CN111116723B - 野生稻抗菌肽OrR214及其应用 - Google Patents
野生稻抗菌肽OrR214及其应用 Download PDFInfo
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- CN111116723B CN111116723B CN202010063138.1A CN202010063138A CN111116723B CN 111116723 B CN111116723 B CN 111116723B CN 202010063138 A CN202010063138 A CN 202010063138A CN 111116723 B CN111116723 B CN 111116723B
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Abstract
本发明属于生物技术领域,具体公开了野生稻抗菌肽OrR214及其应用,申请人通过建立漳浦野生稻cDNA文库,最终分离出野生稻抗菌肽OrR214。申请人发现抗菌肽OrR214对番茄溃疡病菌、青枯病菌、小麦苗枯菌、水稻白叶枯病菌以及稻瘟病菌等多种病菌的生长具有抑制作用,且热稳定性强,对多种酶不敏感,对哺乳动物和人无害,可将其应用于植物病虫害的生物防治,或可作为抗菌剂。
Description
技术领域
本领域属于生物技术领域,具体涉及野生稻抗菌肽OrR214及其应用。
背景技术
水稻是三大主要粮食作物之一,而水稻白叶枯及稻瘟病是水稻上两大重要病害,目前为止依旧很难控制其病害的发生。而引起植物病害的病原微生物有五大类:真菌、细菌、病毒、线虫和寄生性种子植物,其中由细菌和真菌引起的植物病害对人类,牲畜,作物和其他生物的严重病害,造成严重的经济损失。目前已知由细菌引起的植物病害有500种以上,其中细菌性青枯病、水稻白叶枯和溃疡病等是世界性重要病害。
水稻白叶枯病(Bacterial leaf blight,BLB)是由稻黄单胞菌稻致病变种(X.oryzae pv.oryzae)引起的,属细菌性病害,其多在热带和温带发生,产量损失在10%—50%。病菌一般在水稻种子、带病稻草、稻桩和稻李氏禾等田边杂草中越冬,由于水稻苗期温度较低、菌量较少,症状表现不明显。该病发病的最适宜温度为26~30℃,稻苗长势过旺、土壤偏酸等条件极易利于该病发生。种植抗病品种可以很好地控制该病,但是病菌分泌的效应子也会抑制植物抗病性的产生,存在抗病性丧失的风险(Verdier V,et al.2012)。
20世纪初,医学上发现了第一个能有效控制疾病的抗生素之后,在世界范围内开始进入了抗生素开发和应用的时代(Demain and Sergio 2009,Aoki,Kuroda etal.2012)。虽然抗生素的发现和使用对人类健康和生活环境有极大的改善,但是由于长期过度的使用抗生素,病原微生物对抗生素产生了一定的耐药性,导致抗生素的药效达到了一定的临界点,从而产生大多抗生素的效果极显著的降低,甚至失效的现象(Walsh 2000,Lopez,Mathers et al.2006,Huh and Kwon 2011)。耐药性的产生部分是因为抗生素与宿主细胞相互作用,作用专一不具有广谱性,甚至在抗生素之间会产生交叉耐药性(Giuliani,Pirri et al.2007,Moreillon 2008,Maurya,Thota et al.2013),耐药性已经成为全球亟待解决的公共健康和卫生问题之一(Brooks and Brooks 2014,Zhu,Zhang etal.2015)。因此为解决抗生素耐药性残留下来的问题,人们开始将重点转移到开发一种能够代替抗生素的,不会产生耐药性,且作用广泛的新型抗菌物上(Hancock and Sahl 2006,Zhu,Zhang et al.2015)。直到在动物和昆虫中首次发现并分离到抗菌谱广、且不会产生抗药性的抗菌肽,为此抗菌肽被公认为是当今能够代替抗生素的一种很有前途抗菌物质(Michael 2002,Williams and Bax 2009,Nguyen,Haney et al.2011)。抗菌肽种类繁多,天然抗菌肽一般由12-50个左右的氨基酸组成,有些抗菌肽是单一的线性,有些抗菌肽则含有α-螺旋,β折叠等结构,导致即使相同的氨基酸组成由于结构组成不同产生作用结果不一致的抗菌肽(Brown and Hancock 2006,Fjell,Hiss et al.2012)。抗菌肽作为动植物的先天免疫系统的部分组成物质,对于外来微生物的入侵具有一定的抵制防御作用(Lin,Linet al.2010,Li,Zhu et al.2017),其对包括革兰氏阳性和革兰氏阴性细菌、真菌等病原微生物都有广泛的抗菌活性(Wakabayashi,Hiratani et al.1996,Giuliani,Pirri etal.2007),此外抗菌肽对宿主菌和细菌没有特定的靶标位点,且速效,不会产生交叉耐药性(Yamauchi,Tomita et al.1993,Kong,Yang et al.2018)。抗菌肽具有的优良特性激励了许多研究着不断建立并优化从各种生物中分离纯化抗菌肽及其衍生物的方法(Mygind,Fischer et al.2005)。
由于全球抗生素药物的的过度使用,越来越多的超级细菌出现,其不断对传统抗生素产生耐药性。人们迫切地寻找能够代替传统抗生素的药物,这使得抗菌肽成为一种很有潜力的抗菌物质。
发明内容
本发明的目的在于提供一种野生稻抗菌肽OrR214,所述抗菌肽的氨基酸序列为SEQ ID NO.1所示。
本发明的另一个目的在于提供抗菌肽OrR214的应用,该抗菌肽能用于制备成抑菌剂,特别是水稻的防病剂。
为了完成上述目的,本发明采用如下技术方案:
申请人通过构建一个高质量的漳浦野生稻cDNA文库,筛选能够使宿主细胞枯草芽胞杆菌(SCK6)自溶的基因,将出现自溶现象的菌株进行菌液PCR检测插入片段大小,提取质粒,重新转化SCK6感受态细胞,PCR检测插入片段。并在含卡那霉素的LB平板上再次划线,出现自溶现象,则表明转化成功。将其中自溶效果最好的菌株提取质粒,测序,最终获得抗菌肽OrR214,所述抗菌肽的氨基酸序列为SEQ ID NO.1所示。所述的抗菌肽可用任何本领域制备蛋白的方式得到,包括但不限于原核表达,合成等。
编码SEQ ID NO.1所示氨基酸序列的核苷酸序列也属于本发明的保护范围。
抗菌肽OrR214的应用,包括利用该抗菌肽制备成抑菌剂,或是水稻的生防制剂;
所述的抑菌剂可抑制的细菌包括但不限于:番茄细菌性溃疡病菌、青枯病菌、小麦苗枯病菌、水稻白叶枯病菌、稻瘟病菌、水稻细菌性条斑菌。
与现有技术相比,本发明具有以下优点:
本发明筛选出的野生稻抗菌肽OrR214能有效抑制供试的革兰氏阳性细菌和一些革兰氏阴性细菌及真菌的正常生长,特别是该抗菌肽对水稻上的两大重要病害:水稻白叶枯病菌及稻瘟病菌具有一定的抑制作用,还能够抑制小麦苗枯菌、番茄溃疡菌以及难以用化学药物控制的青枯菌,且热稳定性强,对多种酶不敏感。溶血实验及毒性试验证明该基因对哺乳动物和人无害。该抗菌肽基因从野生稻中筛选,在后期研究中有望用于对水稻上重要病虫害防治中的生物防治,或可作为抗菌剂应用。
附图说明
图1为筛选出的OrR214及OrR935自溶菌株与无自溶效果SCK6空载菌株生长不同时间后的菌株效果图;
其中图1中A左上为OrR214菌株(Ⅱ)和无自溶效果SCK6空载菌株(Ⅰ)生长12h状态;图1中A右上为OrR214菌株(Ⅱ)和无自溶效果SCK6(Ⅰ)生长48h状态;
图1中A左下为OrR935菌株(Ⅳ)和无自溶效果SCK6空载菌株(Ⅲ)生长12h状态;图1中A右下为OrR935菌株(Ⅳ)和无自溶效果SCK6(Ⅲ)生长60h状态。
图1中B、C、D分别为SCK6空载菌株、OrR214菌株、OrR935菌株扫描电镜图。
图2为抗菌肽OrR214及OrR935及其对照组的抑菌圈示意图;
其中Ⅰ为OrR935抗菌肽,Ⅱ为OrR214抗菌肽,III为OrR119抗菌肽,IV为OrR1135抗菌肽;Ⅴ为SCK6空载菌株表达蛋白作为对照。
图3为抗菌肽OrR935及SCK6空载菌株表达蛋白对禾谷镰刀及其孢子的抑制示意图;
图3中A为SCK6空载菌株表达蛋白处理的孢子图,B为OrR935抗菌肽处理的孢子图,C为抗菌肽OrR935对禾谷镰刀菌平板抑制图(其中Ⅰ为SCK6空载菌株表达蛋白,Ⅱ为OrR935抗菌肽,III为OrR1135抗菌肽,IV为OrR214抗菌肽),D为禾谷镰刀菌孢子萌发率数据图。
图4为抗菌肽OrR214及OrR935对稻瘟菌抑制图;
其中图4中A为SCK6空载菌株表达蛋白作为对照,B、C分别为含有抗菌肽OrR935、OrR214的PDA平板中稻瘟病菌生长示意图,D为具体的抑制数据。
图5为抗菌肽OrR935、OrR214及SCK6空载菌株表达蛋白对不同酶敏感性示意图;
图5中A为抑菌直径图,B为不同酶处理后抗菌肽对X.oryzae pv.oryzae的抑菌圈直径示意图,C为不同酶处理后抗菌肽对R.solancearum的抑菌圈直径示意图,D为不同酶处理后抗菌肽对C.fangii的抑菌圈直径示意图。
图6为抗菌肽OrR935、OrR214及SCK6空载菌株表达蛋白对温度的稳定性实验示意图;
图6中A为不同温度处理后抗菌肽对C.fangii的抑菌圈直径示意图,B为不同温度处理后抗菌肽对C.michiganensis的抑菌圈直径示意图,C为不同温度处理后抗菌肽对X.oryzae pv.oryzae的抑菌圈直径示意图,D为不同温度处理后抗菌肽对R.solancearum的抑菌圈直径示意图。
图7为抗菌肽OrR935、OrR214及SCK6空载菌株表达蛋白对水稻白叶枯菌在植株上的生防应用示意图。
具体实施方式
本发明所述技术方案,如未特别说明,均为本领域的常规技术;所述试剂或材料,如未特别说明,均来源于商业渠道。
实施例1:
野生稻抗性基因OrR214及OrR935的获得:
1)构建高质量的野生稻cDNA文库,初步筛选出对枯草芽胞杆菌SCK6宿主细胞自身产生一定毒害作用,并能使宿主细胞自溶的基因:取-70℃保存的3000个左右枯草芽胞杆菌野生稻cDNA文库菌株和SCK6菌株解冻备用,在含卡那霉素的LB平板上点样,每12h观察结果,记录后期出现自溶现象的菌株编号。
2)复筛:对于初筛有效果的菌株在含卡那霉素的LB平板上再次进行筛选,每个菌株3个重复,每隔12h观察平板上菌落生长形态,最终确定自溶效果稳定的菌株,如图1中A左上所示,OrR214(Ⅱ)菌株和SCK6空载菌株生长12h均长势良好且表型相近,在图1中A左下所示,OrR935(Ⅳ)菌株和SCK6空载菌株生长12h时均长势良好且表型相近,而在48h-60h后OrR214菌株(图1中A右上)及OrR935号菌株(图1中A右下)均出现自溶现象。
3)自溶菌株稳定性鉴定:将出现自溶现象的菌株进行菌液PCR检测插入片段大小,提取质粒,重新转化SCK6感受态细胞,PCR检测插入片段大小与之前相同,证明转化成功。在含卡那霉素的LB平板上再次点样重复,出现自溶现象,则表明自溶现象为插入基因引起。
4)将其中自溶效果最好的菌株提取质粒,测序鉴定,其中,OrR214菌株中的抗菌肽OrR214对应的核苷酸序列为:
CTAGTAGATATATATACACATGTGTACAATTGTACAAGTAGTGAGAAACACACGCATTGCTATGAAATACGAAAGTCAATTTCC
抗菌肽OrR214对应的氨基酸序列为:
LVDIYTHVYNCTSSEKHTHCYEIRKSIS。
OrR935菌株中的抗菌肽OrR935对应的核苷酸序列为:
CTAGGCGTGCCAGTGAGCTCGACTTTGCGGTTAAATAACACGACAATGAATCCCTGTTTGCCATCC;
抗菌肽OrR935对应的氨基酸序列如下:
LGVPVSSTLRLNNTTMNPCLPS。
实施例2:
抗菌肽OrR214及OrR935的获得:
(1)本发明所用的抗菌肽均为人工合成的方式,抗菌肽OrR214对应的氨基酸为:
LVDIYTHVYNCTSSEKHTHCYEIRKSIS。
抗菌肽OrR935对应的氨基酸为:LGVPVSSTLRLNNTTMNPCLPS。
(2)本发明的抗菌肽,还可采用原核表达的方式制备:
1)抗菌肽OrR214及OrR935粗蛋白的表达与分离纯化
将含有NdeⅠ和XbaⅠ酶切位点的PBE-S载体酶切后线性化,并用T4连接酶将OrR214、OrR935基因分别与其连接,获得目的基因融合表达载体PBE-S-OrR214及PBE-S-OrR935。
将融合表达载体PBE-S-OrR214及PBE-S-OrR935质粒,用热激法转化到大肠杆菌DH5α高效感受态细胞中扩增,提取质粒后再转入到SCK6感受态细胞中获得重组表达菌株OrR214及OrR935。利用SCK6表达载体PBE-S的高效的分泌表达特性,将重组菌株摇培在LB(含卡那霉素)液体培养基72h获得OrR214及OrR935发酵液,离心取上清并用硫酸铵饱和溶液析出抗菌肽,4℃过夜后离心取沉淀,并用PBS(磷酸盐缓冲液)溶解沉淀,于4℃透析48h,可得到两种抗菌肽的粗蛋白,备用。
2)纯化的抗菌肽OrR214及OrR935表达与分离纯化
为了得到可用于纯化的抗菌肽,对上述步骤1)中的基因OrR214及OrR935进行改造,在载体信号肽与目的片段之间插入6x His标签及一段肠激酶序列,然后再转入枯草芽孢杆菌SCK6进行表达,。对改造后的重组抗菌肽OrR214及OrR935、根据方案使用Ni-NTA HisBind Resin试剂盒分离。纯化后的抗菌肽过TEV抗菌肽酶4℃酶切过夜,浓缩回收。即得纯的目的抗菌肽OrR214及OrR935。
实施例3:
抗菌肽OrR214及OrR935抑菌效果:
1)抗菌肽OrR214及OrR935抑制细菌效果:
以1%的接种量在含有普通LB培养基的2mL EP管中分别接种小麦苗枯病菌(C.fangii)、白叶枯病菌(X.oryzaepv.oryzae)、番茄溃病疡菌(C.michiganensis)、青枯病菌(R.solanacearum),水稻细菌性条斑菌(X.oryzaepv.oryzicola)在28℃摇床中150rpm震荡培养过夜;摇培后,取300μl指示菌菌液于10mL摇菌管中,混入4mL温热的半固体牛肉膏培养基,快速混匀,迅速倒入固体LB平板中,放置5min,使培养基充分凝固晾干;将培养皿分为5个区域,每个区域中心放置无菌的滤纸片,使之与培养基充分粘合;吸取浓度为1000μg/mL抗菌肽OrR214及OrR93520μl,缓慢滴在一个区域的滤纸片上,使液体缓慢均匀扩散,按同样的方法将对照SCK6空载菌株的表达蛋白及抗菌肽OrR119及OrR1135滴在另外区域的滤纸片上,无菌工作台内吹干;分别在28℃培养箱倒置培养,7h后观察,通过测定抑菌圈大小来计算抗菌肽OrR214、OrR935的抗菌活性。共进行3次实验,每次实验进行3次重复,测量数据直径(已去除滤纸片直径),取其平均值。结果如图2和表1、2中及图5中A所示,含有抗菌肽OrR214及OrR935的滤纸片周围都出现大的抑菌圈,而对照部分没有明显的抑菌圈出现。抗菌肽OrR214及OrR935对小麦苗枯病(C.fangii)(A)、白叶枯病菌(X.oryzaepv.oryzae)(B)、番茄溃疡病菌(C.michiganensis)(C)、青枯病菌(Rsolanacearum)(D)展现出了广谱的抑菌活性,在4种细菌平板上均展现出了高效的抑制作用。同时抗菌肽OrR214和OrR935对水稻细菌性条斑菌(X.oryzaepv.oryzicola)也展示出高效的抑制作用。
表1抗菌肽OrR214抑制细菌活性
表2抗菌肽OrR935抑制细菌活性
2)抗菌肽OrR214及OrR935抑制真菌效果:
将抗菌肽OrR214、OrR935、SCK6空载的表达蛋白浓度调一致为1800μg/mL,并用细菌过滤器过滤备用,用于抗菌肽OrR935对禾谷镰刀菌的抑菌实验
(1)抗菌肽OrR935对禾谷镰刀菌菌丝的抑制作用
抗菌肽OrR214对禾谷镰刀菌不起抑制作用,因此本实验只考察抗菌肽OrR935对禾谷镰刀菌的作用。
在PDA平板上活化禾谷镰刀菌,用打孔器沿菌丝外缘打孔,放置于PDA平板正中央,于28℃培养。待菌丝生长至1-2cm左右时,将培养皿分为四个区域,每个区域中心放置一个牛津杯,打孔;
吸取抗菌肽OrR935200μl缓慢滴入孔内,将对照SCK6空载菌株的表达蛋白和抗菌肽OrR214、抗菌肽OrR1135滴在另两个区域的孔内,无菌工作台内吹干;28℃培养箱培养,约1-2d后观察。结果如图3中C所示,与对照相比,抗菌肽OrR935周围真菌菌丝生长受到阻止,几乎无法越过孔内,而对照部分菌丝则能正常生长。
(2)抗菌肽OrR935对禾谷镰刀菌孢子萌发的抑制作用
在PDA平板上活化小麦赤霉病病原真菌,用打孔器沿菌丝外缘打孔,放置于PDA平板中央,于28℃培养活化。接种菌丝到CMC产孢培养基中,28℃,220rpm光照摇培产孢5d。收集孢子备用。
取100μl的孢子浓度为2.1x104个/mL和100μl的YPG培养基于1.5mL EP管中。分别向管中加入200μl的OrR935抗菌肽、SCK6空载的表达蛋白。28℃,220rpm摇培,分别在2h,4h,6h,8h,10h,12h时显微观察结果。结果如图3中D所示,在与对照菌株SCK6相比,抗菌肽OrR935能明显抑制禾谷镰刀菌的孢子萌发。
禾谷镰刀菌孢子萌发率(%)
(3)抗菌肽OrR214、OrR935对稻瘟病菌的抑制作用
在PDA平板上活化稻瘟病病原真菌,用打孔器沿菌丝外缘打孔,放置于PDA平板中央,于28℃培养活化。
取200μl纯化后浓度为350μg/mL的抗菌肽于10mL摇菌管中,混入3mL温热的半固体PDA培养基中,快速混匀,迅速倒入固体PDA平板中,放置5min,使培养基充分凝固晾干。用直径为0.5cm的打孔器打取稻瘟菌菌饼,用接种环将稻瘟菌菌饼接种在含抗菌肽的PDA板上,28℃,倒置培养,在5d时观察并记录数据。结果如图4所示,与对照菌株SCK6相比,抗菌肽OrR214能明显抑制稻瘟病菌菌丝生长。
稻瘟病病菌的菌圈直径 单位:cm
实施例4:
1)抗菌肽酶稳定性测定
1.提取抗菌肽:
1)取出抗菌肽置于2mL离心管内,并将所有抗菌肽用磷酸缓冲液调整一致,浓度为1000μɡ/mL,备用。
2.酶处理抗菌肽
1)准备配制以下酶:
脂肪酶(Lipase):20mg/mL为储存浓度;
胰蛋白酶(Trypsin):20mg/mL为储存浓度;
胃蛋白酶(Pepsin):20mg/mL为储存浓度;
木瓜蛋白酶(Papain):20mg/mL为储存浓度;
α-淀粉酶(α-Amylase):20mg/mL为储存浓度;
蛋白酶K(Protease K):20mg/mL为储存浓度;
蛋白酶E(Protease E):20mg/mL为储存浓度。
2)抗菌肽及空载菌株的表达蛋白用上述酶分别处理,混合液中酶使用终浓度为100μɡ/mL。
3)分别将用脂肪酶(Lipase)处理的抗菌肽于30℃处理1h;胰蛋白酶(Trypsin)、胃蛋白酶(Pepsin)处理的抗菌肽于37℃处理1h;木瓜蛋白酶(Papain)、α-淀粉酶(α-Amylase)、蛋白酶K(Protease K)、蛋白酶E(Protease E)处理的抗菌肽于55℃处理1h,后于90℃处理5min,使酶失活,置于4℃备用。
3.抑菌试验
1)指示菌制备:以1%接种量摇培C.fangii、C.michiganensis、X.oryzaepv.oryzae、R.solancearum菌株,28℃,180rpm,12-14h。
2)取200μL上述菌液于10mL摇菌管中,混入4mL 50℃左右的半固体牛肉膏培养基,快速摇匀,迅速倒入固体LB培养基中,轻轻摇晃均匀,放置5min左右,待半固体牛肉膏培养基凝固晾干后,培养皿分为六个区域,每个区域分别代表脂肪酶(Lipase)、胃蛋白酶(Pepsin)、木瓜蛋白酶(Papain)、α-淀粉酶(α-Amylase)、蛋白酶K(Protease K)、蛋白酶E(Protease E),并于每个区域放置一个无菌的滤纸片,吸取与酶混合后的抗菌肽20μL,缓慢滴在一个区域的滤纸片上,使液体缓慢均匀扩散,按同样的方法将空载体菌株的蛋白作为对照,每个抗菌肽四组重复。无菌工作台内吹干后,放置28℃培养箱内培养7h,随时观察并测量抑菌圈直径(已去除滤纸片直径)。结果如图5中B,C,D所示,抗菌肽OrR935、OrR214对脂肪酶(Lipase)、蛋白酶K(Protease K)敏感,而对胃蛋白酶(Pepsin)、木瓜蛋白酶(Papain)等其他蛋白酶均不敏感。以C.fangii、C.michiganensis为例,每种指示菌4次重复,由于数据量较大,在此仅列出部分的具体效果数据,单位(mm)见下表:
2)抗菌肽温度稳定性测定
1.调整抗菌肽浓度:
取出抗菌肽OrR935、OrR214和SCK6空载的表达蛋白分别置于2mL离心管内,并将所有抗菌肽用磷酸缓冲液调整一致,浓度为1000μɡ/mL,备用。
2.不同温度处理抗菌肽
将OrR935、OrR214抗菌肽分别在50℃、80℃、100℃条件下热处理30min,结束后置于4℃,备用。
3.抑菌试验
1)指示菌制备:以1%接种量摇培C.fangii、C.michiganensis、X.oryzaepv.oryzae、R.solancearum菌株,28℃,180rpm,12-14h。
2)取200μL上述菌液于10mL摇菌管中,混入4mL50℃左右的半固体牛肉膏培养基,快速摇匀,迅速倒入固体LB平板中,轻轻摇晃均匀,放置5min左右,待半固体牛肉膏培养基凝固晾干后,培养皿分为四个区域,每个区域分别代表4℃、50℃、80℃、100℃,并于每个区域放置一个无菌的滤纸片,吸取抗菌肽20μL,缓慢滴在一个区域的滤纸片,使液体缓慢均匀扩散,空载体菌株的蛋白作为对照,处理方法相同。无菌工作台内吹干后,放置28℃培养箱内培养7h,,随时观察并测量抑菌圈直径(已去除滤纸片直径),具体结果如图6所示,随着温度升高,抗菌肽抑菌活性有所降低,相比对照仍具体较强的抑菌活性。以C.fangii、C.michiganensis为例,每种指示菌4次重复,由于数据量较大,在此仅列出部分的具体效果数据,单位(mm)见下表:
实施例4:
1)MIC assay
用A microtiter broth dilution method测定抗菌肽的最低抑菌浓度(MIC)。将接种物在LB液体培养基中于28℃孵育至对数生长期后,调整接种物的浓度,制备浓度为1-2×104CFU/mL的细菌接种物,并加入96孔滴定板中。随后加入不同浓度梯度的等量抗菌肽溶液,并将96孔板在28℃温育24h,测定OD600 nm的吸光值。将细菌没有生长的抗菌肽浓度记录为MIC值。
表3抗菌肽OrR214及OrR935的最低抑菌浓度(MIC:μM)
实施例5:
抗菌肽OrR214及OrR935的溶血实验:
对抗菌肽OrR214及OrR935进行哺乳动物红细胞毒性测试。具体方式如下:
1.取新鲜的猪血500μl,4℃离心5000rpm,10min。
2.小心弃上清,用0.2M的PBS缓冲液(PH=7.2)重悬浮沉淀3次,每次均4℃离心3000rpm,5min。
3.用相同的PBS缓冲液将红细胞稀释至0.5%或1%,并将抗菌肽调至不同浓度。
4.取50μl红细胞悬浮液与50μl不同浓度的抗菌肽于96孔细胞培养板中37℃孵育1h,阳性对照为0.1%Triton-100x(完全溶血),阴性对照为上述PBS缓冲液(不溶血)。
5.孵育1h后,4000rpm,离心10min,除去沉淀。
6.取70μl上清于新的96孔细胞培养板中,测定吸光值(猪血红细胞为450nm)。
7.计算公式:溶血率(%)=(实验组A450–阴性对照A450)/(阳性对照A450–阴性对照A450)×100%
为了评估抗菌肽OrR214及OrR935对哺乳动物红细胞的毒性,我们检测了其在1×MIC、1.5×MIC、2×MIC、2.5×MIC、猪红细胞的溶血活性。在共孵育1h后,即使在2×MIC的浓度下溶血活性也较低(表4),且在MIC浓度以下溶血活性几乎为零。实验结果表明,该抗菌肽对哺乳动物细胞相对安全。
表4抗菌肽OrR214的溶血活性
实施例6:
抗菌肽OrR214及OrR935的细胞毒性:
通过改良的标准微量滴定稀释方法,用哺乳期10d左右的小猪红细胞测定纯化后抗菌肽的细胞毒性。通过溴化3-(4,5-二甲基硫代唑-2-基)-2,5-二苯基四唑溴化物(MTT)染料还原测定法测试,对于MTT测定,将(1.0-2.0)×105个细胞/孔接种在96孔板中,然后用不同浓度的肽在37℃,5%CO2中处理24h。然后,取50μL的MTT以0.5mg/mL的终浓度加入96孔板中,将混合物于37℃下温育4h,5000rpm离心5min后,弃去上清液。用100μL溶解液溶解,并使用酶标仪测量450nm处的OD值,结果如表5所示,即使在2.5×MIC浓度下,抗菌肽OrR214及OrR935对哺乳动物细胞几乎无毒性。
表5抗菌肽OrR214及OrR935的毒性
实施例7:
抗菌肽OrR214及OrR935水稻白叶枯菌在植株上的生防应用。
将水稻白叶枯菌于LB培养基中28℃,150rpm摇培至OD600为1.0左右,将抗菌肽浓度调整一致为1500μg/mL,用无菌剪刀剪去10cm左右叶片,保湿培养后,用无菌剪刀蘸取等体积混合的白叶枯菌液和抗菌肽的混合液后,剪去水稻叶尖,以空载为对照。接种7d后开始测量病斑长度,每个处理4片叶片,重复3次。结果如图7所示,对照(图7中A)菌株正常发病,而抗菌肽OrR214(图7中B)及OrR935(图7中C)发病较轻。
序列表
<110> 华中农业大学
<120> 野生稻抗菌肽OrR214及其应用
<160> 4
<170> SIPOSequenceListing 1.0
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<212> PRT
<213> 人工序列(Artificial Sequence)
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His Thr His Cys Tyr Glu Ile Arg Lys Ser Ile Ser
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Claims (4)
1.一种分离的野生稻抗菌肽,所述抗菌肽的氨基酸序列为SEQ ID NO.1所示。
2.编码SEQ ID NO.1所示氨基酸序列的核苷酸序列。
3.权利要求1所述的抗菌肽或权利要求2所述的核苷酸序列在制备抑菌剂中的应用;
所述的抑菌剂的抑制对象为:番茄细菌性溃疡病菌(C. michiganensis )、青枯病菌(R. solanacearum)、小麦苗枯病菌(C. fangii)、水稻白叶枯病菌(X. oryzaepv.oryzae)、稻瘟病菌(Pyricularia oryzae Cav.)、或水稻细菌性条斑菌(X. oryzaepv.oryzicola)。
4.权利要求1所述的抗菌肽或权利要求2所述的核苷酸序列在制备水稻生防制剂中的应用;
所述的生防制剂防治由番茄细菌性溃疡病菌、青枯病菌、小麦苗枯病菌、水稻白叶枯病菌、稻瘟病菌、或水稻细菌性条斑菌引起的疾病。
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