CN105925602B - 山核桃miR169在提前植物花期中的应用 - Google Patents
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Abstract
本发明公开了山核桃miR169在提前植物花期中的应用。应用山核桃miR169,有望调控山核桃的花期,使得山核桃童期缩短,提前开花,结实。
Description
技术领域
本发明涉及基因功能领域,更具体地涉及山核桃miR169在提前植物花期中的应用。
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。通过Solexa测序发现,miR169在山核桃营养生长阶段表达量为1.12,在生殖生长阶段的表达量为8.26,生殖生长是营养生长表达量的7.3倍,表明miR169与花发育存在一定的关系。为了研究miR169的功能,通过转山核桃miR169基因,探索其在植物发育中的作用。
发明内容
本发明的目的在于提供山核桃miR169在提前植物花期中的应用。
本发明提供了山核桃miR169在提前植物花期中的应用,所述山核桃miR169的碱基序列如SEQ ID NO.1所示。
在一个实施例中,所述植物是拟南芥或烟草。
在一个实施例中,所述植物是拟南芥。
在一个实施例中,获得山核桃miR169转基因植株的具体方法包括:将包含所述山核桃miR169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。
由于山核桃miR169本身序列很短(GGCAGTCTCCTTGGCTAATC),只有20bp,而前体序列相对较长,连接到载体上,有利于转化实现,优选地,所述前体基因的碱基序列如SEQIDNO.2所示。
本发明的优点在于,应用山核桃miR169,有望调控山核桃的花期,使得山核桃童期缩短,提前开花,结实。
附图说明
图1为为山核桃花芽的总RNA;
图2为山核桃miR169前体的表达载体菌液PCR检测;
图3为山核桃miR169转基因拟南芥植株的抗性筛选;
图4为野生型(WT)和山核桃miR169转基因拟南芥的叶片数目统计;
图5为观察到的野生型(WT)和山核桃miR169转基因拟南芥的表型。
具体实施方式
1.材料
1.1实验材料
山核桃雌花芽、根、茎、叶、果实均采自浙江省杭州市临安板桥村,选取同一株山核桃树进行采样。样品采下后立即液氮保存,带回实验室并放置在-80℃冰箱保存待用。拟南芥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PE9700 PCR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山核桃花芽总RNA的提取
采用改良CTAB+Trizol法提取花芽样品总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200μL β-巯基乙醇;
(2)取-70℃保存的花芽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,12000rpm 4℃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。12000rpm 4℃离心10min弃掉上清,沉淀加入65℃预热好的SSTE 400μL溶液至全溶;
(8)溶液中加入1mL TRIzol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,8000rpm 4℃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 cDNA合成
使用TAKARA试剂盒Prime ScriptTMⅡ 1st Strand cDNA Synthesis Kit,详细内容如下:
(1)在经过DEPC处理过的PCR管中配置下列反应体系:
(2)65℃保温5min,冰上迅速冷却。(模板RNA变性)
(3)再次配置反应体系如下:
(4)缓慢摇匀。
(5)42℃反应30-60min。
(6)95℃5min酶失活后,冰上冷却。
2.3前体序列的引物设计
依据前体序列:
TCTTGTATGCAAAAATAAATATATATAGTAGTGTTCATGATGAGGGATGTTCAGACGTTGCAGTTCGAAGAAGAGAAGACCTGGCATGAGAATGAAGAGACTGTAAGATTAGCCAAGGAGACTGCCTACGAATCACAGAAAGAGTTTCTAGGGATCCAAACACACAAAACTACTAATTATATCTTGAAGCCGCTTGGAGGAGCAGGCAAGTCATCCTTGGCTACCAAACAAAGACTCTTATCCTCATGATAGATCTCTCCTCTCAAAAATATATTTCCTTA,使用PrimerPrimer 5.0设计引物,引物两端分别加上酶切位点KpnI和XbaI,正向引物:5’-GGGGTACCGATGAGGGATGTTCAGACGT-3,反向引物:5’-GCTCTAGATTGTTTGGTAGCCAAGGATG-3’,克隆所需序列。
2.4 PCR扩增反应
以山核桃cDNA为模板扩增,反应体系如下:
PCR反应条件为:94℃预变性5min,94℃变性30s,56℃退火30s,72℃延伸1min。30个循环后,72℃延伸10min。PCR反应完成后4℃暂时保存,其中退火温度可根据Tm值进行调整。
2.5琼脂糖凝胶电泳检测与胶回收
将PCR反应产物进行1%琼脂糖凝胶电泳,电泳缓冲液为TAE(40mM Tris-乙酸盐,1mMEDTA),核酸燃料为EB。紫外光检测电泳结果并回收PCR产物。利用上海生工公司的DNA胶回收试剂盒进行胶回收,具体步骤如下:
(1)通过琼脂糖凝胶电泳尽可能将目的DNA片段与其他片段分开,用干净的手术刀片将含有目的DNA的琼脂糖凝胶块切下,放入1.5mL离心管中。每个胶块尽量不要超过400mg,否则将会导致溶胶不完全。另外切胶过程应尽可能快,从而减少DNA在紫外线中暴露时间来降低对DNA的损伤。
(2)根据胶块的质量和浓度,每100mg琼脂糖加300~600μL的比例加入Buffer B2。
(3)置于55℃金属浴10min,期间混匀2~3次直至胶块完全溶化为止。
(4)当目的片段<500bp时,可加入1/3体积的Buffer B2的异丙醇,混匀。若≥500bp,则此步骤可以省略。
(5)将溶化好的溶液全部移入吸附柱,8,000xg离心30sec。倒掉收集管中的液体,并将吸附柱放入同一个收集管中。
(6)加入500μL Wash Solution,9000xg离心30sec。再次倒掉收集管中的液体。
(7)重复一次步骤6。
(8)将带有收集管的吸附柱放入离心机,9000xg空离1min。扔掉收集管,并将吸附柱置于灭过菌的1.5mL EP管中。
(9)打开吸附柱的盖子,室温放置10min。为了去除残留的酒精,否则会严重影响回收得率和后续实验结果。
(10)在吸附膜中央加入15-30μL ddH2O。室温静置5min,9000xg离心1min。1.5mLEP管底部收集的DNA溶液即为回收的DNA,-20℃保存。
2.6 DNA回收片段与PMD-19T载体连接
根据PMD19-T载体使用说明书,在灭过菌的PCR反应管中将Vector与回收DNA片段进行T-A克隆连接,反应体系如下:
轻轻混匀后16℃过夜连接10h以上,转化DH5 α感受态。
2.7连接产物转化DH5α感受态
转化感受态具体操作步骤如下:取出适量DH5α感受态细胞并至于冰上冻融。在超净工作台内向冰冷的感受态细胞中加入上述连接混合液,并用移液器吸头轻轻吸打混匀。置于冰上20min。快速平稳放入42℃水浴中60s(热激,使细胞膜开放,重组质粒进入细胞),并迅速放入冰水混合物中,保持2min(使细胞膜关闭)。加入500μL无抗LB液体培养基,37℃恒温振荡培养1h。室温下4000rpm离心5min,弃掉上清(保留少许培养基,约50μL)。超净台内,用枪头轻轻吸打并重悬菌液,涂于氨苄抗性平板。倒置琼脂平板于37℃,平板通常在37℃温育14h。
2.8重组子的筛选和鉴定
用10μL小号枪头挑取平板上至少5个单菌落(每个菌落均做好标记),并置于含有800μL LB培养基(含相应抗生素)的1.5mL EP管中,37℃振荡培养4h左右至培养基浑浊,并对培养基浑浊的菌样进行菌检验证,菌检验证载体构建成功的菌液取1mL送上海生工测序。利用BLAST、CLUSTAL、MEGA4.0等软件对序列进行分析。
2.9表达载体的构建
将测序获得完全正确的序列,提取质粒后用KpnⅠ和XbaⅠ双酶切中间载体和表达载体pCAMBIA13011(pC13011),再用T4连接酶连接经过双酶切的pC13011载体和miRNA169前体片段,然后转化到大肠杆菌中提取质粒酶切验证。
2.10转化农杆菌
2.10.1电击杯的处理
(1)用移液枪吸取ddH2O反复冲洗电击杯3-5次。
(2)用75%的乙醇反复冲洗电击杯3-5次。
(3)将电击杯浸泡在无水乙醇中2h,然后倒掉乙醇,置于超净工作台中让残余酒精挥发。
(4)酒精挥发完全后,盖好电击杯盖子,室温保存备用。
2.10.2电转化
采用仪器为伯乐公司GeneP μ Lser 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)取出保藏的农杆菌GV3101感受态细胞放置冰上融化。
(2)取1μL质粒加入到解冻的感受态细胞中,轻轻混匀。
(3)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。
(4)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。
(5)28℃,220rpm震荡培养2h左右。
(6)取30-40μL菌液,用无菌的涂布棒将菌液均匀的涂在含有相应抗生素YEP平板上,倒置放入28℃培养箱培养2-3天。
2.10.3电转化农杆菌及其检测
将酶切验证正确的pC13011-miRNA169质粒转化到农杆菌GV3101中,再从农杆菌中提取质粒后,转化到大肠杆菌中提质粒,酶切验证。验证正确的含有pC13011-miRNA169质粒的农杆菌,-70℃保存菌种。
2.11拟南芥遗传转化及其转基因植株纯合子的筛选
2.11.1拟南芥的种植
(1)将野生型拟南芥种子均匀撒在1/2MS固体培养基中。
(2)4℃冰箱避光春化处理3天。
(3)3天后,移至培养室培养,条件为温度23℃,光强7000-9000Lx,光照16小时,黑暗8小时。
(4)待拟南芥长出2片子叶和2片真叶后,将其移植到含基质的盆中,继续培养。
2.11.2拟南芥的遗传转化
(1)农杆菌菌液的准备:配制含有卡那霉素和利福平的YEP固体培养基,倒平板,划线以活化保存的农杆菌,28℃倒置培养2天。挑单菌落至1mL含相应抗生素的YEP液体培养基中,28℃培养2天,再转移至100mL相同的培养基中扩大培养,直至培养液浑浊变为橙色,测其OD值达到1.2时停止培养。
(2)4000rpm,离心10min,倒掉上清液,用浸染介质悬浮菌体。
(3)浸花法转化拟南芥
a.选取开花初期的拟南芥,将花序浸入含有目的基因农杆菌的转化介质中,约10-20秒。
b.将浸染过的拟南芥植株平放于一个大容器中,避光培养24小时。
c.第二天,放置正常条件下继续培养。
d.收获拟南芥种子,干燥。
2.11.3拟南芥转基因种子的筛选及种植
(1)将转基因种子均匀撒在1/2MS的固体培养基中(培养基中潮霉素的浓度为50mg/L)。同时将野生型拟南芥种子撒在不含潮霉素培养基中,作为对照。
(2)4℃避光春化处理3天。
(3)3天后将撒有野生型拟南芥种子的培养皿打开,置于培养室中正常培养,而含有转基因种子的培养皿则避光培养。
(4)48小时后,将含有转基因种子的培养皿置于光下正常培养。转基因成功的植株就会生长,反之不生长。
(5)待拟南芥长出2-4片真叶后,移植到含有泥炭的盆中,继续培养。
(6)观察对照拟南芥(野生型WT拟南芥)和山核桃miR169转基因拟南芥的生长情况。
3.实验结果
3.1山核桃花芽的总RNA提取分析
利用改良CTAB+TRizol法提取山核桃花芽及拟南芥叶片总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山核桃miR169前体序列的克隆
根据所设计的引物序列,使用PCR扩增,产物经连接到pMD19-T(simple),菌检PCR进行检测(图2),并送到上海生工测序,验证了山核桃miRNA169前体的存在。
3.3山核桃miR169的功能验证
将所获得前体序列,进行表达载体的构建,将miR169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。对山核桃miR169转基因拟南芥植株进行抗性筛选,筛选到纯合后,对其表型进行观察可以发现转基因植株叶片数目在2016年3月19日-2016年3月25日期间,明显多于野生型(图4);同时观察发现,转基因植株与野生型相比,花期提前(图5)。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (1)
1.山核桃miR169在提前植物花期中的应用,包括:将包含所述山核桃miR169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,其特征在于,所述山核桃miR169的前体基因的碱基序列如SEQ ID NO.2所示。
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