CN116555321A - PpERF98基因在调控桃流胶病抗性中的应用 - Google Patents
PpERF98基因在调控桃流胶病抗性中的应用 Download PDFInfo
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Abstract
本发明涉及PpERF98基因在调控桃流胶病抗性中的应用,本发明通过基因工程手段,在PpERF98基因的开放阅读框序列中获取一段特异性目标片段,并设计目标片段引物进行扩增;通过酶切法将目标片段与病毒介导的基因沉默(VIGS)载体pCaRNA3融合,成功构建VIGS载体;通过农杆菌介导的方法将VIGS载体转化到桃植株,降低PpERF98的表达,获得具有流胶病抗性的植株材料;所述开放阅读框全长序列引物为SEQ ID NO:1和SEQ ID NO:2。本发明通过转基因手段提高桃树对流胶病菌的抗性,显著降低流胶病发病率,减少桃流胶病带来的经济损失。
Description
技术领域
本发明涉及桃流胶病防治方法领域,具体涉及PpERF98基因在调控桃流胶病抗性中的应用。
背景技术
桃树流胶病是一种积累性的枝干病害,在长江流域及其以南地区发生普遍,是我国桃生产中最严重的病害之一。该病引起桃树主干、主枝等部位组织坏死,造成树势衰弱,降低果实产量和品质,缩短桃树的寿命,严重影响经济效益。引起桃流胶病的病原菌大部分属葡萄座腔菌科真菌,该菌属于半活体营养型真菌,可以侵染至桃树的木质部,这导致桃流胶病的防治存在困难。目前,尚未发现抗桃流胶病的抗性材料。因此,解析桃树的抗性基因是桃生产中亟待解决的问题,研究结果将为流胶病的防治和抗流胶病材料的创制提供重要的理论基础。
发明内容
本发明的目的在于克服目前没有行之有效的防治手段和抗流胶病的桃资源的问题,提供PpERF98基因在调控桃流胶病抗性中的应用。
本发明解决上述技术问题的技术方案如下:
PpERF98基因在调控桃流胶病抗性中的应用,其特征在于,所述PpERF98基因的序列为SEQ ID NO:5。
上述PpERF98基因在调控桃流胶病抗性中的应用,是通过基因工程的方法沉默桃PpERF98基因来提高桃的流胶病抗性。
具体应用方法为通过在PpERF98基因的全长序列中选取一段目标片段,设计目标片段的引物对VIGS-PpERF98进行扩增,通过酶切法将目标片段与病毒介导的沉默载体pCaRNA3相连,构建VIGS载体,将VIGS载体导入桃植株来提高桃对流胶病的抗性;所述目标片段的序列为SEQ ID NO:6。
进一步的,所述目标片段的引物对VIGS-PpERF98序列为SEQ ID NO:3和SEQ IDNO:4。
本发明的有益效果为:本发明发现PpERF98基因与桃流胶病抗性有关,可通过基因工程手段沉默桃PpERF98基因来提高流胶病抗性,可显著降低流胶病发病率,减少因桃流胶病带来的经济损失。
附图说明
图1为PpERF98基因全长的电泳图片;
图2为不同物种中ERF98氨基酸序列比对分析示意图;
图3为不同桃品种接种桃流胶病菌后的发病症状和病斑比较示意图;
图4为PpERF98基因在不同桃品种接种流胶病菌后的相对表达量示意图;
图5为桃植株沉默PpERF98基因的沉默效率示意图;
图6为沉默PpERF98基因的桃植株接种流胶病菌的发病症状和病斑面积示意图;
图7为沉默PpERF98基因的桃植株接种流胶病后病程相关基因的相对表达量示意图;
图8为桃植株过表达PpERF98基因的效率示意图;
图9为过表达PpERF98基因的桃植株接种流胶病菌的发病症状和病斑面积示意图;
图10为过表达PpERF98基因的桃植株接种流胶病后病程相关基因的相对表达量示意图;
图11为转基因番茄植株的PpERF98相对表达量示意图;
图12为转基因番茄植株和对照植株接种桃流胶病菌的发病症状示意图;
图13为转基因番茄植株和对照植株接种流胶病菌后病程相关基因的相对表达量示意图;
图14为PpERF98沉默植株和过表达植株水杨酸含量示意图。
具体实施方式
以下对本发明的原理和特征进行描述,所举实例只用于解释本发明,并非用于限定本发明的范围。
我们通过转录组数据筛选出了一个响应桃流胶病菌Lasiodiplodia theobromae菌株的转录因子PpERF98,发现其在不同敏感性桃品种的表达水平与桃对流胶病菌的敏感性呈正相关关系。基于转基因技术,在普通番茄A57中异源过表达该基因,发现转基因植株对桃流胶病菌的抗性降低。目前,桃尚无稳定的转基因遗传体系,本发明采用VIGS技术通过农杆菌介导的侵染体系在桃苗植株中沉默目的基因,发现沉默PpERF98的桃植株对流胶病菌的抗性增强。
主要实验过程如下:
1、PpERF98基因的全长克隆
基于前期得到的转录组数据,筛选出一个响应桃流胶病菌的ERF家族的转录因子其编号为Prupe.8G224700。将其氨基酸序列经NCBI数据库BlastP比对分析,发现与拟南芥中ERF98同源性最高(67.74%),因此命名为PpERF98。
以接种流胶病菌的桃当年生枝条韧皮部组织为样本,利用EASY spin Plus植物RNA快速提取试剂盒(Aidlab,Beijing)提取总RNA,Nanodrop one(Thermo,USA)对RNA浓度和质量进行检测,利用反转录试剂盒RT Reagent Kit with gDNA Eraser(TaKaRa,Dalian,China)将合格的RNA样本反转录得到cDNA,最后使用Phanta Max Super-Fidelity DNA Polymerase(Vazyme,Nanjing)进行开放阅读框片段扩增,其扩增引物根据在NCBI上获得序列信息利用Primer 5.0软件设计全长扩增引物SEQ ID NO:1和SEQ ID NO:2,其引物序列分别为:
PpERF98-Full length-F:5’–ATGCACTATATATCTTGCATGTCACAA--3’
PpERF98-Full-length-R:5’--CTAATGGGTTGGTTTCCCCTGTCTA--3’
扩增获得的产物利用DNA凝胶回收试剂盒(Sangon Biotech,Shanghai)回收目的片段、利用pEASY-Blunt Zero Cloning Kit(Transgene,Beijing)连接载体得到阳性克隆后,送TSINGKE公司(TSINGKE,Wuhan)进行测序获得序列,PpERF98的全长序列SEQ ID NO:5为:
ATGCACTATATATCTTGCATGTCACAAGCACAACCCATATTTTTGCCACCAAATTTGAATAAAGACCTGTTTCTGCTTAACATGGAAGGGAAGGGAGTGGAGAACCAGCAGAAGGAGCAGACTAAGGTAAGAGATCAAACCCGGTATCGAGGGATTCGGAGGCGACCGTGGGGCAAGTTTGCTGCTGAAATACGTGACCCTTCAAGAAATGGGGCACGCCTATGGCTAGGCACATTTGAGACAGCTGAAGAGGCAGCTAGGGCTTATGATCGAGCTGCTTTCGGCTTCCGGGGTCATTTGGCCATCCTCAACTTCCCTAATGACTACCAGTATCATAACCCATCAAGCTCTTTGATCAGCACTTCATCCTCTTCATCATCATCTCCATTTTCTGCTGCTGATATTGGAAAGAGTACTAATTTTGGCAGAGGCCAAGAAGAAGAAGAAGTTATAGAGTTTGAGTACCTGGACAACATGGTTTTGGAGGAGCTTCTTGACACAAAAGAGGATCATCATAGACAGGGGAAACCAACCCATTAG
该序列全长540bp,电泳照片见图1。利用MEGA7对PpERF98的蛋白质序列与拟南芥、梅、杏、苹果以及柑橘等物种的ERF98蛋白质序列进行多序列比对,发现在N端包含ERF家族特征的AP2结构域,C端包含EDLL的酸性激活域(图2)。
2、PpERF98的相对表达量分析
桃枝条接种的方法参考高磊(2016)。在接种后1、2、3d时间点观察枝条的发病症状;在0、6、12、24、48、72h的时间点取病斑周围0.5-1cm处的韧皮部组织,置于-80℃冷冻备用。使用EASY spin Plus植物RNA快速提取试剂盒(Aidlab,Beijing)提取植物总RNA,Nanodrop one(Thermo,USA)检测RNA浓度和质量;cDNA合成使用反转录试剂盒RT Reagent Kit with gDNA Eraser(TaKaRa,Dalian,China)。qRT-PCR使用HieffTM qPCR SYBR&Green Master Mix(Low Rox Plus)(YEASEN,Shanghai),仪器为QuantStudio 6(ABI,USA)。候选基因利用Primer5软件设计定量引物,以管家基因PpTEF2(Translation enlongation factor 2)(Tong et al2009)作为桃内参基因,计算方法参考Livak and Schmittgen(2001)。序列特异性引物如下:
PpERF98荧光定量引物序列:
PpERF98-F:5’--TGATCAGCACTTCATCCTCTTCA--3’
PpERF98-R:5’--CTCCTCCAAAACCATGTTGTCC--3’
桃内参基因PpTEF2的引物序列(Tong et al 2009):
PpTEF2-F:5’--AGCAAGTCACCCAACAAGCATA--3’
PpTEF2-R:5’--CCAACCAAACTCTTCAGCCAAT--3’
使用桃流胶病菌L.theobromae分别离体接种桃‘春雪’和‘大红袍’当年生枝条,结果显示‘春雪’和‘大红袍’对流胶病菌的敏感性有显著差异,具体表现为前者的病斑显著大于后者,确认‘春雪’的敏感性大于‘大红袍’(图3)。对接种流胶病菌后的两个桃品种枝条中的PpERF98的相对表达水平进行分析,发现其与桃品种对流胶病菌的敏感性呈正相关(图4)。
3、VIGS介导的桃苗PpERF98基因沉默
在PpERF98的全长序列中选取100bp长度的特异性目标片段,目标片段的序列为SEQ ID NO:6:
AAGAAGAAGAAGAAGTTATAGAGTTTGAGTACCTGGACAACATGGTTTTGGAGGAGCTTCTTGACACAAAAGAGGATCATCATAGACAGGGGAAACCAAC
设计特异性引物,对VIGS-PpERF98目标片段进行扩增,通过Thermo公司FastDigestXbal快切酶和TaKaRa公司T4 DNA Ligase将其与病毒载体pCaRNA3相连,得到VIGS重组载体PNRSV-PpERF98。VIGS-PpERF98片段扩增的引物序列为SEQ ID NO:3和SEQ IDNO:4:
VIGS-PpERF98-F:5’--GCTCTAGACAAGAAGAAGAAGAAG--3’
VIGS-PpERF98-R:5’--GCTCTAGAGTTGGTTTCCCCTGTCTAT--3’
参考Cui and Wang(2017)的方法进行桃苗进行VIGS瞬时转化。将PNRSV-PpERF98和pCaRNA1&2分别转入农杆菌GV3101感受态,阳性检测后,分别将携带PNRSV-PpERF98和pCaRNA1&2的农杆菌菌液进行过夜培养,再利用重悬液MMA(10mmol/L MES,10mmol/LMgCl2,200μmol/L乙酰丁香酮,pH=5.6~5.7)调至OD约为1.0后,按照体积比1:1混匀后将菌液注射至苗龄6-8片叶的桃幼苗叶片中,避光2d进行正常培养,以pCaRNA3和pCaRNA1&2为阴性对照,PNRSV-PpPDS和pCaRNA1&2为阳性对照。4周后对沉默桃苗完全展开未注射的功能叶进行qRT-PCR分析,结果显示,该方法可以有效沉默桃苗PpERF98基因的表达水平(图5)。
4、沉默桃植株对流胶病菌菌株L.theobromae的抗性鉴定
以沉默桃植株和对照的离体叶片为材料,参考赵丽娜(2012)的方法进行桃流胶病菌菌株L.theobromae的叶片接种。接种1和2d后观察发病症状,结果显示,沉默PpERF98桃植株病斑面积显著小于对照,对流胶病菌的抗性显著增强(图6)。
5、沉默毛桃植株中病程相关基因的qRT-PCR分析
在上述接种后1和2d的时间点取沉默毛桃和对照叶片接种点周围5mm的组织,进行病程蛋白相关基因的相对表达量分析。结果显示,沉默毛桃植株叶片中的依赖于水杨酸病程相关基因PpPR1、PpPR2均显著上调表达(图7)。沉默桃苗中PpERF98,可能通过提高PpPR1、PpPR2病程相关蛋白基因的表达,使沉默毛桃植株对流胶病菌的抗性增强。
桃病程蛋白相关基因引物:
PpPR1-F:5’--TGACAAGGTGTGTGGGCATT--3’
PpPR1-R:5’--CGGATCATAGTTGCACCCGA--3’
PpPR2-F:5’--ACAGGAGGACCATTGGCTTG--3’
PpPR2-R:5’--ACGGCCATGGTATGAAGCTC--3’
6、超表达载体的构建
通过Gateway载体同源重组技术,利用Phanta Max Super-Fidelity DNAPolymerase(Vazyme,Nanjing)进行两轮PCR反应给目的片段加接头,第一轮PCR的模板为含目的基因的质粒,引物序列:
attB-PpERF98-F:
5’--AAAAAGCAGGCTCCATGCACTATATATCTTGCATGTCACAAG--3’
attB-PpERF98-R:
5’–AGAAAGCTGGGTTCTAATGGGTTGGTTTCCCCTGTCTA--3’
第二轮PCR的模板上一轮产物,引物序列:
attB-F:5’--GGGGACAAGTTTGTACAAAAAAGCAGGCT--3’
attB-R:5’--GGGGACCACTTTGTACAAGAAAGCTGGGT--3’
然后使用Thermo公司Gateway试剂盒进行BP反应和LP反应,将PpERF98全长片段先后连接至入门载体pDONR207和过表达载体pK7WG2D,完成过表达载体的构建pK7WG2D-PpERF98。
将过表达载体pK7WG2D-PpERF98通过热激发转入大肠杆菌DH5α,通过康为世纪公司2×Es Taq MasterMix(Dye)进行PCR阳检后测序。通过全式金生公司EASY pure PlasmidMiniPrep Kit提取质粒,最后通过热激发转入转化农杆菌GV3101(WEIDI,Shanghai)。
7、桃苗瞬时过表达PpERF98和接种处理
选取8-10片完全展开叶片的桃幼苗进行农杆菌注射。用1mL注射器从叶背面分别将含有pK7WG2D和pK7WG2D-PpERF98农杆菌液注射到桃叶片中,每颗桃苗注射4-5片完全展开的功能叶并做好标记,完成注射后,22℃黑暗放置2d后进行接种处理。接种1和2d后观察发病症状,结果显示,农杆菌介导的瞬时过表达可以显著提高桃叶片中PpERF98的转录水平(图8),过表达桃植株病斑直径显著大于对照,对流胶病菌菌株的抗性显著减弱(图9),其PpPR1、PpPR2均显著下调表达(图10)。
8、番茄遗传转化及T2代植株的阳性筛选
参考欧阳波等(2003)的方法进行农杆菌介导的番茄子叶遗传转化,通过qRT-PCR对T0代植株进行相对表量进行分析,其结果如图11,选择PpERF98相对表达量最高的转基因株系继续筛选,得到T2代种子。通过喷施卡纳霉将T2代种子播种9d后,连续4d早晚在叶面喷施卡纳霉素,剔除叶片出现黄化畸形的非阳性植株,再通过艾德莱公司EASYspin Plusplant DNA Kit提取单株DNA,以载体引物进行PCR检测,结果显示阳性植株出现预期大小的目标条带。
过表达载体pK7WG2D的阳检引物序列:
pK7WG2D-F:5’--TTTCATTTGGAGAGGACTCC--3’
pK7WG2D-R:5’--TAACGTGACTCCCTTAATTC--3’
9、T2代转基因及野生型番茄对桃流胶病菌菌株L.theobromae的抗性鉴定
以T2代转基因番茄和野生番茄对照株系的第二复叶的离体叶片为材料,在叶背面接种桃流胶病菌菌株L.theobromae。接种3和5d后观察发病症状,结果显示,T2代转基因株系OX#1和OX#2对桃流胶病菌菌株L.theobromae的抗性显著降低,其叶片背面出现较多的侵染点,病斑面积增加(图12)。
10、转基因番茄中病程相关蛋白基因的qRT-PCR分析
在上述接种后24和48h的时间点取T2代转基因番茄和对照叶片接种点周围5mm的组织,置于-80℃液氮冷冻,进行病程蛋白相关基因的qRT-PCR分析。结果显示,接种桃流胶病菌后,T2代转基因番茄叶片中的依赖于水杨酸病程相关基因SlPR1、SlPR2、SlPR5和SlPRNP24的表达均受到抑制(图13)。PpERF98可能通过抑制这些病程相关蛋白基因的表达,使T2代转基因番茄株系对桃流胶病菌的抗性减弱。
番茄病程蛋白相关基因引物序列:
SlPR1-F:5’--GATGTGGGACGATGAGAAGCAATG--3’
SlPR1-R:5’--GTTGCATCGAACCCTAGCACAACCT--3’
SlPR2-F:5’--CAGATTTCACTTCCGTATGCTCTT--3’
SlPR2-R:5’--CCATCCACTCTCTGACACAACAAT--3’
SlPRNP24-F:5’--GAGGGGAACTAAGATGGCACGTAT--3’
SlPRNP24-R:5’--CTCCACCACAATCACCAGTCTGAC--3’
SlPR5-F:5’--AACTGCCCCTACACCGTTTG--3’
SlPR5-R:5’--GCCCAAAACCACCAACTCTG--3’
番茄内参引物序列:
SlActin-F:5’--ATGGCAGACGGAGAGGATATTCA--3’
SlActin-R:5’--GCCTTTGCAATCCACATCTGCTG--3’
11、PpERF98过表达或者沉默影响植物的水杨酸积累
桃幼苗沉默PpERF98后,水杨酸含量显著上升,而过表达后,水杨酸含量显著低于对照组,这表明PpERF98可能通过调控植物水杨酸途径来影响桃对流胶病菌的抗性(图14)。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (4)
1.PpERF98基因在调控桃流胶病抗性中的应用,其特征在于,所述PpERF98基因的序列为SEQ ID NO:5。
2.根据权利要求1所述PpERF98基因在调控桃流胶病抗性中的应用,其特征在于,通过基因工程的方法沉默桃PpERF98基因来提高桃的流胶病抗性。
3.根据权利要求1所述PpERF98基因在调控桃流胶病抗性中的应用,其特征在于,具体应用方法为通过在PpERF98基因的全长序列中选取一段目标片段,设计目标片段的引物对VIGS-PpERF98进行扩增,通过酶切法将目标片段与病毒介导的沉默载体pCaRNA3相连,构建VIGS载体,将VIGS载体导入桃植株来提高桃对流胶病的抗性;所述目标片段的序列为SEQID NO:6。
4.根据权利要求3所述PpERF98基因在调控桃流胶病抗性中的应用,其特征在于,所述目标片段的引物对VIGS-PpERF98序列为SEQ ID NO:3和SEQ ID NO:4。
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