CN109055371B - 光皮桦miR169c的前体基因及其在提前植物开花中的应用 - Google Patents
光皮桦miR169c的前体基因及其在提前植物开花中的应用 Download PDFInfo
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Abstract
本发明公开了一种光皮桦miR169c的前体基因,所述前体基因的序列如下:(a)由SEQ ID NO:1所示的核苷酸序列,或(b)与SEQ ID NO:1所示的核苷酸序列至少具有70%、至少具有75%、至少具有80%、至少具有85%、至少具有90%、至少具有95%、至少具有96%、至少具有97%、至少具有98%或至少具有99%同源性且经剪切后生成光皮桦miR169c的核苷酸序列。本发明提供了光皮桦miR169c的前体基因在提前植物开花中的应用。应用光皮桦miR169c的前体基因,有望人为地提前植物开花。
Description
技术领域
本发明涉及一种光皮桦miR169c的前体基因及其在提前植物开花中的应用。
背景技术
MicroRNA(miRNA)是一类广泛存在于动植物中,含有茎环结构的miRNA前体,经过Dicer加工之后的一类非编码的小RNA分子(18-25个核苷酸)。miRNA通过在转录水平或者转录后水平对基因组上的靶基因抑制其翻译或者切割靶基因来调控基因表达,在基因表达中起负调控其靶基因作用。miRNA在植物从成花诱导到花器官特征属性形成的整个花发育过程均发挥着关键作用。
miR169是一种广泛存在于单子叶植物和双子叶植物中的miRNA。在拟南芥中,miR169的靶基因为NF-Y家族成员,通过抑制NF-YA基因转录来调控胁迫应答,同时miR169过表达促进拟南芥提前开花。然而,光皮桦miR169c的功能作用尚未可知。
发明内容
本发明的目的在于提供了一种光皮桦miR169c的前体基因,所述前体基因的序列如下:
(a)由SEQ ID NO:1所示的核苷酸序列,或
(b)与SEQ ID NO:1所示的核苷酸序列至少具有70%、至少具有75%、至少具有80%、至少具有85%、至少具有90%、至少具有95%、至少具有96%、至少具有97%、至少具有98%或至少具有99%同源性且经剪切后生成光皮桦miR169c的核苷酸序列。
在一个实施方案中,所述光皮桦miR169c的序列为SEQ ID NO:2。
本发明还提供给了上述前体基因的重组载体、转基因细胞系或重组菌。
在一个实施方案中,所述重组菌为上述所述前体基因***表达载体得到的重组菌。
本发明还提供给了上述前体基因在提前植物开花中的应用。
在一个实施方案中,所述植物为拟南芥或烟草。
在一个实施方案中,所述植物是拟南芥。
在一个实施方案中,上述应用包括将包含所述光皮桦miR169c的前体基因连接到载体上,通过农杆菌介导转化到拟南芥、筛选、培养和获得转基因株系。
本发明提供了光皮桦miR169c的前体基因在提前植物开花中的应用。应用光皮桦miR169c的前体基因,有望人为地提前植物开花。
附图说明
此处所说明的附图用来提供对本申请的进一步理解,构成本申请的一部分,本申请的示意性实施例及其说明用于解释本申请,并不构成对本申请的不当限定。在附图中:
图1为本发明的光皮桦叶片的总RNA;
图2为光皮桦miR169c前体的表达载体菌液PCR检测;
图3为光皮桦miR169c转基因拟南芥植株种子的抗性筛选;
图4为过表达miR169c的拟南芥PCR检测;
图5为观察到的野生型与光皮桦miR169c转基因的拟南芥的开花表型(Col-0为野生型,OEmiR169c为转基因植株)。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
为了使本发明的目的、技术方案及优点更加清楚明白,下面结合附图和具体实施例对本发明作进一步详细说明。应当理解,此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明。
1.材料
1.1实验材料
实验材料取自浙江省临安浙江农林大学智能实验室的组培苗,品种光皮桦G49-3,样品采下后立即液氮保存,放置在-80℃冰箱保存待用。拟南芥野生型种子使用哥伦比亚生态型(Columbia-0)。
1.2实验试剂与仪器
DNA聚合酶、各种限制性内切酶、T4连接酶、Marker和TRIzol试剂均购自宝生物工程(大连)有限公司;质粒提取试剂盒及DNA胶回收试剂盒购自上海生工生物工程股份有限公司。PCR仪为美国PE9700PCR仪,超净工作台购自苏州诚净净化科技有限公司。
1.3引物合成及测序
引物合成及测序均由生工生物工程(上海)股份有限公司完成。
2.方法
2.1光皮桦总RNA的提取
采用改良CTAB+Trizol法提取光皮桦叶片的RNA(Total RNA),步骤如下:
(1)在10mL离心管中加入3mL 65℃预热的CTAB提取缓冲液(10%CTAB,10%PVP40,1.0M Tris-HCl(pH 8.0),5M NaCl,0.6M EDTA(pH 8.0)),加入200μLβ-巯基乙醇;
(2)取-70℃保存的叶片2g,放入液氮充分预冷的研钵中,于液氮中充分研磨至百色粉末;
(3)将白色粉末迅速转入预热的提取液中,立即充分振荡混匀,65℃水浴30min,期间振荡3-4次;
(4)在混合物中加入等体积的25:24:1(酸性饱和酚:氯仿:异戊醇),约3ml。混合均匀后14000rpm离心10min(常温);
(5)取上清转入新的10ml离心管中,加入等体积的氯仿/异戊醇(24:1)上下颠倒混匀,12000rpm4℃离心10min后取上清;
(6)重复步骤4一次,直至中间层消失;
(7)上清转移至1.5mL离心管中,加入等体积的异丙醇后放置于-20℃冰箱冷冻30min。12000rpm4℃离心10min弃掉上清,沉淀加入65℃预热好的SSTE400μL溶液至全溶;
(8)溶液中加入1mLTRIzol试剂,室温静置5min。再加入200μL氯仿摇匀,室温静置2-3min;
(9)4℃12000rpm离心15min;
(10)吸取上清,加入等体积异丙醇,室温静置10min;
(11)4℃12000rpm离心10min,弃掉上清,肉眼可见RNA沉于管底;
(12)加入1mL预冷的75%乙醇洗涤沉淀,温和振荡,8000rpm4℃离心5min,弃掉上清;
(13)重复步骤11,并将沉淀真空干燥7-10min;加入适量DEPC水溶解沉淀,-80℃保存备用。
2.2基因克隆
光皮桦的RNA(Total RNA)用适量DEPC ddH2O稀释后,参照PrimeScriptTM RTReagent Kit(Perfect Real Time)(TaKaRa)的说明书进行反转录cDNA第一链的合成。配制下列基因克隆PCR体系(20ul):
反应程序为:94℃,3min;94℃,30sec,58℃,30sec,72℃,1.5min,循环37次;72℃,8min。
基因克隆所用引物(表1)。
2.3目的片段回收
利用AxyPrepTM DNA凝胶回收试剂盒(AXYGEN)进行目的片段回收。
(1)在紫外灯下切含有基因目的片段的琼脂糖凝胶,称取凝胶重量,该重量作为一个凝胶体积(如100mg=100ul)。
(2)加入Buffer DE-A溶液,约3个凝胶体积,放入75℃水浴锅加热6-8min,期间每隔2-3min摇晃1次,至凝胶完全融化。
(3)加入Buffer DE-B溶液,约1/2的Buffer DE-A体积,混匀;当目的片段长度小于400bp时,再加入适量异丙醇,约1个凝胶体积。
(4)将上一步骤的混合液吸到DNA制备管(置于2ml离心管中),12000rpm1min,去滤液。
(5)加500ul Buffer W1于置备管中,12 000rpm 1min,弃滤液。
(6)加700ul Buffer W2于置备管中,12 000rpm 1min,弃滤液,重复1次。
(7)将置备管置于2ml离心管中,12 000rpm 1min,弃滤液。
(8)将置备管放入干净的1.5ml离心管,向置备管中央加25-30ul已预热至65℃的ddH2O,室温静置1-3min,12 000rpm 1min,洗脱DNA。
(9)取2-3ul DNA溶液用于琼脂糖凝胶电泳进行检测,剩余溶液放置-20℃保存。
2.4目的片段连接
混合下列溶液,轻轻混匀,短暂离心。PCR:25℃ 5-30min(根据目的基因大小而定)。
2.5目的片段转化
(1)将5ul连接产物,加入到50ul感受态TransT1中,冰浴25-30min。
(2)42℃水浴热激40sec。置于冰上2min。
(3)加入300-500ul无激素LB液体培养基,200rpm 37℃培养30-60min。
(4)4000rpm 2min,去部分上清,留50-100μl菌液,加入10μl异丙基硫代半乳糖苷(IPTG,100mg/ml)和8μl 5-溴-4-氯-3-吲哚-α-D-半乳糖苷(X-gal,100mg/ml),混匀。
(5)将剩下的溶液涂于具有卡那霉素(Kan,50mg L-1)固体培养基中,37℃倒置培养过夜。
2.6目的片段PCR验证
(1)挑取1.3.2中步骤5的白色单菌落,置于相同浓度抗生素的液体LB培养基500ul中,37℃培养3-6h,取1ul,用于菌液PCR。
(2)用基因克隆引物进行菌液PCR检测,反应体系(10ul)如下:
(3)PCR程序为:94℃ 3min;94℃ 30sec,57℃ 30sec,72℃ x min(依据***片段长度来定),循环30次;72℃ 10min。
(4)琼脂糖凝胶电泳检测PCR产物,选取阳性克隆送往生工生物工程(上海)股份有限公司测序。
利用上述克隆得到的BlmiR169前体premiR 169c,构建miR169的超表达载体。
2.7入门载体构建
(1)配制下列PCR体系(20ul):
(2)PCR程序为:94℃ 5min;94℃ 30sec,58℃ 30sec,72℃ 1min循环35次;72℃5min。
(4)保证产物为单一条带后,取1ul PCR产物用于下列组分的配制,剩余PCR产物保存于-20℃冰箱。
(5)将上述混合液混匀,并短暂离心。然后在PCR仪上按条件反应:25℃ 5-20min。
(6)取2ul步骤5中的反应液,加入50ul Trans1T1感受态中,轻柔混匀,冰浴25-30min。
(7)置于42℃水浴锅中热激40秒,加250ul液体培养基,200rpm 37℃孵育45-60min。
(8)4000rpm离心3min去部分上清液,剩余菌液涂于具有壮观霉素(Spec,50mg L-1)的LB固体培养基中,培养过夜。
(9)用基因特异引物进行菌液PCR检测,并测序确认。
2.8表达构建
遗传转化表达载体选用的是pGWB8(35S pro,C-6xHis),构建MiR169(BlmiR169c)表达载体。
(1)配制下列混合溶液,25℃ 1h,72℃ 10min;
(2)取2ul用于连接转化,然后挑克隆,菌液PCR检测;
(3)PCR体系:
(4)PCR程序:94℃ 5min;94℃ 30sec,56℃ 30sec,72℃ x min(根据目的基因片段长度)循环35次;72℃ 5min;
(5)琼脂糖凝胶电泳检测PCR产物,转化至大肠杆菌后选取阳性克隆送往生工生物工程(上海)股份有限公司测序;
(6)提取测序结果正确的菌株质粒,待用。
2.9电转化
采用仪器为伯乐公司GenePμLser Xcell电穿孔仪进行感受态细胞的转化。主要参数为:电脉冲2.5μF、电压2.5kV、电阻200Ω。操作步骤如下:
(1)配置YEP固体和液体培养基(表)
(2)YEP固体培养基微凉时加利福平和卡那霉素抗生素(50mg.L-1Rif和50mg.L- 1Kan),至超净工作台上倒平板。
(3)取出在-80℃冰箱保藏的农杆菌GV3101感受态细胞放置冰上融化;
(4)取1μL重组质粒加入到解冻的感受态细胞中,轻轻混匀;
(5)用ddH2O和75%的乙醇反复清洗电击杯3-5次,超净工作台晾干;
(6)取出在-80℃冰箱保藏的农杆菌GV3101感受态细胞放置冰上融化;
(7)取1μL重组质粒加入到解冻的感受态细胞中,轻轻混匀;
(8)将混合液加到电击杯中(-20℃预冷),在电脉冲为2.5μF、电压2.5kV、电阻200Ω的参数条件下电击;
(9)取出电击杯,迅速加入800μL预热的无抗的YEP液体培养基,悬浮细胞后,转移到1.5mL的离心管中;
(10)28℃,220rpm震荡培养2h左右;
(11)取40μL菌液,均匀地涂布在含有利福平和卡那霉素抗性的YEP平板上,倒置放入28℃培养箱培养2-3d。
2.10电转化农杆菌及其检测
含表达载体35Spro-miR169的大肠杆菌提取质粒转化到农杆菌GV3101中,再从农杆菌中提取质粒后,利用基因引物检测。验证正确的含有35Spro-miR169质粒的农杆菌,-70℃保存菌种。
2.11花序浸染法转化拟南芥的操作步骤
(1)野生型拟南芥种子(COL-0)点播在1/2MS培养基中,待长至四片子叶时移至已灭菌土壤穴盆中,培养室中培养。培养条件为:白天24℃/15h,夜间24℃/9h。3周左右减去拟南芥主茎,抑制顶端优势促进生殖生长。待拟南芥抽出多条侧枝并进入盛花期后采用花序浸染法侵染拟南芥。侵染前将土壤浸透。
(2)配置拟南芥浸润培养基(1/2MS液体培养基,PH5.7)。将含有目的表达载体的农杆菌划板活化(有抗生素的YEP培养基),挑取单克隆接种于200mL加抗生素的YEP液体培养基(50mg.L-1Rif和50mg.L-1Kan),28℃恒温摇床,200rpm,培养2d,至终浓度OD600=0.8-1.0。
(3)收集菌株:将菌液倒入50mL灭菌离心管中,4000-6000rpm离心十分钟,弃上清,用等体积的浸润培养基重悬菌体,并加入0.02%表面活性剂silwet。
(4)将盛花期的拟南芥花序,浸泡于重悬液中40秒后轻轻擦干植株叶片的遗留液体。
(5)侵染后的植株套上塑料袋,暗培养12-24h,之后养7-10d,进行二次侵染。
(6)侵染后的植株,正常培养,种子成熟前3天减少浇水量,按单株收取T0代种子装入1.5mL离心管中,存放于4℃冰箱中。
2.12拟南芥转基因阳性株筛选
(1)实验试剂:75%的乙醇、无水乙醇、无菌水、含有抗生素的1/2MS固体培养基(Hyg浓度为30mg·L-1)
(2)取适量的待筛选转基因拟南芥种子,加入1.5Ml的离心管中,加入75%的乙醇,充分振荡10min。倒去上层溶液后,加入无水乙醇振荡1min,重复操作三次。倒于灭菌的滤纸,在超净工作台吹干。
(3)将吹干的拟南芥种子,均匀的播种于固体培养基中,封好培养皿,4℃春化培养1d,后放置于长日照培养箱(日照16h/黑夜8h)。
(4)观察培养皿中拟南芥发芽情况,至幼苗有4-5片子叶时选择根系良好,叶片绿色的苗,移栽至基质中生长。
(5)待拟南芥有一定的生物量时,提取叶片DNA,进行阳性检测。
(6)观察对照拟南芥(野生型Col-0拟南芥)和光皮桦miR169c转基因拟南芥的生长情况。
3.实验结果
3.1光皮桦的叶片总RNA提取分析
利用改良CTAB+TRizol法提取光皮桦叶片的总RNA(见图1),并通过紫外分光光度计测定每个RNA样品260nm及280nm的吸光度,并以此计算RNA的浓度及纯度,并在1%琼脂糖凝胶电泳检测RNA的完整性。所提RNA的光密度比值均在2.0-2.2之间,说明总RNA基本无糖类、酚及蛋白质的污染;电泳结果则显示RNA样品的18s和28s两条带非常清晰,可推断RNA没有降解,符合下步实验的要求。
3.2光皮桦miR169c前体序列的克隆
根据所设计的引物序列,使用PCR扩增,菌检PCR进行检测(图2),并送到生工生物工程(上海)股份有限公司测序,验证了光皮桦miR169c前体的存在。
3.3光皮桦miR169c的功能验证
将所获得前体序列,进行表达载体的构建,将光皮桦miR169c前体序列连接到pC13011表达载体上,转化到DH5α感受态细胞中,将连接成功的单克隆提取质粒,转化到土壤农杆菌GV3101中,进行拟南芥转基因。对光皮桦miR169c拟南芥植株种子进行抗性筛选(如图3),将筛选出的转基因拟南芥体取DNA,进行PCR检测(如图4,PCR扩增获得光皮桦miR169c前体基因)。移栽种植,幼苗培养20天后观察野生型、35Spro-miR169c植株表型,发现过表达miR169c相比野生型植株提前开花结实(图5)。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应以权利要求的保护范围为准。
序列表
<110> 浙江农林大学
<120> 光皮桦miR169c的前体基因及其在提前植物开花中的应用
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 174
<212> DNA
<213> 人工序列
<400> 1
ctcgcatgta cggcgtagaa agtattatgc agccaaggat gacttgccga ctcttgctag 60
ctaggtagtt tgtttcctct ttataattca agtagtacta ctgatccaaa aaccgatcga 120
tcaaccaagt cggcaagttg tcgttggcta catgtttctt tcttctccgc atgc 174
<210> 2
<211> 21
<212> DNA
<213> 人工序列
<400> 2
agccaaggat gacttgccga c 21
<210> 3
<211> 21
<212> DNA
<213> 人工序列
<400> 3
ctcgcatgta cggcgtagaa a 21
<210> 4
<211> 21
<212> DNA
<213> 人工序列
<400> 4
gcatgcggag aagaaagaaa c 21
Claims (3)
1.光皮桦miR169c的前体基因在提前植物开花中的应用,其特征在于,所述前体基因的序列为SEQ ID NO:1。
2.根据权利要求1所述的应用,其特征在于,所述植物为拟南芥或烟草。
3.根据权利要求1所述的应用,其特征在于,将所述光皮桦miR169c的前体基因连接到载体上,通过农杆菌介导转化到拟南芥、筛选、培养和获得转基因株系。
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