CN114214325A - 光皮桦miR156a前体基因及在促进植物分枝形成中的应用 - Google Patents
光皮桦miR156a前体基因及在促进植物分枝形成中的应用 Download PDFInfo
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Abstract
本发明提供一种光皮桦的miR156a前体基因在调控植物分枝数量中的应用。本发明提供的光皮桦miR156a前体基因,该前体基因序列如SEQ ID NO:2所示。本发明还提供了光皮桦miR156a前体基因在促进植物分枝形成中的应用。通过光皮桦miR156a前体基因的过量表达,有望人为地促进植物分枝的形成,增加侧枝的数量,在调控植物株形及产量等方面有重要的应用前景。
Description
技术领域
本发明属于植物生物技术领域,涉及一种光皮桦miR156a前体基因及在促进植物分枝形成中的应用。
背景技术
microRNA(miRNA)是一类长度约为20-24nt内源非编码单链RNA分子,具有调控基因表达的功能,在植物的生长发育、信号转导和胁迫响应等过程中起着关键作用。miRNA基因以单拷贝、多拷贝或基因簇等多种形式存在于基因组中,而且绝大部分定位于基因间隔区。在植物中,细胞核内编码miRNA的基因在转录后经过一系列加工成为成熟的双链miRNA分子,该双链miRNA与沉默复合体RISC结合,通过剪切靶mRNA或抑制靶mRNA翻译来负调控基因表达。
MicroRNA156s(miR156s)是植物microRNAs中最为保守成员之一,在种子萌发、发育阶段转变、分蘖、花青素合成和赤霉素信号转导等方面有重要的调控作用。研究也显示,不同的miR156基因通过靶向不同的基因,在植物中发挥不同的功能。miR156在树木中调控分枝形成的作用尚未见有报道,因此,利用基因工程技术,将从光皮桦中克隆获得miR156a前体基因转入其它植物中,对于研究其功能具有重要意义,同时极具应用前景。
发明内容
为解决上述问题,本发明的目的在于提供一种光皮桦miR156a前体基因及其促进在植物分枝形成中的应用。
本发明提供一种光皮桦miR156a成熟miRNA,其序列如SEQ ID NO:1所示。
本发明还提供一种编码所述光皮桦miR156a成熟miRNA的前体基因。
在一个实施方案中,该光皮桦miR156a前体基因的序列如SEQ ID NO:2所示。
本发明提供了含有上述基因的重组载体、宿主细胞、转基因细胞系或重组菌。
在一个实施方案中,该重组菌为将上述基因插入表达载体得到的重组菌。
另一方面,本发明提供了一种光皮桦miR156a前体基因在促进植物分枝形成中的应用,该基因的序列如SEQ ID NO:2所示的核苷酸序列。
在一个实施方案中,该光皮桦miR156a前体基因经剪切后的成熟序列为SEQ IDNO:1。
在一个实施方案汇总,将所述前体基因转入寄主植物基因组中,并在转基因植物中超量表达,促进植物分枝形成。
在一个实施方案中,该植物为烟草或拟南芥。
在一个实施方案中,该植物为烟草。
在一个实施方案中,包括:将包含上述光皮桦miR156a前体基因连接到过表达载体上,利用农杆菌介导转化到烟草,通过筛选和培养获得转基因株系。
本发明还提供一种转基因植株的构建方法,采用农杆菌介导的方法,将含有所述前体基因的过表达载体转入植物基因组中,筛选获得转基因植株,所述的转基因植株与野生型相比,转基因植株分化生长出更多的分枝。
本发明提供了光皮桦miR156a前体基因在促进植物分枝形成中的应用。通过光皮桦miR156a前体基因的过量表达,有望人为地促进植物分枝的形成,增加侧枝的数量,在调控植物株形及产量等方面有重要的应用前景。
附图说明
图1为本发明的光皮桦叶片的基因组DNA。
图2为光皮桦miR156a前体的表达载体菌液PCR检测。
图3为光皮桦miR156a前体转基因烟草植株的抗性筛选。
图4为转基因烟草miR156a前体表达水平检测。
图5为过量表达miR156a烟草与野生型烟草分枝数量的比较。
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。
实验材料取自浙江农林大学林木遗传育种实验室的组培苗,品种光皮桦优3,叶片样品采下后立即液氮速冻并放置在-80℃冰箱保存待用。烟草野生型采用本生烟(Nicotiana benthamiana)。
DNA聚合酶、DNA Marker和TRIzol试剂均购自宝生物工程(大连)有限公司;十六烷基三甲基溴化铵(CTAB)、β-疏基乙醇、聚乙烯吡咯烷酮(PVP)、NaCl、Tris-HCl等DNA提取试剂购自上海生工生物工程股份有限公司;质粒提取试剂盒、DNA胶回收试剂盒购自爱思进生物技术有限公司,无缝克隆试剂盒购置海南你行生物技术有限公司。PCR仪为美国伯乐PCR仪,超净工作台购自苏州诚净净化科技有限公司。
引物合成及测序均由北京擎科生物技术有限公司杭州分公司完成。
实施例1光皮桦miR156a前体基因克隆
利用改良CTAB法提取光皮桦叶片基因组DNA(图1),通过Nanodrop分光光度计测定每个DNA样品的OD260、OD280,并以此计算DNA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DNA的OD260/OD280比值均在1.80-1.90之间,浓度均在200ng/μL以上,说明DNA无多糖、酚及蛋白质的污染;琼脂糖凝胶电泳结果显示DNA样品条带清晰,大小在10kb以上,可推断DNA没有降解,符合后续实验的要求。
将光皮桦的基因组DNA用适量ddH2O稀释至50ng/μL后,进行PCR扩增。基因克隆PCR体系如下(25μL):
反应程序为:94℃,3min;94℃,30sec,60℃,20sec,72℃,20sec,循环35次;72℃,5min。
基因克隆所用引物为Primer1:5’-AGGCATCATGTTGACAGAAGAG-3’,Primer2:5’-ATGGAAGTGATGGTGACAGAAG-3’。
通过琼脂糖凝胶电泳进行PCR产物检测(图2),然后利用AxyPrep DNA凝胶回收试剂盒(爱思进)进行目的片段回收。将目的片段连接到pEASY-T1 Cloning Vector后,转化Trans-T1感受态细胞,挑取阳性克隆鉴定和测序,序列如SEQ ID NO:2所示,该光皮桦miR156a前体基因经剪切后的成熟序列如SEQ ID NO:1所示。
实施例2表达载体构建
根据无缝克隆Nimble Cloning试剂盒(海南你行生物科技有限公司)说明书构建入门载体。
(1)提取测序正确的阳性克隆质粒,利用高保真taq酶PCR扩增光皮桦miR156a前体基因片段,配制如下PCR体系(25μL):
(2)PCR程序为:94℃,3min;94℃,30sec,60℃,20sec,72℃,20sec,循环35次;72℃,5min。基因克隆所用引物为NC_Primer1:5’-AGTGGTCTCTGTCCAGTCCTAGGCATCATGTTGACAGAAGAG-3’,NC_Primer2:5’-GGTCTCAGCAGACCACAAGTATGGAAGTGATGGTGACAGAAG-3’。
(3)回收PCR产物,利用无缝克隆方法将miR156a前体基因构建至表达载体pNC-Cam1304-35S,配制反应体系(10μL)如下:
(4)将上述混合液充分混匀,在PCR仪上按50℃反应30min。
(5)取2-5μL步骤4中的反应液,加入50μL DH5α感受态中,轻柔混匀,冰浴25-30min。
(6)置于42℃水浴锅中热激60s,加250μL LB液体培养基,200rpm 37℃孵育45min。
(7)4000rpm离心3min去部分上清液,余下100μL菌液涂于LB固体培养基(含50mg/L卡那霉素)上,37℃培养过夜。
(8)用基因特异引物NC_Primer1和NC_Primer2进行菌液PCR检测,并测序确认。
(9)提取测序结果正确的菌株质粒,待用。
实施例3化学转化农杆菌及其检测
(1)取-80℃保存的农杆菌感受态GV3101于室温或手心片刻待其部分融化,处于冰水混合状态时插入冰浴中。
(2)取1.0μL质粒DNA加入50μL感受态中,用手拨打管底混匀,依次于冰上静置5分钟、液氮5分钟、37℃水浴5分钟、冰浴5分钟。
(3)加入700μL无抗生素的LB液体培养基,于28℃振荡培养2~3小时。
(4)6000rpm离心1min,留取100μL左右上清轻轻吹打重悬菌块涂布于LB(含50mg/L卡那霉素和50mg/L利福平)平板上,倒置放于28℃培养箱培养2-3天。
(5)挑取单克隆,通过PCR鉴定阳性农杆菌,-80℃保存菌种。
实施例4叶圆盘法转化烟草
(1)取保存于-80℃的菌种,划线于LB平板,倒置放于28℃培养箱培养3天,从平板上挑取含有目的基因的单菌落,接种到3-5ml YEB液体培养基中(含50mg/L卡那霉素和50mg/L利福平)于恒温摇床上28℃,180rpm摇培过夜至OD 600为0.6-0.8。
(2)培养过夜的菌液按1%-2%的比例,转入新配置的无抗生素的YEB培养基中,在与上述相同的条件下培养6h左右,待OD600为0.2-0.5时即可用于转化。
(3)取烟草无菌小苗的幼嫩、健壮叶片,去主脉,在无菌条件下用打孔器将其打成5mm直径的叶圆盘,放入固体诱导培养基MS1(MS+1mg/L 6-BA+0.1mg/L NAA)中预培养48h。
(4)将叶圆盘在稀释的农杆菌中浸泡30-60s,用无菌滤纸吸干。
(5)将侵染后的叶圆盘放入固体诱导培养基MS1(MS+1mg/L6-BA+0.1mg/L NAA)中,黑暗28℃共培养7天,至有微菌落产生。
(6)取出叶圆盘,用含500mg/L羧苄青霉素的无菌水漂洗5次左右,无菌滤纸吸干。
(7)将叶圆盘置于诱导出芽的固体选择培养基MS2(MS1+30mg/L潮霉素+400mg/L羧苄青霉素)中,在25-28℃,16h/8h光暗条件下选择培养。
(8)20天后转入MS3(MS1+30mg/L潮霉素+300mg/L羧苄青霉素)固体选择培养基中,培养2周后转入MS4(MS1+30mg/L潮霉素+200mg/L羧苄青霉素)固体选择培养基中培养(图3)。
(9)待不定芽长到1cm左右,将不定芽移至1/2MS固体培养基中(含200mg/L羧苄青霉素)生根培养,约5-10天长出不定根。
(10)将生根的小苗移入基质中,在培养室中培养40-50天,开花结种按单株收集种子。
实施例5转基因烟草纯合株系获取
(1)取适量的转基因烟草种子,加入75%乙醇,充分振荡10min。弃去75%乙醇后,加入无水乙醇振荡1min,重复操作3次。加入无菌水振荡1min,重复3次,将无菌水吸去。
(2)将灭菌后的烟草种子,均匀的播种于固体培养基(MS+30mg/L潮霉素)上,封好培养皿,置于光照培养箱萌发。
(3)烟草萌发后长至有4-5张叶片时,选择生长良好的幼苗,移栽至基质中培养。
(4)待烟草长至8-10张叶片时,提取叶片DNA,进行PCR检测。
(5)按上述方法培养3代,得到miR156a前体基因的转基因烟草的纯合株系。
实施例6转基因烟草miR156a表达水平检测及分枝性状观察
(1)取野生型和转基因烟草纯化株系种子,用无菌水浸泡2小时。
(2)将浸泡后的种子播种于基质上,培养箱内25℃,16h/8h光暗条件下萌发。
(3)萌发一周后,将小苗移栽至育苗盆内,每个株系至少移栽10株,培养室内25-28℃,16h/8h光暗条件下培养40-50天。
(4)取烟草叶片提取RNA,通过定量PCR对miR156a前体基因的表达进行检测(图4)。定量PCR结果显示不同,转基因株系miR156a前体基因表达水平存在差异。
(5)观察野生型和转基因烟草植株的形态,统计各个植株的分枝数量,比较野生型和转基因植株间分枝数量的差异。观察野生型和转基因植株在生长40天后的植株形态和分枝数量,发现野生型植株的平均分枝数量为4.4个;而转基因植株分枝数量随着miR156a表达水平的提高而明显增加,其中表达水平最低的株系植株平均分枝数为6.8个,表达水平最高的株系植株平均分枝数为12.8个,表明miR156a过量表达植株可以分化生长出更多的分枝(图5)。
综上所述,转入光皮桦miR156a前体基因使得植物的生长发育受到影响,促进分枝的形成,植株可产生更多的分枝。
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。
序列表
<110> 浙江农林大学
<120> 光皮桦miR156a前体基因及在促进植物分枝形成中的应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> 光皮桦(Betula luminifera)
<400> 1
ttgacagaag agagagagca c 21
<210> 2
<211> 107
<212> DNA
<213> 光皮桦(Betula luminifera)
<400> 2
aggcatcatg ttgacagaag agagagagca caacccgcca ttggctaaag agagtctttg 60
tctttgttgg gagtgtgctt tctcttcttc tgtcaccatc actccat 107
<210> 4
<211> 22
<212> DNA
<213> 人工序列
<400> 4
aggcatcatg ttgacagaag ag 22
<210> 4
<211> 22
<212> DNA
<213> 人工序列
<400> 4
atggaagtga tggtgacaga ag 22
<210> 5
<211> 42
<212> DNA
<213> 人工序列
<400> 5
agtggtctct gtccagtcct aggcatcatg ttgacagaag ag 42
<210> 6
<211> 42
<212> DNA
<213> 人工序列
<400> 6
ggtctcagca gaccacaagt atggaagtga tggtgacaga ag 42
Claims (10)
1.光皮桦miR156a成熟miRNA,其序列如SEQ ID NO:1所示。
2.编码权利要求1所述光皮桦miR156a成熟miRNA的前体基因。
3.如权利要求2所述的前体基因,其特征在于,序列如SEQ ID NO:2所示。
4.含有权利要求2或3所述前体基因的载体。
5.含有权利要求4所述载体的宿主细胞。
6.含有权利要求2或3所述前体基因的工程菌。
7.权利要求2或3所述前体基因在促进植物分枝形成中的应用。
8.如权利要求7所述的应用,其特征在于,将所述前体基因转入寄主植物基因组中,并在转基因植物中超量表达,促进植物分枝形成。
9.如权利要求5或6所述的应用,其特征在于,所述植物为烟草或拟南芥,优选为烟草。
10.一种转基因植株的构建方法,其特征在于,采用农杆菌介导的方法,将含有权利要求2或3所述前体基因的过表达载体转入植物基因组中,筛选获得转基因植株,所述的转基因植株与野生型相比,转基因植株分化生长出更多的分枝。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106191065A (zh) * | 2016-08-03 | 2016-12-07 | 吉林大学 | 蓝莓miR156基因及其克隆产物和应用 |
CN109055371A (zh) * | 2018-07-06 | 2018-12-21 | 浙江农林大学 | 光皮桦miR169c的前体基因及其在提前植物开花中的应用 |
CN110373413A (zh) * | 2019-06-24 | 2019-10-25 | 浙江农林大学 | 光皮桦miR169a的前体基因及其在降低植物低氮胁迫耐受性中的应用 |
-
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106191065A (zh) * | 2016-08-03 | 2016-12-07 | 吉林大学 | 蓝莓miR156基因及其克隆产物和应用 |
CN109055371A (zh) * | 2018-07-06 | 2018-12-21 | 浙江农林大学 | 光皮桦miR169c的前体基因及其在提前植物开花中的应用 |
CN110373413A (zh) * | 2019-06-24 | 2019-10-25 | 浙江农林大学 | 光皮桦miR169a的前体基因及其在降低植物低氮胁迫耐受性中的应用 |
Non-Patent Citations (4)
Title |
---|
JUNHONG ZHANG等: "Genome-wide mining for microRNAs and their targets in Betula luminifera using high-throughput sequencing and degradome analyses", TREE GENETICS & GENOMES, vol. 121, pages 99 * |
XIU-YUN LI等: "Molecular Characterization of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) Gene Family in Betula luminifera", FRONTIERS IN PLANT SCIENCE, vol. 9, pages 608 * |
YING PAN等: "Identification of heat-responsive miRNAs to reveal the miRNAmediated regulatory network of heat stress response in Betula luminifera", TREES, vol. 31, pages 1635 * |
张俊红等: "光皮桦开花发育的miR156调控机制", 万方会议论文数据库(中国林学会林木遗传育种分会第七届全国林木遗传育种学术大会会议论文集), pages 442 * |
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