CN117603968A - 一种苹果早期落叶病菌跨界传递milRNA及其防治方法 - Google Patents
一种苹果早期落叶病菌跨界传递milRNA及其防治方法 Download PDFInfo
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Abstract
本发明涉及一种苹果早期落叶病菌跨界传递milRNA(AltmilR823)及其防治方法。本发明提供所述跨界AltmilR823在感病品种接菌后的原位杂交,通过加有5,6‑FAM修饰的锁核酸探针,在接菌后的GL‑3叶片中检测到AltmilR823。本发明还提供所述AltmilR823的靶基因MdPECL4在改良苹果品种的应用,通过将MdPECL4过表达在植物表达载体上,导入苹果早期落叶病感病苹果中,可提高植物的抗病性。由于MdPECL4是通过过表达苹果自身基因发挥功能,相比转入外源抗性基因的苹果植株更安全,且更易于被消费者接受。
Description
技术领域
本发明涉及植物分子生物学领域,具体说是一种苹果早期落叶病菌跨界传递milRNA及其防治方法。
背景技术
milRNA介导的跨界调控在植物与病原体相互作用中起着重要作用。真菌milRNA已被证明通过调节大丽花轮枝菌、稻瘟病菌和苹果腐烂病中的致病基因以参与植物侵染过程。然而,植物病原真菌的milRNAs介导的跨界调控的功能和机制在很大程度上仍是未知的。苹果早期落叶病是苹果目前感染的较为严重的真菌性病害,包括苹果斑点落叶病(Alternaria sp.Mali,ALT1)、苹果褐斑病(Marssonina leaf spot,MLS1)及苹果炭疽叶枯病(Glomerella leaf spot,GLS1)。苹果早期落叶病侵染苹果叶片后,会使苹果在生长早期发生落叶的现象,对苹果的产量形成巨大的影响,因此,探究苹果早期落叶病的致病机制及防治方法十分必要。
发明内容
针对现有技术中存在的缺陷,本发明的目的在于提供一种苹果早期落叶病菌跨界传递的milRNA及其防治方法。本发明通过比较苹果接种苹果斑点落叶病Alternariasp.mali(ALT1)前后milRNA的表达差异,得到了一种苹果早期落叶病菌跨界传递milRNA,命名为AltmilR823。研究发现,与未接种ALT1、GLS1(Glomerella leaf spot)与MLS1(Marssonina leaf spot)的GL-3相比,在接种了ALT1、GLS1与MLS1后的GL-3中AltmilR823的表达均上升。苹果叶片注射瞬时过表达AltmilR823载体可增强苹果对ALT1、GLS1与MLS1的易感性。另外,在接种了ALT1、GLS1与MLS1的GL-3的叶片中可以观察到AltmilR823的存在。随后,在GL-3的基因组DNA中扩增得到了AltmilR823的靶基因MdPECL4。进一步通过5‘RACE验证AltmilR823在10-11位点靶向切割其靶基因MdPECL4。过表达靶基因后,在GL-3中接种ALT1、GLS1与MLS1后,AltmilR823的表达量下降。因此发明人通过过表达靶基因MdPECL4开发了抗早期落叶病的分子手段。
为达到以上目的,本发明采取的技术方案是:
一种苹果早期落叶病菌跨界传递的milRNA(命名为AltmilR823),其特征在于,其核苷酸序列如SEQ ID NO:1所示。
一种苹果早期落叶病菌跨界传递的milRNA的初级转录本(命名为AltMILR823),其特征在于,其核苷酸序列如SEQ ID NO:2所示。
一种检测苹果早期落叶病菌跨界传递milRNA的原位杂交方法,其特征在于,包括如下步骤:
S1:取接菌48h后叶片和未接菌叶片,先分别在RNase浸泡15min,然后用pH 7.4的1×PBS洗涤3次;将处理后的叶片样品放置于4%多聚甲醛真空浸润15min,至样品下沉,变成半透明状,然后放到4℃冰箱过夜;
S2:所有样品用1×PBS冲洗3次,每次间隔5min。冲洗后的样品在添加20μg/mL蛋白酶K和0.5%Triton-X的TE缓冲液中37℃处理2-3h,以消化细胞内多余的RNA酶和蛋白质,并渗透细胞以进入探针;
S3:经S2处理的样品在pH 7.4的1x PBS中冲洗3次,每次冲洗间隔5分钟;在室温下用0.2%甘氨酸浸泡5分钟,以终止蛋白酶活性;经甘氨酸处理后,样品用4%多聚甲醛固定5分钟,再次在1x PBS缓冲液中冲洗;
S4:将LNA和FAM修饰后的探针用50%甲酰胺中85℃变性3min,然后立即冰浴5min;
S5:将经S4处理的20μL浓度为35μM的探针溶液和作为无探针对照的20μL的50%甲酰胺溶液,分别连同80μL的杂交缓冲液加入到装有经S3处理的样品管中,50℃,300rpm过夜孵育;
S6:将0.2×SSC缓冲液加入经S5处理的样品管中,50℃,300rpm清洗30min,上述清洗重复一次;
S7:将包含0.1%碘化丙啶的0.2×SSC缓冲液加入经S6处理的样品管中,37℃,300rpm清洗5min;
S8:将包含0.1%碘化丙啶的0.2×SSC缓冲液加入经S7处理的样品管中,室温,300rpm清洗5min;
S9:用0.2×SSC缓冲液漂洗经S8处理的样品,去色。
在上述方案的基础上,所述的经LNA和FAM修饰后的探针的序列如SEQ ID NO:3所示。
一种苹果早期落叶病菌跨界传递milRNA的靶基因(命名为MdPECL4),其特征在于,其核苷酸序列如SEQ ID NO:4所示。
一种植物表达载体,其特征在于,所述植物表达载体为包含检测早期落叶病菌跨界传递的milRNA的pFGC5941载体。
一种工程菌,其特征在于,包含上述植物表达载体(该植物表达载体为包含检测早期落叶病菌跨界传递的milRNA的pFGC5941载体)。
一种植物表达载体,其特征在于,包含上述靶基因的pFGC5941载体。
一种工程菌,其特征在于,包含上述的植物表达载体(该植物表达载体为包含上述靶基因的pFGC5941载体)。
一种提高苹果植株对苹果早期落叶病菌抗性的方法,其特征在于,包括如下步骤:
S1:通过CTAB方法提取感病苹果总RNA;
S2:将经S1提取的总RNA逆转录成cDNA;
S3:利用S2得到的逆转录的模板和RT-PCR引物克隆权利要求5所述的靶基因;
S4:将S3得到的靶基因插入pFGC5941载体上的BamHI和NcoI两个酶切位点中,转入大肠杆菌DH5α中;
S5:提取经S4构建的过表达靶基因载体质粒;
S6:将S5提取的质粒转入农杆菌中,涂布于加有抗生素的固体YEP培养基上,将平板置于28℃恒温培养箱中倒置培养24-48h;上述抗生素包含50mg/L Kana,20mg/L Rif;
S7:从经S6培养的农杆菌菌板挑取单斑,加入到2ml包含50mg/LKana,20mg/L Rif的YEP液体培养基中,28℃,180rpm培养过夜;
S8:取80μL经S7培养的农杆菌菌液,加入到4ml包含50mg/LKana,20mg/L Rif及10μM乙酰丁香酮的YEP液体培养基中,28℃,180rpm培养12-16h;
S9:将经S8培养的农杆菌菌液室温10000rpm离心1min,除去培养基;用1-2ml悬浊液经涡旋震荡悬浮上述菌液;取经震荡悬浮的菌液10μL加入990μL悬浊液得菌体悬浊液,用分光光度计测定其OD600,将菌体悬浊液调整至OD600=1.0,室温静置2-5h;上述悬浊液中包括:pH调至5.2的10mM MES-KOH,10mM MgCl2,100μM乙酰丁香酮;
S10:将S9得到的菌液使用前经涡旋震荡或移液枪吸打悬浮起菌体,然后用去针头的1mL无菌注射器吸取上述菌液,避开叶脉,用上述1mL注射器针头在苹果叶片上开小孔后,将菌体液注射入叶片中,每个叶片注射1-2个孔;
S11:注射农杆菌4天后观察叶片。
本发明所述的一种苹果早期落叶病菌跨界传递milRNA及其防治方法,其有益效果为:
本发明提供的AltmilR823的靶基因MdPECL4在改良苹果品种的应用,通过将MdPECL4过表达在植物表达载体上,导入苹果早期落叶病感病苹果中,可提高植物的抗病性。由于MdPECL4是通过过表达苹果自身基因发挥功能,相比转入外源抗性基因的苹果植株更安全,且更易于被消费者接受
附图说明
本发明有如下附图:
图1为本发明实施例1中AlrmilR823接种ALT1、GLS1、MLS1菌48小时后,在GL-3组培苗中的表达情况;
图2为本发明实例1中AltmilR823的初级转录本AltmilR823接种ALT1、GLS1、MLS1菌48小时后,在GL-3组培苗中的表达情况及过表达AltmilR823载体图;
图3为本发明实例1在感病品种GL-3接菌48小时后通过整体原位杂交方法检测叶片中AltmilR823的表达情况;
图4为本发明实例2中靶基因扩增及过表达MdPECL4载体图;
图5为本发明实例2中AltmilR823在感病品种GL-3中对MdPECL4切割位点检测的5’RACE结果;
图6为本发明实施例1在感病品种GL-3中过表达AltmilR823使GL-3对早期落叶病易感性增强实验结果;
图7为本发明实例2在感病品种GL-3中过表达MdPECL4使GL-3对早期落叶病抗性增强情况。
具体实施方式
以下结合附图对本发明作进一步详细说明。
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实例1与实例2均按照制造厂商说明书建议的条件。
实施例1:AltmilR823的发现
以感病品种GL-3为实验材料,筛选接种苹果斑点落叶病Alternaria sp.mali(ALT1)前后表达差异2倍以上的milRNA,与miR Base(http://www.mirbase.org/)和苹果基因组(https://www.rosaceae.org/)中比对,获得一个新的milRNA,将其命名为AltmilR823(SEQ ID NO:1),并获得AltmilR823的初级转录本AltmilR823(SEQ ID NO:2)。由于该milRNA为ALT1中的milRNA跨界传送到苹果叶片中,因为本发明利用整体原位杂交的方法在接菌ALT1、GLS1、MLS1的GL-3叶片48小时后检测AltmilR823的传递情况。milRNA产生作用是通过切割靶基因的方式进行的,本发明通过psRNAtarget(https://www.zhaolab.org/psRNATarget/)网站预测AltmilR823在苹果中的靶基因为MdPECL4。通过5’RACE实验确定AltmilR823是通过在10-11位点切割其靶基因MdPECL4(SEQ ID NO:4)。在感病品种GL-3中,瞬时过表达MdPECL44天后,接种ALT1、GLS1、MLS1菌,于48小时后统计病斑面积,发现瞬时过表达MdPECL4可使感病品种GL-3对早期落叶病的抗性增强。可利用MdPECL4的特性,开发选育抗早期落叶病的苹果品种。
具体方法如下:
l、植物总RNA提取:
(1)苹果GL-3各组织样品在液氮中快速研磨;
(2)往植物组织中加入990μL预热后的CTAB溶液和10μL的β-巯基乙醇,涡旋30s后65℃水浴10min;
(3)加入1000μL CI(氯仿/异戊醇体积比=24∶1),上下颠倒混匀;
(4)4℃12000rpm 10min;
(5)吸取800μL上清液,加入等体积的CI,上下颠倒混匀;
(6)4℃12000rpm 10min;
(7)吸取上清约650μL,加入1000μL异丙醇;
(8)-20℃沉淀30min;
(9)4℃12000rpm 10min;
(10)倒净上清液,加入1ml75%乙醇洗涤沉淀;
(11)4℃12000rpm 10min;
(12)倒净上清液,4℃12000rpm 2min;
(13)吸干上清液,加入40μL RNase-free H2O溶解沉淀;
(14)1%琼脂糖凝胶电泳检测完整性,紫外分光光度计测260nm处吸光度计算RNA浓度。
2、milRNA逆转录反应体系如下:
冰上加样并混匀,短暂离心,42℃,30min,75℃,5min,冷却后于-80℃保存。
3、逆转录反应体系如下:
总RNA中的DNA去除
RNA 2μg
gDNA purge 1μL
DEPC水 补足至10μL
冰上加样并混匀,短暂离心,42℃,5min,然后马上置于冰上。
cDNA的第一链合成
上述体系加入10μL 2×Mix,50℃,15min,75℃,5min,冷却后于-80℃保存。
4、RT-PCR反应体系:
上述体系以95℃,3min;95℃,1min;60℃,30s;72℃,30s;72℃,10min;16℃,1min;35个循环。
1%琼脂糖凝胶电泳检测。
5、荧光定量反应体系:
设计AltmilR823特异引物F:GCTCAGACGTGCGTCATTGA,R:AGTGCAGGGTCCGAGGTATT。
设计MdPECL4特异引物F:TCCGAGGGCGATCTATTCCT,R:ACAGTTACCACCTGCTCTGC。
上述反应体系在Applied Biosystems 7500进行荧光定量,PCR反应程序如下:95℃,3mins;95℃10sec,60℃,30sec,共40个循环。结果利用2-ΔΔCt方法进行统计分析(Livakand Schmittgen,2001)。
6、milRNA Northern法检测:
合成5’端修饰地高辛标记的AltmilR823、18S rRNA和U6探针(AltmilR823-probe、18S rRNA-probe和U6-probe),取20μg RNA(CTAB提取)加入2×loading buffer,95℃5min,然后4℃冷却,上样于15%聚丙烯酰胺凝胶(含7M尿素),1×TBS buffer中220V电泳1h;1×TBS buffer中220mA,冰上电转于尼龙膜上;1200mJ紫外交联3mins,利用地高辛杂交检测试剂盒(Mylab corporation)进行预杂交、杂交、洗膜和信号检测。
接种苹果早期落叶病病原菌(ALT1、GLS1、MLS1)48小时后,AltmilR823的含量在GL-3中如图1所示:图1A为接种ALT1、GLS1、MLS1 48小时后,AltmilR823在感病品种GL-3中的RT-PCR结果,以不接菌表达量作为对照(0小时)。图1B为接种ALT1、GLS1、MLS1 48小时后,AltmilR823在感病品种GL-3中的荧光定量PCR结果,以不接菌表达量作为对照(0小时);图1C为接种ALT1、GLS1、MLS1 48小时后,AltmilR823在感病品种GL-3中的Northern结果,以不接菌表达量作为对照(0小时)。
7、过表达AltmilR823载体的构建(图2C为载体示意图):
(1)利用RT-PCR扩增AltmilR823初级转录本AltMILR823序列。
(2)设计AltMILR823克隆引物,F:TCGTTGAGCGCATCAGGGT;R:GGTATCGTCGCCCTCCTCAA。
(3)PCR反应程序如下:94℃预变性3min;94℃,40s;61℃,30s;72℃,1min,共35个循环;最后72℃延伸2min。
(4)PCR产物检测:根据目标片段大小制作1%琼脂糖凝胶,0.1%TAE电泳缓冲液,70-110v电压电泳约15min,溴化乙啶染色,紫外灯下检测PCR产物片段大小。结果如图2所示:图2B为接种ALT1、GLS1、MLS1 48小时后,AltMILR823在感病品种GL-3中的RT-PCR结果,以不接菌表达量作为对照(0小时);另外,图2A为预测的AltMILR823的二级结构示意图。
(5)利用BamHI和NcoI两个酶切位点将AltMILR823序列连接至pFGC5941载体上,转入大肠杆菌DH5α中,涂板挑斑测序,测序序列正确后提取质粒,进行瞬时表达。
具体操作如下:
酶切反应体系:
上述反应体系置于37℃水浴中,水浴24h后,利用琼脂糖凝胶电泳进行回收产物检测。挑选大小正确的片段利用诺维赞公司回收试剂盒进行回收。
T4连接酶连接体系:
上述反应体系置于16℃金属浴中,24h后将上述20μL连接体系转至大肠杆菌DH5α中。
8、日的片段琼脂糖凝胶回收
使用诺维赞公司回收试剂盒,方法参照说明书。
(1)切取含有目的序列片断的琼脂糖凝胶,尽量切除多余凝胶,加入3个凝胶体积的Buffer B2,间断混合,直至凝胶块完全熔化。
(2)加200μL体积的异丙醇,混合均匀。
(3)将混合液转移到柱形分离纯化柱中,12,000×g离心60s,倒掉收集管中的废液。
(4)向纯化柱中加入300μl Buffer B2,12,000×g离心30s,倒掉收集管中的废液。
(5)向纯化柱中加入500μl Wash Buffer,12,000×g离心30s,倒掉收集管中的废液。
(6)重复(5)一次。
(7)空离2min后置于通风橱吹5min。
(8)取一新的1.5ml离心管,将纯化柱放于新1.5ml离心管中,加入55℃预热好的TEBuffer 30μL,室温放置2min,12,000×g离心60s。
(9)弃去吸附柱,回收产物置于-20℃冰箱中保存。
9、大肠杆菌DH5α转化流程:
将连接后的载体10μL加入30μL大肠杆菌DH5α中,冰浴30min,42℃热激30s,冰浴2min后添加液体LB培养基200μL,置于37℃摇床,200rpm摇40-60min后,室温10,000rpm离心1min。吸去上清200μL,利用剩余液体悬浮菌体,涂布于LB+Amp固体培养基上,至于37℃烘箱过夜培养。
10、挑斑:
挑取过夜培养后的单一菌落于200μL LB+Amp液体培养基中,置于37℃摇床,200rpm摇3h。
11、菌液PCR:
上述体系以95℃,3min;95℃,1min;60℃,30s;72℃,30s;72℃,10min;16℃,1min;35个循环。
1%琼脂糖凝胶电泳检测,0.1%TAE电泳缓冲液,70-110v电压电泳约15min,溴化乙啶染色,紫外灯下检测PCR产物片段大小。
挑选条带大小正确的菌液送至生工生物工程(上海)股份有限公司,测序结果通过DNAMAN进行比对。
12、整体原位杂交检测AltmilR823:
具体方法如下:
(1)取接菌(48h)后叶片和未接菌叶片
第一组样品:先在RNase浸泡15min,然后用1×PBS(PH7.4)洗涤3次(目的是去除/减少外部miRNA污染的可能);
(2)固定:放置于4%多聚甲醛真空浸润15min(至样品下沉,变成半透明状),然后放到4度冰箱过夜;
(3)固定后,所有样品用1×PBS冲洗3次,每次间隔5min;
(4)将固定后样品放置于添加20μg/mL蛋白酶K和0.5%Triton-X的TF缓冲液中37℃处理2-3h,目的是消化细胞内多余的RNA酶和蛋白质,并渗透细胞以进入探针;
(5)在1x PBS(pH 7.4)中冲洗3次,间隔5分钟;
(6)在室温下用0.2%甘氨酸5分钟,以停止蛋白酶活性;
(7)甘氨酸处理后,样品用4%多聚甲醛固定5分钟,再次在1x PBS缓冲液中冲洗;
(8)LNA和FAM修饰后的探针(35μM)在50%甲酰胺中85度变性3min,然后立即冰浴5min;
(9)将20μL的这些溶液和20μL的50%甲酰胺作为无探针对照,连同80μL的杂交缓冲液加入到装有样品的管子中,50度300rpm过夜孵育;
(10)第二天,0.2×SSC缓冲液加入样品管中,50度300rpm清洗30min,重复一次;
(11)0.2×SSC缓冲液(包含0.1%碘化丙啶)加入样品管中,37度300rpm清洗5min;
(12)0.2×SSC缓冲液(包含0.1%碘化丙啶)加入样品管中,室温300rpm清洗5min;
(13)用0.2×SSC缓冲液漂洗去色;
(14)将样品放在载玻片上,用confocal microscope观察,LNA探针荧光团在488nm处被激发。
如图3所示:其中图3A为WT代表不接菌的GL-3叶片的原位杂交;图3B为接种ALT1的GL-348小时后叶片的原位杂交;图3C为接种GLS1的GL-348小时后叶片的原位杂交;图3D为接种MLS1的GL-348小时后叶片的原位杂交。CK代表没有添加探针的空白对照,Negative代表添加正义链探针的阴性对照,Positive代表添加18S rRNA探针的阳性对照,AltmilR823代表添加AltmilR823探针实验结果。
实施例2:在感病品种GL-3中过表达MdPECL4。
1、MdPECL4的克隆:
(1)CTAB提取GL-3总RNA;
(2)逆转录成cDNA;
(3)设计RT-PCR引物:F:AGCATCAAAGTTTAGAGGCATTGTT;R:TTCTTTTCTCTATGCACGTTTAACG。
(4)PCR结束后,1%琼脂糖凝胶电泳检测,0.1%TAE电泳缓冲液,70-110v电压电泳约15min,溴化乙啶染色,紫外灯下检测PCR产物片段大小,结果见图4A(在感病品种GL-3中克隆到的Altmi lR823的靶基因MdPECL4的RT-PCR胶图)。
2、miRNA靶基因5’RACE分析。
具体流程如下:
(1)总RNA经基因特异性引物逆转录,获得cDNA。
(2)加乙醇沉淀1小时,离心4℃,12000rpm,用DEPC水溶解,将cDNA纯化,去除其中的dNTP及其他离子。
(3)纯化后的cDNA进行末端磷酸化酶(TdT)处理。
体系如下:
混匀后37℃处理30min。
(4)加入150μL无RNase水后,再加入等体积200μL CI混匀,4℃,12,000rpm离心10min。取上清加入等体积CI颠倒混匀,4℃,12,000rpm离心10min。
(5)取上清,加入5μL的3M NaOAC(PH5.2)和2.5倍体积的无水乙醇,-20℃沉淀1小时。
(6)4℃,12,000rpm离心20min;弃上清,用75%的冷乙醇清洗沉淀,吹干后溶于20μL无菌水中,-70℃保存备用。
(7)TdT处理后的加C尾产物为模板进行PCR反应,引物使用3’锚定引物和逆转录时使用的特性引物。
(8)PCR反应体系:
反应条件:94℃,5min;94℃,30s;60℃,30s;72℃,1min;72℃,10min;16℃,10min;35个循环。
(9)PCR产物进行下一轮的巢式PCR反应,引物为锚定引物中的保守部分及逆转录引物内侧的基因特异性引物。
(10)PCR产物经琼脂糖凝胶回收,T-A克隆到Pmd18-T中,送生工生物工程(上海)股份有限公司进行测序,实验结果如图5(AltmilR823在感病品种GL-3中对MdPECL4切割位点检测的5’RACE结果)所示。
3、质粒提取(Vazyme,FastPure Plasmid Mini Kit,DC201)
(1)取实例1中构建的OE-MILR823载体的大肠菌液1-5ml过夜培养(12-16h)的菌液,加入2ml离心管中,10000rpm离心1min。弃培养基,吸尽残液;
(2)向离心管中加入250μl Buffer P1(已加入RNase A),涡旋振荡混匀;
(3)向步骤2中加入250μl Buffer P2,温和的上下颠倒混匀8-10次;
(4)向步骤3中加入350μl Buffer P3,立即温和地上下颠倒8-10次,12000rpm离心10min;
(5)将FastPure DNA Mini Columns吸附柱置于Collection Tube 2ml收集管中。将步骤4上清用移液器小心转移至吸附柱中,12000rpm离心30sec。倒掉收集管中的废液,把吸附柱重新放回收集管中;
(6)加入600μl Buffer PW2(已用无水乙醇稀释)至吸附柱中。12000rpm离心30sec。弃废液,将吸附柱重新放回收集管中;
(7)重复步骤6;
(8)将吸附柱放回收集管中12000rpm离心1min干燥吸附柱;
(9)把吸附柱置于一个新的灭菌的1.5ml离心管中。加入30-100μl ElutionBuffer至柱吸附柱的膜中央。室温静置2min,12000rpm离心1min洗脱DNA;
(10)弃去吸附柱,DNA产物保存于-20℃。
4、农杆菌转化:
(1)取一管制备好的感受态细胞,冰上完全溶解后轻轻悬浮细胞;
(2)加入5-10μL植物表达载体质粒,轻轻混匀,冰上放置30min;
(3)液氮中冷激1min;
(4)37℃热激5min,冰上放置2min;
(5)加入500μL YEP液体培养基,于28℃,140rpm振荡培养4-6h;
(6)室温4000rpm离心3min,去除约400μL上清液,用剩余的培养基悬浮细胞;
(7)将细菌涂布在加有抗生素(50mg/L Kana,20mg/L Rif)的固体YEP培养基上;
(8)将平板于28℃倒置培养24-48h。
5、农杆菌介导的瞬时表达:
苹果GL-3组培苗培养于25℃,50%湿度的光照培养箱中,昼夜长度保持为16h/8h,光照强度为200μmol·m-2·s-1。待植株5-6片真叶展开时对每个单株编号并取一片真叶保存后进行农杆菌注射。农杆菌注射方法参见Bai等(Bai et al.2011)。
(1)预培养:YEP液体培养基2ml(50mg/L Kana,20mg/L Rif),农杆菌菌斑或甘油菌28℃,180rpm培养过夜;
(2)本培养:YEP液体培养基4ml(加对应的抗生素和10μM乙酰丁香酮),加入1/50体积的菌液(80μL),28℃,180rpm培养12-16h;
(3)室温10,000rpm,1min离心,除去培养基,用1-2ml悬浊液悬浮菌体(可以涡旋震荡);
取10μL菌液加入990μL悬浊液中,用分光光度计测定OD600,将菌体悬浊液调整至OD600=1.0。室温静置2-5h;
悬浊液:10mM MES-KOH(pH 5.2),10mM MgCl2,100μM乙酰丁香酮);
(4)菌液使用前涡旋震荡或用移液枪吸打悬浮菌体,用没有针头的1ml注射器吸取菌液;
(5)避开叶脉,用注射器针在叶片上开小孔后,用装有菌液的注射器压住小孔,另一只手的手指在叶片反方向压住小孔,慢慢用力,将菌液注射入叶片中,注射好的部分的颜色会变浅。每个叶片注射1-2个孔;
(6)注射农杆菌4天后观察叶片。
实验结果如图6所示:图6A为农杆菌介导的瞬时表达感病品种GL-3组培苗4天,接菌48小时后通过荧光定量PCR检测AltmilR823表达量结果;图6B为农杆菌介导的瞬时表达感病品种GL-3组培苗4天,接菌48小时后通过荧光定量PCR检测MdPECL4表达量结果;图6C为农杆菌介导的瞬时表达感病品种GL-3组培苗4天,接菌48小时后病叶及病斑面积统计情况;WT代表不进行瞬时表达GL-3组培苗;EV代表pFGC5941空载进行瞬时表达金冠组培苗;OE-AltMILR823代表进行瞬时过表达OE-AltMILR823的pFGC5941载体的GL-3组培苗。
6、构建过表达MdPECL4载体
将MdPECL4插入pFGC5941载体上的BamHI和NcoI两个酶切位点中,构建形式见图4B,转入大肠杆菌DH5α中,涂板挑斑测序,测序序列正确后提取质粒。
7、GL-3中过表达MdPECL4
构建完成的MdPECL4质粒转入农杆菌中,进行瞬时表达,培养4天,结果见图7C:农杆菌介导的瞬时表达感病品种GL-3组培苗4天,接菌48小时后病叶及病斑面积统计情况;WT代表不进行瞬时表达GL-3组培苗;EV代表pFGC5941空载进行瞬时表达金冠组培苗;OE-MdPECL4代表过表达MdPECL4的pFGC5941载体进行瞬时表达GL-3组培苗。
另外,图7A为农杆菌介导的瞬时表达感病品种GL-3组培苗4天,接菌48小时后通过荧光定量PCR检测MdPECL4表达量结果;图7B为农杆菌介导的瞬时表达感病品种GL-3组培苗4天,接菌48小时后通过荧光定量PCR检测AltmilR823表达量结果。
荧光定量PCR使用的引物:
AltmilR823特异性茎环引物:
5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCCTCC-3’
AltmilR823荧光定量引物:
F:GCTCAGACGTGCGTCATTGA,
R:AGTGCAGGGTCCGAGGTATT。
内参18S rRNA特异性茎环引物:
5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCTGTA-3’
内参18S rRNA荧光定量引物:
F:5’-ATTACGGAACCTCTCGGGGT-3’;
R:5’-AGTGCAGGGTCCGAGGTATT-3’。
MdPECL4荧光定量引物:
F:TCCGAGGGCGATCTATTCCT,
R:ACAGTTACCACCTGCTCTGC。
内参U6荧光定量引物:
F:5’-TTGGGGACATCCGATAAAATTG-3’,
R:5’-AAAAATTTGGACCATTTCTCG-3’。
基因克隆引物:
AltmilR823特异性茎环引物:
5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCCTCC-3’
AltmilR823 RT-PCR引物:
F:GCTCAGACGTGCGTCATTGA,
R:AGTGCAGGGTCCGAGGTATT
内参18S rRNA RT-PCR引物:
F:5’-GAACCTCTCGGGGTTACAGC-3’;
R:5’-GCATTTCGCTGCGTTCTTCA-3’。
AltMILR823 RT-PCR引物:
F:TCGTTGAGCGCATCAGGGT,
R:GGTATCGTCGCCCTCCTCAA。
MdPECL4 RT-PCR引物:
F:AGCATCAAAGTTTAGAGGCATTGTT,
R:TTCTTTTCTCTATGCACGTTTAACG。
内参U6 RT-PCR引物:
F:5’-TTGGGGACATCCGATAAAATTG-3’,
R:5’-AAAAATTTGGACCATTTCTCG-3’。
整体原位杂交探针序列:
LNA-milR823:5,6-FAM-ccctcct caAtgAcgCac gtctga
LNA-positive:5,6-FAM-gaacc tcTcgGggTt acagc
LNA-negative:5,6-FAM-tggtgg atCttCtgTag gcaggc
其中大写字母为添加锁核酸修饰碱基。
5’RACE引物序列:
Adap t or:TAATACGACTCACTATAGGGGGGGGGG
F:TAATACGACTCACTATAGGG
R 1:TTCTTTTCTCTATGCACGTTTAACGTGAA
R2:CTATGCACGTTTAACGTGAAATAATTAATCGG
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
本说明书中未作详细描述的内容属于本领域专业技术人员公知的现有技术。
Claims (10)
1.一种苹果早期落叶病菌跨界传递milRNA,其特征在于,其核苷酸序列如SEQ ID NO:1所示。
2.如权利要求1所述一种苹果早期落叶病菌跨界传递milRNA的初级转录本,其特征在于,其核苷酸序列如SEQ ID NO:2所示。
3.一种检测苹果早期落叶病菌跨界传递milRNA的原位杂交方法,其特征在于,包括如下步骤:
S1:取接菌48h后叶片和未接菌叶片,先分别在RNase浸泡15min,然后用pH 7.4的1×PBS洗涤3次;将处理后的叶片样品放置于4%多聚甲醛真空浸润15min,至样品下沉,变成半透明状,然后放到4℃冰箱过夜;
S2:所有样品用1×PBS冲洗3次,每次间隔5min。冲洗后的样品在添加20μg/mL蛋白酶K和0.5%Triton-X的TE缓冲液中37℃处理2-3h,以消化细胞内多余的RNA酶和蛋白质,并渗透细胞以进入探针;
S3:经S2处理的样品在pH 7.4的1x PBS中冲洗3次,每次冲洗间隔5分钟;在室温下用0.2%甘氨酸浸泡5分钟,以终止蛋白酶活性;经甘氨酸处理后,样品用4%多聚甲醛固定5分钟,再次在1x PBS缓冲液中冲洗;
S4:将LNA和FAM修饰后的探针用50%甲酰胺中85℃变性3min,然后立即冰浴5min;
S5:将经S4处理的20μL浓度为35μM的探针溶液和作为无探针对照的20μL的50%甲酰胺溶液,分别连同80μL的杂交缓冲液加入到装有经S3处理的样品管中,50℃,300rpm过夜孵育;
S6:将0.2×SSC缓冲液加入经S5处理的样品管中,50℃,300rpm清洗30min,上述清洗重复一次;
S7:将包含0.1%碘化丙啶的0.2×SSC缓冲液加入经S6处理的样品管中,37℃,300rpm清洗5min;
S8:将包含0.1%碘化丙啶的0.2×SSC缓冲液加入经S7处理的样品管中,室温,300rpm清洗5min;
S9:用0.2×SSC缓冲液漂洗经S8处理的样品,去色。
4.如权利要求3所述的一种检测苹果早期落叶病菌跨界传递milRNA的原位杂交方法,其特征在于,所述的经LNA和FAM修饰后的探针的序列如SEQ ID NO:3所示。
5.一种植物表达载体,其特征在于,所述植物表达载体为包含权利要求1所述一种检测早期落叶病菌跨界传递milRNA的pFGC5941载体。
6.一种工程菌,其特征在于,包含权利要求5所述的植物表达载体。
7.一种苹果早期落叶病菌跨界传递milRNA的靶基因,其特征在于,其核苷酸序列如SEQID NO:4所示。
8.一种植物表达载体,其特征在于,包含权利要求7所述靶基因pFGC5941载体。
9.一种工程菌,其特征在于,包含权利要求8所述的植物表达载体。
10.一种提高苹果植株对苹果早期落叶病抗性的方法,其特征在于,包括如下步骤:
S1:通过CTAB方法提取感病苹果总RNA;
S2:将经S1提取的总RNA逆转录成cDNA;
S3:利用S2得到的逆转录的模板和RT-PCR引物克隆权利要求5所述的靶基因;
S4:将S3得到的靶基因插入pFGC5941载体上的BamHI和NcoI两个酶切位点中,转入大肠杆菌DH5α中;
S5:提取经S4构建的过表达靶基因载体质粒;
S6:将S5提取的质粒转入农杆菌中,涂布于加有抗生素的固体YEP培养基上,将平板置于28℃恒温培养箱中倒置培养24-48h;上述抗生素包含50mg/L Kana,20mg/L Rif;
S7:从经S6培养的农杆菌菌板挑取单斑,加入到2ml包含50mg/L Kana,20mg/L Rif的YEP液体培养基中,28℃,180rpm培养过夜;
S8:取80μL经S7培养的农杆菌菌液,加入到4ml包含50mg/L Kana,20mg/L Rif及10μM乙酰丁香酮的YEP液体培养基中,28℃,180rpm培养12-16h;
S9:将经S8培养的农杆菌菌液室温10000rpm离心1min,除去培养基;用1-2ml悬浊液经涡旋震荡悬浮上述菌液;取经震荡悬浮的菌液10μL加入990μL悬浊液得菌体悬浊液,用分光光度计测定其OD600,将菌体悬浊液调整至0D600=1.0,室温静置2-5h;上述悬浊液中包括:pH调至5.2的10mM MES-KOH,10mM MgCl2,100μM乙酰丁香酮;
S10:将S9得到的菌液使用前经涡旋震荡或移液枪吸打悬浮起菌体,然后用去针头的1mL无菌注射器吸取上述菌液,避开叶脉,用上述1mL注射器针头在苹果叶片上开小孔后,将菌体液注射入叶片中,每个叶片注射1-2个孔;
S11:注射农杆菌4天后观察叶片。
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