CN114015691B - PnSE基因上游启动子及其在响应外源激素中的应用 - Google Patents

PnSE基因上游启动子及其在响应外源激素中的应用 Download PDF

Info

Publication number
CN114015691B
CN114015691B CN202111337068.5A CN202111337068A CN114015691B CN 114015691 B CN114015691 B CN 114015691B CN 202111337068 A CN202111337068 A CN 202111337068A CN 114015691 B CN114015691 B CN 114015691B
Authority
CN
China
Prior art keywords
pnse
gene
promoter
upstream
upstream promoter
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202111337068.5A
Other languages
English (en)
Other versions
CN114015691A (zh
Inventor
夏鹏国
张宇
郑宇婕
胡婉莹
梁宗锁
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang Sci Tech University ZSTU
Original Assignee
Zhejiang Sci Tech University ZSTU
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhejiang Sci Tech University ZSTU filed Critical Zhejiang Sci Tech University ZSTU
Priority to CN202111337068.5A priority Critical patent/CN114015691B/zh
Publication of CN114015691A publication Critical patent/CN114015691A/zh
Application granted granted Critical
Publication of CN114015691B publication Critical patent/CN114015691B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8291—Hormone-influenced development
    • C12N15/8293—Abscisic acid [ABA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8291—Hormone-influenced development
    • C12N15/8297—Gibberellins; GA3
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
    • C12Y114/13132—Squalene monooxygenase (1.14.13.132)

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Endocrinology (AREA)
  • Medicinal Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明公开了PnSE基因上游启动子及其在响应外源激素中的应用，该PnSE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PnSE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PnSE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

PnSE基因上游启动子及其在响应外源激素中的应用
技术领域
本发明涉及植物生物技术领域，尤其涉及PnSE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，角鲨烯环氧化酶(Squalene epoxidase,SE)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PnSE基因上游启动子及其在响应外源激素中的应用，该PnSE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PnSE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PnSE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PnSE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PnSE基因上游启动子在响应外源激素中的应用，所述PnSE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PnSE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PnSE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PnSE基因上游启动子的顺式作用元件分布图。
图2为PnSE基因上游启动子的转录起始位点示意图。
图3为PnSE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 PnSE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PnSE基因上游2000bp为研究对象，采用primer premier 5.0设计引物(SE-F：5’-TGGGCACCAAACAAC AAATACCGAA-3’，SE-R：5’-CGAGAGGCAGCCAGGAGGATAAAGA-3’)。
PCR扩增时退火温度为61℃。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：0.5μL pMD19-TVector、4.5μL回收产物和5.0μL SolutionⅠ。
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PnSE上游启动子的基因序列。
实施例2 PnSE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，PnSE基因上游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。CGTCA基序和TGACG基序分别位于-1350bp和-628bp处，它们与MeJA反应有关。在-1211bp和-929bp处，分别是参与ABA反应的ABRE顺式作用元件。对GA有反应的P-box位于-721bp处。
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PnSE基因上游启动子包含3个转录起始位点(图2)。
表1 PnSE基因上游启动子的顺式作用元件
实施例3 PnSE基因上游启动子对外源性激素的响应
对PnSE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’-AACTGCAGTGGGCACCAAACAACAAATACCGAA-3’；下游引物：5’-CGGGATCCCGAGAGGCAGCCAGGAGGATAAAGA-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(CodeNo.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PnSE基因上游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PnSE基因上游启动子能特异性、显著地响应外源GA和ABA信号。
SEQUENCE LISTING
<110>浙江理工大学
<120>PnSE基因上游启动子及其在响应外源激素中的应用
<130>
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 1492
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 1
tgggcaccaa acaacaaata ccgaacgccc aaaccccata tagccaagac cattttagac 60
cactttaatg ccaccattcc tccaccactt tcatttgcta cccaccaaaa caattaattc 120
ttacaaaaaa tatctttaag acaaaaaata acgtttgaga cacaaactat tctataaata 180
ttttataaac tgtagatgtg acatacttca cctcagcata ttgtgtgggg ccgaaaacta 240
tattgaaata atttgttgat caatatgctg aggtggagta taacacatca gcagtttata 300
aaatagtttg tgtctgtagc actaccctaa gacaaaatag tggtagcctc acacggacaa 360
agatcttctc cttttcaaat aaatggagac tctttatttt aggatctaaa tttaaaatgt 420
gttaaaatat gacatcatga cgacatgaga gagattattt taaatttaaa taatcataaa 480
atgaggagtc tctatttatt tgaaatggga gaatcctaat atgccccaca catgattttg 540
gtaattacca cccaccgtat tcctcttgcc tcttacacgt aataaaatga caaatttttt 600
ttttgctctt gcagaaattt ttttatgtct gaatatatat gctttgaagt gttgttatgc 660
tgcgtgcaaa taataataat aaaaatataa ttataaccaa acgcacatta tttaatttaa 720
tttacatttt gaatatttat tctttagttg tgaaaaaata aaaatgaggg accgacggtt 780
tgttttttcc tttagaaagt aggctataaa atgcgccatt ttgggacgat tgcttcactc 840
aaaattgcag cagaacaaac gtgaaaggca ttgtagagag agagagagag agttgttcag 900
agagagaaac gtgaacgtcc aaccaacacc 930
<210> 2
<211> 25
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 2
tgggcaccaa acaacaaata ccgaa 25
<210> 3
<211> 25
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 3
cgagaggcag ccaggaggat aaaga 25
<210> 4
<211> 33
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 4
aactgcagtg ggcaccaaac aacaaatacc gaa 33
<210> 5
<211> 33
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 5
cgggatcccg agaggcagcc aggaggataa aga 33

Claims (1)

1.PnSE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PnSE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。
CN202111337068.5A 2021-11-12 2021-11-12 PnSE基因上游启动子及其在响应外源激素中的应用 Active CN114015691B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111337068.5A CN114015691B (zh) 2021-11-12 2021-11-12 PnSE基因上游启动子及其在响应外源激素中的应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111337068.5A CN114015691B (zh) 2021-11-12 2021-11-12 PnSE基因上游启动子及其在响应外源激素中的应用

Publications (2)

Publication Number Publication Date
CN114015691A CN114015691A (zh) 2022-02-08
CN114015691B true CN114015691B (zh) 2024-01-23

Family

ID=80063705

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111337068.5A Active CN114015691B (zh) 2021-11-12 2021-11-12 PnSE基因上游启动子及其在响应外源激素中的应用

Country Status (1)

Country Link
CN (1) CN114015691B (zh)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114107294A (zh) * 2021-11-12 2022-03-01 浙江理工大学 PnSS基因上游启动子及其在响应外源激素中的应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101687106B1 (ko) * 2015-06-29 2016-12-15 제주대학교 산학협력단 감귤 유래 CuCRTISO-like 프로모터 및 이를 포함하는 재조합 벡터
CN113598003A (zh) * 2021-09-15 2021-11-05 昆明理工大学 一种提高三七皂苷合成的方法及应用

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114107294A (zh) * 2021-11-12 2022-03-01 浙江理工大学 PnSS基因上游启动子及其在响应外源激素中的应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
三七皂苷合成关键基因PnSS与PnSE转录调控的分子机制;郑宇婕;中国优秀硕士学位论文全文数据库 农业科技辑(第3期);第20页图2.2 *

Also Published As

Publication number Publication date
CN114015691A (zh) 2022-02-08

Similar Documents

Publication Publication Date Title
CN103103194B (zh) 茉莉酸甲酯应答的人参PgPDR3基因启动子及其应用
CN103088027B (zh) 一种调控人参皂苷积累的pdr转运蛋白基因启动子及其应用
Takahashi et al. Location of the cis-acting auxin-responsive region in the promoter of the par gene from tobacco mesophyll protoplasts.
WO1991009948A1 (en) Rice actin gene and promoter
CN106497939A (zh) 一种三七转录因子基因PnMYB1及其应用
CN115819528B (zh) 一种桑黄bHLH类转录因子Pb-bHLH6及其编码基因与应用
CN107955067B (zh) 参与桃黄酮醇生物合成调控的两个myb转录因子及其应用
CN114350680B (zh) 一种增加桂花挥发性有机物质合成相关OfMYB1R70基因及其编码蛋白和应用
CN114369603B (zh) 抑制桂花香气物质合成相关OfMYB1R基因及其编码蛋白和应用
CN114107294B (zh) PnSS基因上游启动子及其在响应外源激素中的应用
CN114164219B (zh) 一种增强桂花香气物质合成相关OfMYB1R114基因及其编码蛋白和应用
CN114736910A (zh) 人参PgRb1-057-001基因及其应用
DE69836136T2 (de) Rekombinantes konstrukt für eine erhöhte expression von genen in pflanzen.
CN113943733B (zh) 落叶松内源启动子及应用
Fan et al. In vitro transcription of plant RNA polymerase I‐dependent rRNA genes is species‐specific
CN114015691B (zh) PnSE基因上游启动子及其在响应外源激素中的应用
CN101302530A (zh) 一种通路克隆入门载体及其构建方法和应用
CA2674170C (en) Artificial dna sequence with optimized leader function in 5&#39; (5&#39;-utr) for the improved expression of heterologous proteins in plants
CN103103193B (zh) 一种人参pdr跨膜转运蛋白基因启动子及其应用
CN102533804B (zh) 白沙蒿Δ12脂肪酸脱氢酶(As FAD2)基因及用途
Iwamoto et al. Strong expression of the rice catalase gene CatB promoter in protoplasts and roots of both a monocot and dicots
CN112458091B (zh) 水稻组成型表达启动子Os02g0752800及应用
CN114214332A (zh) 一种天目地黄花青素相关基因RcMYB1及其应用
CN114058632A (zh) 基因PnCOX11及其在调控三七皂苷合成中的应用
CN108004241B (zh) 烟草硝酸还原酶nia2启动子、其表达载体及其应用

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant