CN111944818B - 一种花生过敏原基因Arah1的启动子Arah1-P及其克隆和应用 - Google Patents
一种花生过敏原基因Arah1的启动子Arah1-P及其克隆和应用 Download PDFInfo
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Abstract
本发明涉及生物技术领域，特别涉及一种花生过敏原基因Arah1的启动子Arah1‑P及其克隆和应用。上述花生过敏原基因Arah1的启动子Arah1‑P，其核苷酸序列如SEQ IN NO：1所示。上述花生过敏原基因Arah1的启动子Arah1‑P能驱动外源基因在植物种子中特异表达。上述花生过敏原基因Arah1的启动子Arah1‑P的植物表达载体为pBI121‑Arah1‑P。本发明花生过敏原基因Arah1的启动子Arah1‑P，对于改良花生种子品质以及花生种子作为“生物反应器”的研究具有重要的应用价值。
Description
技术领域
本发明涉及生物技术领域，特别涉及一种花生过敏原基因Arah1的启动子Arah1-P及其克隆和应用。
背景技术
花生是世界重要的油料作物，其种子富含脂肪和蛋白质，是理想的“生物反应器”。启动子可以驱动外源基因在宿主植物中表达，是花生遗传改良重要的分子工具。自1993年获得转基因花生以来，陆续出现了以抗逆、品质改良等为目标的转基因花生的报道。然而，目前可以利用的花生启动子资源较为匮乏，且大多数启动子功能未被鉴定。
花生是世界八大类过敏食物之一，目前，人们已经发现17种花生过敏原蛋白。其中，花生过敏原基因Arah1是蛋白含量最高，且被90％的花生过敏患者的血清所识别，是花生主要过敏原之一。基因启动子是基因的一个组成部分，控制基因表达(转录)的起始时间和表达的程度。启动子就像“开关”，决定基因的活动。对于花生过敏原基因Arah1启动子的研究对于消除过敏原，改良花生品质以及花生种子作为“生物反应器”的研究具有重要的应用价值。
发明内容
本发明提供一种花生过敏原基因Arah1的启动子Arah1-P及其克隆和应用。
本发明的技术方案为：
一种花生过敏原基因Arah1的启动子Arah1-P，其核苷酸序列如SEQ ID NO：1所示。
一种花生过敏原基因Arah1的启动子Arah1-P在驱动外源基因在植物种子中特异表达的应用。
进一步的，所述外源基因为GUS基因。
本发明所达到的有益效果为：
该启动子驱动外源基因GUS在转基因拟南芥种子中表达。本发明丰富了花生种子特异表达启动子资源，其克隆鉴定将对于花生过敏原基因Arah1启动子的研究，对于消除过敏原，改良花生品质以及花生种子作为“生物反应器”的研究具有重要的应用价值。
附图说明
图1是半定量RT-PCR检测Arah1基因在花生根、茎、叶、花、入土前果针、成熟种子果壳及成熟种子的表达情况，Actin是内标基因。
图2是GUS组织化学染色，其中A：成熟的转基因拟南芥种子，能染较深的蓝色；B：萌发的转基因拟南芥真叶以及胚轴和胚根也能被染成染色；C：植株长出真叶后，子叶仍然能被染上较浅的蓝色。
具体实施方式
下面结合附图和具体实施方式对本发明技术方案作进一步详细的说明。
实施例1
本实施例具体包括以下试验过程：
1.1试验材料
植物材料为花生品种“花育9303”和拟南芥(生态型Co10)。大肠杆菌DH5α感受态、DNA分子量标记、PCR mix等购自北京全式金生物有限公司；高保真酶Phusion购自NewEngland Biolabs公司；X-Gluc购自北京索莱宝科技有限公司；限制性内切酶BamHI和HindIII购自Fermentas公司；T4 DNA连接酶购自宝生物工程(大连)有限公司；质粒小提试剂盒和胶回收试剂盒购自天根生化科技(北京)有限公司；农杆菌菌株GV3101以及超表达载体pBI121为本实验室保存。
1.2 Arah1基因内源表达分析
设计基因特异引物QF和QR(如表1所示)，提取花生栽培种“花育9303”根、茎、叶片和成熟种子的RNA，反转录成cDNA。利用半定量RT-PCR方法检测其在根、茎、叶片、入土前果针、成熟种子果壳和种子中的表达情况，以花生基因Actin作为内参基因(表1)。PCR反应条件：94℃预变性30min；94℃变性30s，58℃退火30s，72℃30s，26个循环；72℃后延伸10min，用1％琼脂糖凝胶进行电泳。
图1实验结果显示：该基因只在种子中有很强的表达信号，在根、茎、叶片、入土前果针、成熟种子果壳均不表达。这表明该基因在种子中特异表达。
该启动子DNA序列(SEQ ID NO：1)如下：
1.3花生幼嫩叶片DNA提取及启动子片段的克隆
本发明用植物DNA提取试剂盒提取“花育9303”幼嫩叶片基因组，以此为模板，使用高保真酶Phusion，利用特异引物P5F和P6R(表1)进行PCR扩增。
PCR反应体系：ddH2O 31μL，5x HF buffer(含Mg2+)10μL；dNTP(2.5mM)2μL；P5F和P6R(5μM)各2μL；DMSO 0.6μL；Phusion酶0.5μL；基因组模板2μL。
PCR反应条件：98℃预变性30sec；98℃变性10sec，58℃退火10sec，72℃50sec，30个循环；72℃后延伸10min。用1％琼脂糖凝胶进行电泳，电泳结束后，切取含目的条带的凝胶，用琼脂糖凝胶回收试剂盒(北京天根生化科技有限公司)回收PCR产物。将其回收产物与pEASY-Blunt simple载体(北京全式金公司)连接、热激法转化大肠杆菌DH5a(北京全式金公司)，将阳性克隆送上海桑尼生物科技有限公司测序，经测序正确后，命名为pEASY-Arah1-Pd。
表1本发明用到的引物
1.4启动子序列及顺式作用元件分析
将扩增到的启动子序列经PLACE(http://www.dna.affrc.go.jp/PLACE/signalscan.htm1)和PlantCARE(http://bioinformatics.psb.ugent.be/webtools/plantcare/htm1/)在线分析，该启动子在-147(-)、-244(+)和-286(+)位置DNA负链有一个RY重复序列(CATGCA)，该序列常存在于种子或胚特异启动子序列中。这表明：该启动子能调控下游基因在种子中特异表达。
1.5Arah1-P启动子驱动GUS报告基因表达载体的构建
以pEASY-Arah1-Pd质粒为模板，以SUN699F和SUN670R引物进行PCR扩增(PCR扩增条件与第一次扩增相同)(该引物带有一段与植物表达载体pBI121酶切位点HindI和BamHI附近一致的DNA序列，可以用于无缝克隆)，得到的片段经无缝克隆技术连接到经HindI和BamHI酶切的植物表达载体pBI121质粒。筛选阳性克隆，并测序正确后，得到的质粒命名为pBI121-Arah1-P。此时，该载体中原来的烟草花叶病毒CaMV 35S启动子被Arah1-P启动子替代(Arah1-P：：GUS)。
1.6农杆菌介导的拟南芥遗传转化、筛选及分子鉴定
利用热激法将构建好的质粒pBI121-Arah1-P转化到农杆菌GV3101，然后利用花絮侵染法转化拟南芥。收集农杆菌侵染后的拟南芥T0代种子，在无菌超净工作台上操作，用70％酒精处理1min，2.6％次氯酸钠处理10min，再用无菌水冲洗4-5次，分散于含卡那霉素50μg mL-1的MS培养基上。待T1代卡那霉素抗性的拟南芥幼苗长出2片子叶后，选取绿色健康的幼苗移栽到蛭石中。待生长大约三周后，随机选取了10株正常生长的拟南芥，提取叶片DNA，以GUS-F和GUS-R为引物(表1)，对GUS基因进行PCR检测，筛选阳性转基因植株。
1.7 GUS组织化学染色
配制GUS染色液(0.1M磷酸钠缓冲液；10mM EDTA；0.5mM铁氰化钾；0.5mM亚铁氰化钾；1mM X-Gluc；0.1％Triton X-100)；将试验材料在90％丙酮中(冰浴)固定15-20min；然后在染色液(不含X-Gluc)中漂洗3次；将材料置于GUS染色液中，37℃放置12-16h；先用70％酒精脱色半小时，再用100％酒精脱色；镜检照相。
将转基因拟南芥种植于MS培养基，GUS组织化学染色结果发现(图2)：转基因拟南芥成熟的种子、萌发后的子叶、胚轴、胚根以及长出真叶后残留的子叶均能够被染上蓝色(图2)。结果表明启动子可以驱动外源报告基因GUS在拟南芥种子特异表达，可以用于植物种子遗传改良。
以上实施例仅用以说明本发明的技术方案，而非对其进行限制；尽管参照前述实施例对本发明进行了详细的说明，对于本领域的普通技术人员来说，依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或替换，并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
序列表
<110> 山东省花生研究所（山东省农业科学院花生工程技术研究中心）
<120> 一种花生过敏原基因Arah1的启动子Arah1-P及其克隆和应用
<130> 2020
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1492
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ggaacacacc atatagaatt aagatttttg aggaacaaaa ctttttattt taaaaattta 60
gttcaaaata tcacagtata tatatatata tgaaaaaaaa ttgtggcaaa aatatataat 120
gcactggaaa tatggcctag agggaattaa agcatatgaa tggagcaatc tgaaagtgtt 180
gagttcttct aattaagtat gtatgtctgt ttctgttgtt atataattgt tgttagatgt 240
gctaagttta tgtgatatga gatagtttaa tgttcttatt taagaataat cattgattgt 300
ttaatatctt tgatgataat gtcaaaagca ataattaatt aaattggtga gtattattta 360
gtgtatcttt ctccttttca taggtctgat ttgtaatgaa ctatctaaag aaaaaggcaa 420
agaaaaagta cacacaaaaa actcatattc tgtgtcttaa ttagaaaaag aaatcgatgt 480
gtaatattgc tcgaaataat gaagcacttt catgatgatt tgataaataa ttttataaaa 540
tatttctgtt ggggataata attatatatt gggcaaatat gctaataacc ggattttcct 600
atggaactaa ctttgattat ttaaccaata atgtctagga aaatagtaga taaacaaaag 660
cttaattatg aagaaaaaga gaaacttaaa cgaaaagata aatgttggtt aaaactgttg 720
attattccaa tctttcccaa tagaataaat tatcccccaa ttattttatc aatatgttaa 780
ttaggaacat tttggagatt attgtatcat aatttttaat taagaatgtg attactttgt 840
ttccatagtt ggattatctt ttgtcggcct taggttgtgt tattaatgct tctcatacgg 900
tagcacaact ctgtagctag agcttgcgat tatattgaac agacgtaggt gatgtttaaa 960
attttggtta atgaagacat atatgattta gcctgtgata cgcagatacg ctaaaatgca 1020
aggcaatacc gtaaggatag ctgaatgggt gatgatattt tatcaaaaga caaaaggaat 1080
ccatcctgta tgtgcagtgg aaccttttcc cagacaatgt ccaagttcat catgagagcc 1140
cagcgccgcg tccatcacca tacatatcca aagcaaaaaa attaactaat actgaacccc 1200
aattgccatg catattaaca cgtacactct ctatgtcaat gtgtccacca tgcaccacaa 1260
tccatccatc tcttgtttat ctctaagcca agacctagta atgttgttgc cacctcacat 1320
ttctcaattt caacactcgt caacctgcat gccactccaa tgcccaagtg tacatccata 1380
tcatcctaac gcaactatat aaataccact cacctccctc catcatttca ccatccacac 1440
tactcataat aatacatatt catcaatcat ctatataagt agttgcagga gc 1492
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tttctgcaac gcatgccaag 20
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tttgcttcaa gtcgtccggt 20
<210> 4
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ttggaatggg tcagaaggat gc 22
<210> 5
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
agtggtgcct cagtaagaag c 21
<210> 6
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
agaacccaaa agccaatcg 19
<210> 7
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
cggttcctgt tgacaactc 19
<210> 8
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
accatgatta cgccaagctt ggaacacacc atatagaatt aag 43
<210> 9
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gactgaccac ccggggatcc gctcctgcaa ctacttatat agatg 45
<210> 10
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gtgacaaaaa ccacccaag 19
<210> 11
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ctttcttgtt accgccaac 19
Claims (2)
1.一种花生过敏原基因Arah1的启动子Arah1-P，其特征在于：其核苷酸序列如SEQ IDNO：1所示；所述启动子可驱动外源基因在植物种子中特异表达。
2.根据权利要求1所述的一种花生过敏原基因Arah1的启动子Arah1-P在驱动外源基因在植物种子中特异表达的应用，其特征在于：所述外源基因为GUS基因。
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| 《花生Oleosin启动子和Ara h1启动子功能鉴定及转录因子WRI1基因的in silico分析》;鲁亚萍;《中国优秀硕士学位论文全文数据库 农业科技辑》;20130228(第2期);第D047-158页 * |
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