Ile Ala Leu Leu Lys Ala Thr Gln Ser Glu Ala Ile
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Arg Asn Tyr Tyr His Asp His His Val Ile Gln Asp Leu Leu Val Pro
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Leu Tyr Lys Val Gly Asp Cys Leu Glu Trp Val His Arg Glu Met Glu
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Lys Pro Met Ile Tyr Pro Glu Pro Gly Phe Glu Ala His Arg Arg Gln
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Gly Asp Thr Glu Tyr Ala Gln Met Tyr Thr Asp Ile Gly Val Tyr Tyr
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Val Pro Gly Ala Val Leu Arg Gly Glu Pro Phe Asp Gly Ala Glu Lys
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Cys Arg Gln Leu Glu Leu Trp Leu Ile Glu Asn His Gly Phe Gln Ala
485 490 495
Gln Tyr Ala Val Thr Glu Leu Thr Glu Lys Asn Phe Trp Arg Met Phe
500 505 510
Asp Asn Thr Leu Tyr Glu Gln Cys Arg Lys Lys Tyr Lys Ala Ile Gly
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Thr Phe Met Ser Ala Tyr Tyr Lys Ser Lys Lys Gly Arg Lys Thr Glu
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Lys Glu Val Gln Glu Ala Glu Gln Glu Lys Ala Glu Gln Glu Thr Pro
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Glu Val Asp Glu
Claims (7)
1.一种烟草δ(24)-固醇还原酶基因,其特征在于,核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的烟草δ(24)-固醇还原酶基因,其特征在于,烟草δ(24)-固醇还原酶基因的编码蛋白,氨基酸序列如SEQ ID NO.2所示。
3.根据权利要求1或2所述的烟草δ(24)-固醇还原酶基因,其特征在于,烟草δ(24)-固醇还原酶基因的PCR扩增制备方法,包括如下步骤:
(1)提取基因组,并反转录为cDNA备用;
(2)设计PCR扩增用引物,并进行PCR扩增,具体引物序列设计如下:
NtDHCR24-F:5’-AAGTTGGAGATTGTCTTGAGTG-3’,
NtDHCR24-R:5’-CAGTTGGCGGCATTTCTC-3’。
4.根据权利要求3所述的烟草δ(24)-固醇还原酶基因,其特征在于,在步骤(1)中提取基因组时,以烟草品种红花大金元叶片为样品。
5.一种烟草δ(24)-固醇还原酶基因的应用,其特征在于,利用上述权利要求1至4中任一项的烟草δ(24)-固醇还原酶基因,该基因表达的蛋白与植物叶片中豆甾醇含量相关,降低该蛋白表达后,叶片中豆甾醇含量明显降低。
6.根据权利要求5所述的烟草δ(24)-固醇还原酶基因的应用,其特征在于,利用基因沉默技术、或者基因超表达方法,通过调节烟草δ(24)-固醇还原酶基因的表达量,来调节控制烟叶中豆甾醇含量情况。
7.根据权利要求6所述的烟草δ(24)-固醇还原酶基因的应用,其特征在于,通过转基因技术、瞬时表达技术或基因组编辑技术,构建含有烟草δ(24)-固醇还原酶基因的病毒诱导沉默载体、RNAi干涉载体、超表达载体或基因组编辑载体,转化烟草,筛选获得豆甾醇含量变化的烟草新品种。
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