CN113278600A - 烟草3β羟基类固醇脱氢酶/C4脱羧酶基因及其应用 - Google Patents
烟草3β羟基类固醇脱氢酶/C4脱羧酶基因及其应用 Download PDFInfo
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- CN113278600A CN113278600A CN202110580251.1A CN202110580251A CN113278600A CN 113278600 A CN113278600 A CN 113278600A CN 202110580251 A CN202110580251 A CN 202110580251A CN 113278600 A CN113278600 A CN 113278600A
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Abstract
本发明涉及一种烟草3β羟基类固醇脱氢酶/C4脱羧酶基因及其应用,3β羟基类固醇脱氢酶/C4脱羧酶基因的核苷酸序列如SEQ ID NO.1所示。本申请中,通过对特定烟草3β羟基类固醇脱氢酶/C4脱羧酶基因的初步研究,发现其与烟草叶片中甾醇含量高度相关,在本氏烟草中将该基因沉默,烟草叶片中豆甾醇、菜油甾醇含量发生了明显降低,总甾醇含量降低17.9%。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3β羟基类固醇脱氢酶/C4脱羧酶基因及其应用。
背景技术
植物甾醇是生物膜***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。由于甾醇的结构中含有羟基,热解时其母体的菲环结构可形成多环芳烃,有文献报道烟草中的甾醇是卷烟烟气苯并芘的重要前体化合物。因此,降低烟草成熟叶片中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,但栽培烟草中调控甾醇合成的基因却鲜有报道。烟草中影响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3β羟基类固醇脱氢酶/C4脱羧酶基因及其应用即(一种烟草3β-羟基类固醇脱氢酶/C-4脱羧酶基因及其应用),以改善烟草中甾醇含量,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3β羟基类固醇脱氢酶/C4脱羧酶基因,核苷酸序列如SEQ ID NO.1所示,含有1167个碱基,命名为NtNSDHL。
进一步的,烟草3β羟基类固醇脱氢酶/C4脱羧酶基因的编码蛋白,氨基酸序列如SEQ ID NO.2所示,由388个氨基酸残基组成。
进一步的,烟草3β羟基类固醇脱氢酶/C4脱羧酶基因的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:
NtNSDHL-F:5’-AACAAATCCCAAGTCCTCAAAG-3’,
NtNSDHL-R:5’-CTACCTTAGCAGGATATGGC-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3β羟基类固醇脱氢酶/C4脱羧酶基因的应用,利用基因沉默技术,通过调节烟草甾醇麦角固醇合成的表达量,来调节控制烟叶中甾醇含量。
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3β羟基类固醇脱氢酶/C4脱羧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NtNSDHL基因的表达使其沉默,NtNSDHL基因沉默植株中甾醇含量显著下降,进而获得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,通过对特定烟草甾醇麦角固醇合成NtNSDHL的初步研究,发现其与烟草叶片中甾醇含量高度相关,在将该基因沉默后,烟草叶片中甾醇含量发生了明显降低。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,NtNSDHL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NtNSDHL基因克隆及沉默载体的构建过程简要介绍如下。
(1)烟草NtNSDHL基因克隆
根据前期对于烟草基因组及相关NtNSDHL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:
NtNSDHL-F:5’-AACAAATCCCAAGTCCTCAAAG-3’,
NtNSDHL-R:5’-CTACCTTAGCAGGATATGGC-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NtNSDHL基因;
PCR扩增程序为:95℃预变性3min;95℃变性15s,55℃退火15s,72℃延伸1min,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-NtNSDHL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T4 DNA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-NtNSDHL。
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-NtNSDHL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-NtNSDHL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NtNSDHL基因表达情况进行了检测,结果如图1所示,可以看出,TRV2-NtNSDHL的侵染植株中,NtNSDHL的表达量显著降低,qRT-PCR引物如下:
NtNSDHL-F:5’-ACGGTTCCACAGTTGACTCC-3’,
NtNSDHL-R:5’-CTGGTCCTCGTTCAGCTCTC-3’。
进一步地,对实验组(TRV2-NtNSDHL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,实验组中甾醇含量与对照组相比有显著下降,总甾醇含量降低了17.9%,豆甾醇和和菜油甾醇比对照组显著下降。这进一步表明,通过沉默NtNSDHL基因,可对烟草叶片中植物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NtNSDHL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> 烟草3β羟基类固醇脱氢酶/ C4脱羧酶基因及其应用
<130> WPC211447
<141> 2021-05-25
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1167
<212> DNA
<213> 人工序号(NtNSDHL)
<400> 1
atgggggaag gagaagaaga gaaatggtgt gtggtgactg gtggaagagg ctttgctgct 60
cggcatttag tggaaatgct gattcgttat gaaatctatc atgtccgcat tgctgatttg 120
ggtccgtcca ttaaacttga cccgactgag gaaaagggta tacttggtca agccctccaa 180
tcaggccgtg ctgtatatgt atccatggat cttcgtaaca aatcccaagt cctcaaagct 240
tgtgaaggag ctgaggttgt cttccacatg gctgctccag attcatcaat caacaaccac 300
cagctccact attcagttaa tgtgcaagga acccagaata taattgatgc ttgcattgag 360
ctgaaagtga aaagacttat ttacaccagc tctcccagtg tggtgtttga tggagttcat 420
ggaattctaa atggggatga atcactgcca tatcctgcta agcataatga ttcctactct 480
gcaaccaaag ctgaaggaga ggcacttgtt atcaagtcaa atggtaccaa agggctgctg 540
acatgctgca ttagacctag cagtcttttt ggccctggtg ataggctgct cgttccttca 600
ctagttgcag ctgcaaaggc aggaaaatca aagttcatta ttggtgatgg caacaacatg 660
tatgatttca cttacgtgga gaatgtagca catgctcatg tgtgtgcaga acgagctcta 720
gcatcaggag gagcagttgc agagaaagct gctgggaatg catattttgt cacgaacatg 780
gagcccatta agttttggga gtttgtctca cttattcttg aaggtcttgg ctatgacagg 840
ccaaatatta agattcctgc atctgttatg atgccaattg cacatttggt ggagcttagt 900
tataagctgt tagctcctta tggaatgaag gtcccacagt tgactccttc aagaatcaga 960
ctcctgtccc gtagcagaac atttagttgt tcaaaagcaa gtgatcgaat aggatacaca 1020
cctattatct cacttcagga gggccttcgg aggacaattg agtcctatcc acatttgaga 1080
gctgaacatg ggcctggaaa ggaaggtcct tctaaatcat ctgcatctct ttggatgttt 1140
ttcctcatgg taatttctaa tacataa 1167
<210> 2
<211> 388
<212> PRT
<213> 人工序号(NtNSDHL)
<400> 2
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Ile Glu Ser Tyr Pro His Leu Arg Ala Glu His Gly Pro Gly Lys Glu
355 360 365
Gly Pro Ser Lys Ser Ser Ala Ser Leu Trp Met Phe Phe Leu Met Val
370 375 380
Ile Ser Asn Thr
385
Claims (7)
1.一种烟草3β羟基类固醇脱氢酶/C4脱羧酶基因,其特征在于,核苷酸序列如SEQ IDNO.1所示。
2.根据权利要求1所述的烟草3β羟基类固醇脱氢酶/C4脱羧酶基因,其特征在于,烟草3β羟基类固醇脱氢酶/C4脱羧酶基因的编码蛋白,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3β羟基类固醇脱氢酶/C4脱羧酶基因,其特征在于,烟草3β羟基类固醇脱氢酶/C4脱羧酶基因的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:
NtNSDHL-F:5’-AACAAATCCCAAGTCCTCAAAG-3’,
NtNSDHL-R:5’-CTACCTTAGCAGGATATGGC-3’。
4.根据权利要求3所述的烟草3β羟基类固醇脱氢酶/C4脱羧酶基因,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3β羟基类固醇脱氢酶/C4脱羧酶基因的应用,其特征在于,利用上述权利要求1至4中任一项的烟草3β羟基类固醇脱氢酶/C4脱羧酶基因,该基因表达的蛋白与植物叶片中甾醇含量相关,降低该蛋白表达后,叶片中甾醇含量明显降低。
6.根据权利要求5所述的烟草3β羟基类固醇脱氢酶/C4脱羧酶基因的应用,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3β羟基类固醇脱氢酶/C4脱羧酶基因的表达量,来调节控制烟叶中甾醇含量情况。
7.根据权利要求6所述的烟草3β羟基类固醇脱氢酶/C4脱羧酶基因的应用,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3β羟基类固醇脱氢酶/C4脱羧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,筛选获得甾醇含量变化的烟草新品种。
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