CN107164403A - miR319在培育抗灰霉病植物中的应用 - Google Patents
miR319在培育抗灰霉病植物中的应用 Download PDFInfo
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Abstract
本发明公开了一种miR319在培育抗灰霉病植物中的应用。具体为,用于构建转基因植物,所述转基因植物具有抗灰霉病的性能。
Description
技术领域
本发明涉及植物育种,具体涉及miR319在培育抗灰霉病菌植物中的应用。
背景技术
番茄是世界上重要的经济作物之一,在很多国家都有种植,中国更是番茄的种植大国。灰霉病是近10年来危害番茄的主要病害之一,是由灰葡萄孢菌(Botrytis Cinerea)引起的一种世界性重要病害,主要危害果实,也可以危害叶片和茎等部位,发病后迅速传播[1]。近年来,随着保护地番茄栽培面积的不断扩大,为灰霉病菌在土壤中的大量积累及侵染提供了有利的环境,特别是冬春季大棚内高温、高湿的气候条件,更有利于灰霉病的发生和流行,常导致番茄减产20%~40%,严重时可达60%以上[2-3]。因此,番茄灰霉病的危害日趋严重,已成为保护地番茄产业发展的主要限制因素。
利用传统的育种方法和物种间的现有遗传基因通过基因重组技术来改良作物的抗病能力还存在很多局限,发展起来的转基因技术为改良作物的抗病性提供了新的途径,通过外源基因的导入和表达,可以提高许多转基因植物对生物胁迫的耐性。因此,寻求灰霉病菌侵染诱导基因,研究抗病基因的表达、调控和功能,是利用基因工程技术获得抗灰霉病转基因植物的前提,也是生产上亟待解决的突出问题。
近年来,国内外研究人员已经开始在番茄miRNA参与逆境胁迫应答调控领域展开了相关的研究[4]。2010年,Gu等利用基因芯片技术发现番茄miR395、miR779.1、miR840和miR867在感染丛枝菌根的番茄叶片中特异性表达,而miR158、miR862-3p、miR319、miR394和miR399在感染丛枝菌根的番茄根部异常表达[5]。2012年,Zuo等人通过高通量测序发现外源乙烯刺激下分别有5个miRNA在番茄果实成熟及软化的过程中上调而有3个下调[6]。Feng等人分别在2009年和2011年对几种病毒感染引起番茄miRNA的差异表达进行了相关的研究,发现miR159、miR164、miR165/166及miR168等多个miRNA的表达受CMV或TAV病毒感染时而发生变化[7-8]。Afsar等人在2010年发现番茄miR159/319和miR172可能参与番茄缩叶病毒侵染的应答反应[9]。2012年,Chen等人利用miRNA microarray技术对两种花叶病毒(CMV和ToMV)感染引起番茄miRNA的表达谱进行研究表明,发现至少有80个以上的植物已知miRNA的表达受这两种病毒侵染的影响[10]。2012年,Shivaprasad等人发现番茄miR482通过剪切(CC)-NBS-LRR类型的R基因产生的tasiRNA能够抑制番茄对细菌性叶斑病(P.syringae DC3000)的免疫系统[11]。2012年,Jin等利用micorarray技术发现了3个miRNA在灰霉病菌侵染番茄过程中参与应答调控[12]。2015年,Jin等利用高通量测序技术对灰霉病菌侵染应答相关的番茄miRNA进行了系统地识别分析,结果发现有65个miRNA在灰霉病菌侵染过程中表达异常,对其中的部分差异表达miRNA进行了实验验证[13]。
以上研究成果均表明,miRNA可能在植物应答生物胁迫的过程中扮演着重要的角色,因此,识别抗病miRNA并研究其相应的抗病调控机制将有望帮助我们更加深入揭示植物抗并的调控网络,为将来人工培育抗病作物奠定理论基础。
目前已报道具有抗病相关的miRNA基因还不够多;这些抗病相关的miRNA基因通过转基因进行抗病性鉴定,发现很多不具有抗病性,或对转基因植物的抗病性提高不明显。
参考文献
[1]马治贤.温室番茄主要病害识别与综合防治技术.现代园艺,2013,5:95-96;
[2]武哲,孙蕾,刘彦彦,王家旺,张克诚.生物农药武夷菌素对保护地番茄灰霉病的防治效果.中国农学通报,2013,29:173-178;
[3]尉文彬,武玉环,张艳军,谢明.武夷霉素和枯草芽孢杆菌对温室番茄灰霉病的防效.北方园艺,2013,17:118-121;
[4]崔娟娟,栾雨时.番茄响应生物胁迫相关miRNA的研究进展.生命科学研究,2014,18:533-538;
[5]Gu M,Xu K,Chen A,Zhu Y,Tang G,Xu G.Expression analysis suggestspotential roles of microRNAs for phosphate and arbuscularmycorrhizalsignaling in Solanumlycopersicum.Physiolgia Plantarum,2009,138:226-237(Gu M,Xu K,Chen A,Zhu Y,Tang G,Xu G..microRNAs表达分析表明其在番茄中对磷酸盐和菌根霉素信号传导的潜在作用。Physiolgia Plantarum,2009,138:226-237);
[6]Zuo JH,Zhu BZ,Fu DQ,Zhu Y,Ma YZ,Chi LH,Ju Z,Wang YX,Zhai BQ,LuoYB.Sculpting the maturation,softening and ethylene pathway:The influences ofmicroRNAs on tomato fruits.BMC Genomics,2012,13:7(Zuo JH,Zhu BZ,Fu DQ,Zhu Y,Ma YZ,Chi LH,Ju Z,Wang YX,Zhai BQ,Luo YB.塑造熟化,软化和乙烯途径:miRNAs对番茄果实的影响。BMC Genomics,2012,13:7);
[7]Feng J,Wang K,Liu X,Chen S,Chen J.The quantification of tomatomicroRNAs response to viral infection by stem-loop real-time RT-PCR.Gene,2009,437:14-21(Feng J,Wang K,Liu X,Chen S,Chen J.通过茎环法实时定量RT-PCR定量番茄响应病毒感染的miRNAs。Gene,2009,437:14-21);
[8]Feng JH,Liu X,Lai LY,Chen JH.Spatio-temporal expression of miRNAsin tomato tissues upon Cucumber mosaic virus and Tomato aspermy virusinfections.Acta Biochimicaet Biophysica Sinica,2011,43:258-266(Feng JH,Liu X,Lai LY,Chen JH.番茄组织中miRNAs在黄瓜花叶病毒和番茄不全病毒感染中的时空表达。Acta Biochimicaet Biophysica Sinica,2011,43:258-266);
[9]Naqvi AR,Haq QM,Mukherjee SK.MicroRNA profiling of tomato leafcurl new delhi virus(tolcndv)infected tomato leaves indicates thatderegulation of mir159/319and mir172might be linked with leaf curldisease.Virology Journal,2010,7:281(Naqvi AR,Haq QM,Mukherjee SK.番茄叶卷曲新德里病毒(tolcndv)感染的番茄叶的MicroRNA分析表明mir159/319和mir172的调控异常可能与叶卷曲病有关。Virology Journal,2010,7:281);
[10]Chen JH,Feng JL,Liao QS,Chen SN,Zhang JG,Lang QL,Du ZY,Zheng XD,Ouyang P.Analysis of Tomato MicroRNAs Expression Profile Induced byCucumovirus and Tobamovirus Infections.Journal of Nanoscience andNanotechnology,2012,12:143-150(Chen JH,Feng JL,Liao QS,Chen SN,Zhang JG,LangQL,Du ZY,Zheng XD,Ouyang P.黄瓜花叶病毒和烟草花叶病毒感染下番茄microRNAs表达谱分析。Journal of Nanoscience and Nanotechnology,2012,12:143-150);
[11]Shivaprasad PV,Chen HM,Patel K,Bond DM,Santos BA,Baulcombe DC.AmicroRNA superfamily regulates nucleotide binding site-leucine-rich repeatsand other mRNAs.Plant Cell,2012,24:859-874(Shivaprasad PV,Chen HM,Patel K,Bond DM,Santos BA,Baulcombe DC.miRNA超家族调节核苷酸结合位点富含亮氨酸重复序列和其他mRNA的绑定。.Plant Cell,2012,24:859-874);
[12]Jin WB,Wu FL,Xiao L,Liang GW,Zheng YX,Guo ZG,Guo AG.Microarray-based analysis of tomato miRNA regulated by Botrytis cinerea.Journal of PlantGrowth Regulation,2012,31:38-46(Jin WB,Wu FL,Xiao L,Liang GW,Zheng YX,Guo ZG,Guo AG.基因芯片分析灰霉病菌感染的番茄miRNA。Journal of Plant GrowthRegulation,2012,31:38-46);
[13]Jin WB,Wu FL(Co-first author).Characterization of miRNAsassociated with Botrytis cinerea infection of tomato leaves.BMC PlantBiology,2015,15:1。(Jin WB,Wu FL(Co-first author).与番茄叶灰霉病感染相关的miRNA的表征。BMC Plant Biology,2015,15:1)。
发明内容
本发明要解决的技术问题是提供一种miR319在培育抗灰霉病菌植物中的应用;本发明通过转基因技术提高植物对生物胁迫,特别是灰霉病菌胁迫的抗性。
为了解决上述技术问题,本发明提供miR319在培育抗灰霉病植物中的应用。
作为本发明的miR319在培育抗灰霉病植物中的应用:用于构建转基因植物(例如拟南芥),所述转基因植物具有抗灰霉病的性能。
miR319是植物中保守的miRNA,miR319序列(序列可在miRbase数据库中查到):UUGGACUGAAGGGAGCUCCUU。
本发明涉及的技术方案如下:
1、番茄灰霉病菌侵染处理及RNA的提取;
2、过表达miR319拟南芥突变株的构建;
3、miR319的抗病性分析。
本发明的有益效果主要体现在:将miR319在拟南芥中过表达,可显著提高拟南芥对灰霉病菌的抗性。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细说明。
图1为定量PCR检测灰霉病菌侵染番茄后不同时间点miR319的表达图;
图1中,纵坐标代表相对表达量,横坐标代表时间。
图2是miR319过表达载体图解。
图3是PCR检测转基因阳性植株;
WT代表非转基因植株,Line1~Line15分别代表转基因植株。
图4是定量PCR检测miR319在正常及转基因拟南芥的表达;
WT:非转基因植株野生型;Line4、5、10、13:转基因植株。
图5为灰霉菌病侵染后野生型和转基因植株叶片的症状和对应的台盼蓝染色图;
A.灰霉菌病侵染后野生型和转基因植株叶片的症状,B:台盼蓝染色;
WT:非转基因植株;Line4、5、10、13:转基因植株。
具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
实施例1:番茄灰霉病菌侵染处理及RNA的提取
本实施例对一月龄的番茄材料microTom进行接种灰霉病菌处理,即用1×106spores ml-1孢子喷洒番茄幼苗,接菌后分别在0h、0.5h、1h、2h、6h和12h收集番茄叶片,并提取总RNA。
本实施例采用Trizol试剂盒提取番茄叶片总RNA,其具体步骤如下:
1)、液氮速冻的叶片100mg用塑料棒于Eppendorf管中磨碎,加入1ml的Trizol,剧烈混匀后室温放置5min。
2)、在管中再加入400μl酚:氯仿:异戊醇(25:24:1),剧烈混匀后室温放置5min。
3)、4℃10000g离心10min,吸取上清液600μl至另一新的Eppendorf管,加入600μl氯仿,轻柔混匀,室温放置10min。
4)、4℃10000g离心10min,吸取上清液500μl至另一新的Eppendorf管,加入850μl无水乙醇,轻柔混匀后,-20℃放置8h以上。
5)、4℃12000g离心10min,弃上清液,加入1000μl 70%乙醇,轻柔颠倒Eppendorf管,室温放置5min。
6)、4℃12000g离心10min,弃上清液,放置超净台上自然风干。
7)、用50μl经DEPC处理后的ddH2O溶解沉淀,保存于-20℃。
8)、取1μlDNA样品稀释50倍,以紫外分光光度计测样品的OD260和OD280,通过OD260计算RNA样品浓度及纯度。依据浓度,将样品统一稀释至50ng/ul,取1μl用于反转录反应。
实施例2:在灰霉病菌侵染胁迫下番茄miR319的表达动态
本实施例对实施例1提取的样品总RNA进行定量RT-PCR分析,其具体步骤如下:
按照Takara公司购买的试剂盒PrimeScriptTMRT reagent Kit with gDNAEraser(Perfect Real Time)说明书进行操作,试剂盒中反转录引物为Random 6mers和Oligo dT Primer混合的RT Primer Mix,反应条件为:37℃15min;85℃5sec。
反转录产物取1μg作为模版,其他试剂及容量按照Takara公司提供的SYBR GreenII说明书配制反应体系,使用ABI的定量PCR仪,反应条件:94℃3min;94℃30sec,58℃30sec,72℃30sec,40个循环。实验结果参见图1。
根据图1,可得知:在灰霉病菌侵染后,通过RT-PCR检测,发现不同时间点miR319的表达量有所下调,说明miR319有可能参与机体对灰霉菌病的应答。
实施例3:过表达miR319拟南芥突变株的构建
本实施例在实施例2的检测结果基础上,进一步构建了过表达miR319的拟南芥突变株,其具体步骤如下:
1、miR319组成型表达质粒的构建
扩增拟南芥miR319前体片段,通过分子克隆及DNA测定鉴定正确后,通过与35S启动子融合构建成目标miRNA的植物表达载体。构建策略是:切除pCAMBIA1301载体上的GUS基因,然后将目标片段(miRNA前体片段)插入到原来GUS基因的位置上。最终构建的载体简图如图2所示。
2、miR319组成型表达质粒转化拟南芥
(1)将上述构建的重组质粒导入农杆菌GV3103,然后将含有表达载体的阳性农杆菌菌落接种于5m含有25ug/ml Rif、50ug/ml Gen、50ug/ml Kan抗性的LB液体培养基中,28℃培养2d。
(2)按照1:100比例,移取上一步的培养物于500ml含有25ug/ml Rif、50ug/mlGen、50ug/ml Kan抗性的LB液体培养基中扩大培养,28℃培养16-24h。
(3)测得此时菌液的OD值在1.5-2.0之间,室温4000g×10min离心收集菌体。
(4)用5%等体积的蔗糖溶液重悬菌体,并往其中加入100ul Silwet-L77,混匀,转移至500ml大烧杯中。
(5)倒置处于花期的野生型拟南芥,将其花及茎部分浸入农杆菌悬浮液中并轻轻搅动10s左右。
(6)为了保持一定的湿度,用塑料袋将浸染过的拟南芥覆盖住16-24h。
(7)将处理过的植物移回温室中继续培养1个月,收集种子。
3、转基因拟南芥的筛选
(1)将上述收集的种子经过消毒后均匀涂布到含有50ug/ml Kan抗性的1/2MS培养基上。
(2)将培养皿置于长日照条件(16h光照/8h黑暗,22℃)培养2周,让种子发芽和小苗生长,然后筛选能够正常生长的转基因阳性苗转移到湿土中。
(3)将土中的拟南芥置于温室短日照培养3-4周,再转换成长日照条件诱导其开花。
(4)得到转基因株系Line1-Line15种子T1代种子,经过抗生素筛选后种植,得到T2代种子,再次使用抗生素对T2代种子进行筛选后种植,收获其中的T3代突变株种子,产生3:1的分离比,播种后待其长大,通过PCR技术筛选出PCR阳性的转基因植株,其中WT(野生型)为对照,如图3所示。
实施例4:miR319在转基因拟南芥和野生型中的表达鉴定
本实施例采用定量PCR来检测实施例3所得转基因拟南芥miR319的表达情况,野生型拟南芥作为对照。结果如图4所示,目标miRNA在转基因拟南芥中得到过量表达。
实施例5:灰霉病菌处理野生型和转基因拟南芥
将野生型拟南芥和实施例4所得的miR319高表达的T3代突变株种子种到土里,一个月后进行接菌处理,结果如图5所示,转基因拟南芥叶片没有出现明显症状;而在对照组的非转基因拟南芥的叶片却出现病斑。进一步台盼蓝染色表明,转基因拟南芥的叶片中基本没有菌丝或菌丝增殖较少;而在对照组的非转基因拟南芥的叶片中菌丝增长明显。表明miR319是一个对灰霉病菌具有较强抗性的miRNA基因。
最后,还需要注意的是,以上列举的仅是本发明的若干个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
<110> 浙江理工大学
<120> miR319在培育抗灰霉病植物中的应用
<160> 1
<210> 1
<211> 21
<212> RNA
<213> 人工序列
<220>
<223> miR319
<400> 1
uuggacugaa gggagcuccu u 21
Claims (2)
1.miR319在培育抗灰霉病植物中的应用。
2.根据权利要求1所述的miR319在培育抗灰霉病植物中的应用,其特征是:用于构建转基因植物,所述转基因植物具有抗灰霉病的性能。
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CN112695035A (zh) * | 2021-01-22 | 2021-04-23 | 浙江理工大学 | 一种RNA抑菌剂miRNA157d-3p及作物病菌抑制剂 |
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CN112646819B (zh) * | 2021-01-17 | 2023-04-18 | 浙江师范大学 | 基因增强对番茄灰霉病抗性的用途 |
CN112695035A (zh) * | 2021-01-22 | 2021-04-23 | 浙江理工大学 | 一种RNA抑菌剂miRNA157d-3p及作物病菌抑制剂 |
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