CN114107294B - PnSS基因上游启动子及其在响应外源激素中的应用 - Google Patents
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Abstract
本发明公开了PnSS基因上游启动子及其在响应外源激素中的应用，该PnSS基因上游启动子核苷酸序列如SEQ ID NO.1所示。本发明克隆了三七中PnSS基因的上游序列并进行了测序，通过在线数据库对获得的基因上游序列进行分析，并运用瞬时表达研究外源激素(赤霉素和脱落酸)上游启动子顺式作用元件的作用，发现PnSS基因上游启动子具有响应外源激素的作用，能够为今后研究启动子的顺式作用元件与相关转录因子的相互作用提供基础。
Description
技术领域
本发明涉及植物生物技术领域，尤其涉及PnSS基因上游启动子及其在响应外源激素中的应用。
背景技术
启动子是一个位于结构基因上游5′端的DNA序列，RNA聚合酶可以特异性地识别并与之结合。它是基因的重要组成部分，控制着生物中基因表达的起始时间和程度。启动子主要由核心启动子区、上游控制元件和响应元件组成。近年来，靶基因转录水平的调控一直是研究的热点方向，启动子功能的研究已成为表达调控研究的重要组成部分。通过将部分启动子片段与报告基因(包括β-葡萄糖醛酸酶基因和绿色荧光蛋白基因)融合并构建植物表达载体，然后转化模式植物(拟南芥或烟草)，检测报告基因在转基因植物中的表达，以确认启动子功能。
三七[Panax notoginseng(Burk.)F.H.Chen]为五加科(Araliaxeae)人参属(Panax)多年生直立草本植物，是我国传统名贵药材之一。三七皂苷(Panax notoginsengsaponins,PNS)是三七主要的药用活性成分，由多种四环三萜皂苷组成。已有研究表明三七总皂苷在中枢神经系统、心脑血管系统、血液系统、免疫系统以及抗纤维化、抗衰老、抗肿瘤等方面均具有较好的药理活性。由于三七对种植环境要求苛刻，生长周期长，轮作障碍严重，其产量难以满足市场的需求，严重制约了三七产业的可持续发展。
研究表明，许多关键酶基因的启动子区域包含多个顺式作用元件，可以响应生物和非生物信号调节。其中，激素信号的调节在植物生长和调节中起着重要作用。三七皂苷主要由甲羟戊酸(MVA)途径合成，角鲨烯合成酶(Squalene synthase,SS)是其中关键酶，对植物体内的三萜类和甾醇类化合物有着重要的调控作用。
发明内容
本发明提供了PnSS基因上游启动子及其在响应外源激素中的应用，该PnSS基因上游启动子中包含有能够响应外源激素的顺式作用元件，可为今后研究启动子的顺式作用元件与相关转录因子的相互作用提供基础。
具体技术方案如下：
本发明提供了PnSS基因上游启动子，其核苷酸序列如SEQ ID NO.1所示。
本发明提供了一种包含如上所述PnSS基因上游启动子的表达载体。
本发明提供了一种包含如上所述PnSS基因上游启动子的转化子。
进一步地，所述转化子为根癌农杆菌株EHA105。
本发明还提供了PnSS基因上游启动子在响应外源激素中的应用，所述PnSS基因上游启动子的核苷酸序列如SEQ ID NO.1所示；所述外源激素为ABA或GA。
与现有技术相比，本发明具有以下有益效果：
本发明克隆了三七中PnSS基因的上游序列并进行了测序，通过在线数据库对获得的基因上游序列进行分析，并运用瞬时表达研究外源激素(赤霉素和脱落酸)上游启动子顺式作用元件的作用，发现PnSS基因上游启动子具有响应外源激素的作用，能够为今后研究启动子的顺式作用元件与相关转录因子的相互作用提供基础。
附图说明
图1为PnSS基因上游启动子的顺式作用元件分布图。
图2为PnSS基因上游启动子的转录起始位点示意图。
图3为PnSS基因上游启动子对外源性GA和ABA信号的响应。
具体实施方式
下面结合具体实施例对本发明作进一步描述，以下列举的仅是本发明的具体实施例，但本发明的保护范围不仅限于此。
实施例1 PnSS基因上游启动子的克隆
使用CTAB方法提取三七基因组DNA，经1.0％琼脂糖凝胶电泳和核酸浓度检测仪检测其完整性及浓度。根据已发表的三七全基因组序列，以PnSS基因上游2000bp为研究对象，采用primer premier 5.0设计引物(SS-F：5’-CGCACACATCCACAAGG-3’，SS-R：5’-TGAATGACGAGGCCAAA-3’)。PCR扩增时退火温度为55℃。PCR产物在1.0％琼脂糖凝胶上进行电泳分析。将扩增产物用Tiangen TIANgel Midi Purification Kit(DP190123)试剂盒进行切胶回收，随后将回收产物连接到pMD19-T载体上并16℃孵育过夜，其连接体系为：0.5μLpMD19-T Vector、4.5μL回收产物和5.0μL Solution Ⅰ。取5μL连接产物加入到大肠杆菌DH5α感受态细胞中，轻轻混匀，冰上放置30min，42℃热激60s，迅速放入冰中冰浴2min，加入700μL LB培养基，于37℃摇床内200rpm震荡摇菌1h，在超净台中吸取200μL涂布于含100mg/L氨苄青霉素的LB固体培养基板上，于37℃培养箱中培养12h，挑取单克隆于LB液体培养基(含100mg/L氨苄青霉素)中，37℃震荡摇菌5h，进行菌液PCR验证，将验证正确的送去测序，进而得到PnSS上游启动子的基因序列。
实施例2 PnSS基因上游启动子序列分析
在线PlantCARE数据库(http://bioinformatics.psb.ugent.be/webtools/plantcare/html)和PLACE数据库(http://www.dna.affrc.go.jp/PLACE/signalscan.html)用于预测和分析顺式调控元件。结果发现SS启动子上含有多种顺式作用元件，如生长调节元件、光响应元件、脱落酸(ABA)响应元件、赤霉素(GA)响应元件、MYB结合元件和厌氧元件等(表1)。其顺式作用元件的分布如图1所示。在SS启动子的-148bp位置包含一个MBS顺式作用元件，该元件参与干旱诱导。参与脱落酸反应的顺式作用元件位于-892bp。预测结果中有多种光响应基序，包括Box 4、G-Box、TCT和ATCT基序。
转录起始位点使用在线网站(http://www.fruitfly.org/seq_tools/promoter.html)进行预测，结果发现PnSS启动子只含有1个转录起始位点(图2)。
表1 PnSS启动子的顺式作用元件
实施例3 PnSS基因上游启动子对外源性激素的响应
对PnSS上游启动子序列和植物表达载体PCAMBIA0390序列上酶切位点的分布情况进行分析，设计带有PstI、BamHI酶切位点的PCR引物(上游引物：5’-AACTGCAGCGCACACATCCACAAGG-3’；下游引物：5’-CGGGATCCTGAATGACGAGGCCAAA-3’)，用于构建重组植物表达载体。重组载体用TIANGEN的TIANpure Mini Plasmid Kit II(Code No.DP107)进行提取，然后转化根癌农杆菌株EHA105。将含有重组质粒的菌株EHA105培养到OD600＝0.4～0.6，在注射缓冲液中解毒2h，然后将OD600调整至0.8用于注射烟草叶片。注射后的烟草在25℃下光照16h、黑暗8h培养72h。然后分别喷洒GA(200mg/L)和ABA(100mg/L)。在透明塑料袋中培养24小时后收集烟叶，放入液氮中快速冷冻。以未注射的烟叶作为阴性对照。
使用GUS提取液提取烟草总蛋白，用酶标仪在595nm处测定吸光度，检测蛋白含量。将400μLGUS提取液，100μL蛋白样品和500μL 2mM MUG于1.5mL离心管中混合，37℃水浴。在5个时间点(0min、15min、30min、45min和60min)，将200μL的反应液加入到800μL的0.2MNa2CO3溶液中，室温黑暗保存。用酶标仪进行荧光扫描(激发光：360nm，发射光：460nm)，并计算单位时间荧光强度的变化率。变化率除以蛋白质总量即为GUS蛋白酶活性水平。
GUS酶活性分析结果如图3所示。与对照相比，添加了PnSS启动子的烟草叶喷洒GA和ABA后，GUS蛋白酶活性显著提高，表明PnSS启动子能特异性、显著地响应外源GA和ABA信号。
序列表
<110> 浙江理工大学
<120> PnSS基因上游启动子及其在响应外源激素中的应用
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1492
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gataatttac ataatactcc tatatatata tatgtgattt aaaagtgaaa accacataaa 60
ataatttaga aaaaatgtgg cataattttt ttaataaaaa aattgtggtt ttcacttttt 120
atgtcaatac aacaaaaatg agaaccagat gttttaaact ttaatatgcc agccattaat 180
ttagtgggca aatcaattat ttggatatcg acaaaagtac caagggttga tgagaggcat 240
ctaggagtcc tcgctggaaa gaaacactgt taattaatgt gatgagaaga cagtgtttgg 300
gctcctaaaa taaaatatga gttcttacag gaaaacggga catgaaatgt atttaaattt 360
taattattta tatttttatt ctttttagat gtcaaaatac acatatgtga cctaattttt 420
tgtcccattt tcttgttcac caaaagtttt tgataaaata ttcattatta acttagtggt 480
caaatactaa taattaaaga tggaaaataa aataataaaa aaaggaattt gtctcaaata 540
aatgagagtc atgcatatgt tttctttatt tcttttattt tattttcttt tttcaaaggt 600
cacgttaaat ttttaaaaaa tgaatgaact ttttaaaata aaattatctt ttaaaacgta 660
cgaaataaca cttaacaact cgataaatat acgatgaaca aacttgataa ataaataatc 720
atgaatatta atgttgaatt cgctagatat actaattgta gtaacatttt gtcataagca 780
atcttgtatc tctagtctct aattaatgtt tatcttgact taataaaata aaatttataa 840
ataacaaagt tcttgggatt aaattttagt gtaaaaaaat aaaataacaa aattatttta 900
acaaccaact tatattttaa tttataaatt atttaaaaat aaatcattcc cgaactccca 960
tcttctaacg agcaacttga gttccgtatt tacttattag ctttttctcg tacttccgat 1020
attattttga actacttaga taatttatta aaatgagtaa aaaaaataaa aatcacttgt 1080
attttatgaa ataaatattc aaaataaata attatttcct cgccgtttct tttctctccg 1140
ctgtctccgt ctctctgtcg tattcgtttt gcatctttat cgaatccgtg agtctctgtc 1200
tttcaatttc cgtcttcatt ttgacaaatc aaaattcctc ccaaacagaa atcactaatt 1260
ttatcccctc aaaatatatg gaaggagtac taaactcgtc caaacaataa ttcactattc 1320
cattttcatt tccatttcac attccaactg caaattaagc aagcttatta taaagtgagc 1380
aaattcgttt gaaaaatctc aatgttcttt ataatcattc aattaattgc tttagatctg 1440
ttcatttcat tcattttttt tgtgcttggc gaaacagata tatagagaga aa 1492
<210> 2
<211> 17
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cgcacacatc cacaagg 17
<210> 3
<211> 17
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tgaatgacga ggccaaa 17
<210> 4
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
aactgcagcg cacacatcca caagg 25
<210> 5
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cgggatcctg aatgacgagg ccaaa 25
Claims (1)
1.PnSS基因上游启动子在响应外源激素中的应用，其特征在于，所述PnSS基因上游启动子的核苷酸序列如SEQ ID NO.1所示；所述外源激素为脱落酸或赤霉素。
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| CN106222172A (zh) * | 2016-08-11 | 2016-12-14 | 河南农业大学 | 一种烟草gcn2启动子及其应用 |
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| CN106222172A (zh) * | 2016-08-11 | 2016-12-14 | 河南农业大学 | 一种烟草gcn2启动子及其应用 |
| CN111826376A (zh) * | 2020-06-24 | 2020-10-27 | 广东工业大学 | 一种植物启动子及其应用 |
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