CN110063363B - 抗菌肽o3tr和c12o3tr在制备防治柑橘果实采后绿霉病的药物中的应用 - Google Patents
抗菌肽o3tr和c12o3tr在制备防治柑橘果实采后绿霉病的药物中的应用 Download PDFInfo
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Abstract
本发明公开了抗菌肽O3TR和C12O3TR在制备防治柑橘果实采后绿霉病的药物中的应用。研究结果显示,抗菌肽O3TR和C12O3TR能够有效防治柑橘果实采后绿霉病,其杀菌能力强,瞬时杀菌能力优于已有文献报道能有效控制柑橘果实采后绿霉病的抗菌肽PAF56和BP21,长效杀菌能力优于抗菌肽PAF56,且抑菌稳定性好,不受温度、pH、Na+、K+等的影响。本发明拓宽了抗菌肽O3TR和C12O3TR在果蔬保鲜领域的应用范围,也为柑橘果实采后绿霉病的防治提供了新的解决方法。
Description
技术领域
本发明涉及抗菌肽O3TR和C12O3TR在果蔬保鲜领域的应用。
背景技术
柑橘果实在采后贮藏运输过程中易受指状青霉(Penicillium digitatum)侵染而引起绿霉病病害,导致大量的经济损失。在现有的柑橘采后病害防治方法中,化学杀菌剂的使用最为普遍,但其毒性大,对人体健康和环境存在潜在威胁,且易使病原微生物对其产生抗性,因而使用逐渐受到限制。抗菌肽(antimicrobial peptides, AMPs)是一类有抗菌活性的小分子多肽的总称,广泛存在于各种生物体内,因作用机制不同于化学杀菌剂和抗生素,病原微生物不易对其产生抗性,对环境无污染,使其成为替代化学杀菌剂和抗生素的理想资源。近年来,抗菌肽在医药、农业、食品及畜牧等行业受到越来越多的关注,但在果蔬采后病害控制方面的研究较少。
发明内容
本发明的目的在于筛选能够有效防治柑橘果实采后绿霉病且杀菌能力强、稳定性好的抗菌肽,以拓宽抗菌肽在果蔬保鲜领域的应用范围,同时为柑橘果实采后绿霉病的防治提供新的解决方法。
经研究,本发明提供如下技术方案:
1、抗菌肽O3TR在制备防治柑橘果实采后绿霉病的药物中的应用;所述抗菌肽O3TR的氨基酸序列如SEQ ID No.1所示,其C端酰胺化修饰。
2、抗菌肽C12O3TR在制备防治柑橘果实采后绿霉病的药物中的应用;所述抗菌肽C12O3TR的氨基酸序列如SEQ ID No.1所示,其N端十二烷酰化修饰,C端酰胺化修饰。
本发明的有益效果在于:本发明公开了抗菌肽O3TR和C12O3TR在制备防治柑橘果实采后绿霉病的药物中的应用。研究结果显示,抗菌肽O3TR和C12O3TR能够有效防治柑橘果实采后绿霉病,其杀菌能力强,瞬时杀菌能力优于已有文献报道能有效控制柑橘果实采后绿霉病的抗菌肽PAF56和BP21,长效杀菌能力优于抗菌肽PAF56,且抑菌稳定性好,不受温度、pH、Na+、K+等的影响。本发明拓宽了抗菌肽O3TR和C12O3TR在果蔬保鲜领域的应用范围,也为柑橘果实采后绿霉病的防治提供了新的解决方法。
附图说明
图1为七种抗菌肽对柑橘果实采后绿霉病的控制效果,其中A为发病率,B为病斑直径。
图2为接种P. digitatum 6天后对照组与七种抗菌肽处理组的柑橘果实对比。
图3为五种抗菌肽对柑橘果实采后绿霉病的控制效果对比,其中A和B为抗菌肽与病原菌混合后立即接种,A为发病率,B为病斑直径;C和D为抗菌肽与病原菌混合16 h后接种,C为发病率,D为病斑直径。
图4为接种P. digitatum 6天后对照组与五种抗菌肽0 h(抗菌肽与病原菌混合后立即接种)和16 h(抗菌肽与病原菌混合16 h后接种)处理组的柑橘果实对比。
图5为温度对抗菌肽O3TR(A)、C12O3TR(B)和Jelleine-I(C)抑菌稳定性的影响。
图6为pH对抗菌肽O3TR(A)、C12O3TR(B)和Jelleine-I(C)抑菌稳定性的影响。
图7为金属离子对抗菌肽O3TR(A1,A2)、C12O3TR(B1,B2)和Jelleine-I(C1,C2)抑菌稳定性的影响。
图1和图3中,标有相同小写英文字母(a、b、c)的组之间不存在显著差异(p>0.05);标有不同小写英文字母(a、b、c)的组之间存在显著差异(p<0.05)。
具体实施方式
为了使本发明的目的、技术方案和优点更加清楚,下面将结合附图对本发明的优选实施例进行详细的描述。
本发明先从已知的大量抗菌肽中筛选出文献报道具有广谱和高效抑菌性的七种阳离子短链抗菌肽:O3TR(人工序列)、C12O3TR(人工序列)、dhvar5(来源于人类乳铁蛋白)、hLF(1-11) (来源于人类乳铁蛋白)、LDP-NLS(人工序列)、Jelleine-I(来源于意大利蜜蜂(Apis mellifera))和Osmin(来源于红壁蜂(Osmia rufa)),然后使用固相合成方法合成这七种抗菌肽,通过果实试验明确这七种抗菌肽对柑橘果实采后绿霉病的控制效果,筛选出确实能够有效控制柑橘果实采后绿霉病的抗菌肽,并与文献报道的能有效控制柑橘果实采后绿霉病的抗菌肽PAF56和BP21进行瞬时杀菌能力和长效杀菌能力对比,筛选出杀菌能力更强的抗菌肽,对其进行抑菌稳定性测试,最终筛选出杀菌能力强、抑菌稳定性好的抗菌肽。
一、实验材料
1、抗菌肽
委托南京金斯瑞公司使用固相合成方法合成纯度>90%的如下抗菌肽:
O3TR:氨基酸序列如SEQ ID No.1所示,其C端酰胺化修饰(OOWW-NH2,O表示鸟氨酸);
C12O3TR:氨基酸序列如SEQ ID No.1所示,其N端十二烷酰化修饰,C端酰胺化修饰((C12H23O)-OOWW-NH2);
dhvar5:氨基酸序列如SEQ ID No.2所示(LLLFLLKKRKKRKY);
hLF(1-11):氨基酸序列如SEQ ID No.3所示(GRRRRSVQWCA);
LDP-NLS:氨基酸序列如SEQ ID No.4所示(KWRRKLKKLRPKKKRKV);
Jelleine-I:氨基酸序列如SEQ ID No.5所示,其C端酰胺化修饰(PFKLSLHL-NH2);
Osmin:氨基酸序列如SEQ ID No.6所示(GFLSALKKYLPIVLKHV);
PAF56:氨基酸序列如SEQ ID No.7所示(GHRKKWFW);
BP21:氨基酸序列如SEQ ID No.8所示,其N端乙酰化修饰,C端酰胺化修饰(Ac-FKLFKKILKVL-NH2)。
将合成的抗菌肽分别用无菌超纯水配成1 mmol/ L的母液,-40℃贮藏。
2、病原菌
指状青霉(P. digitatum)分离于发病柑橘果实,并通过分子生物学方法鉴定。
用马铃薯葡萄糖琼脂培养基(PDA)于25℃培养病原菌,7天后收集孢子并用无菌水调至所需浓度。
二、实验方法
1、抗菌肽对柑橘果实采后绿霉病的控制作用
柑橘果实随机分组:无菌水对照组和抗菌肽处理组,每组15个果实;每组果实以2%次氯酸钠溶液浸泡2 min后清洗以除去表面残留污物,果实表面赤道部位用75%酒精擦拭消毒后,用灭菌打孔器在果实赤道部位等距打两个孔(深3 mm,直径4 mm);无菌水对照组接种10 μL无菌水于每孔中;抗菌肽处理组将新鲜P. digitatum孢子悬浮液与抗菌肽溶液混合后立即接种或16 h后接种10 μL于每孔中,P. digitatum孢子和抗菌肽的终浓度分别为1×104 CFU/mL和100 μmol/L;待孔中液体被完全吸收后,用聚乙烯薄膜袋(170 mm×140mm)将果实单果包装,置于25℃、相对湿度为90-95%的环境中贮藏;每天统计发病率并测量病斑直径。实验重复3次。
2、不同温度对抗菌肽抑菌活性的影响
将不同浓度的抗菌肽溶液分别在40℃、50℃、60℃、70℃、80℃静置5 min后快速冷却至常温(25℃),以常温(25℃)静置未加热的抗菌肽溶液为对照;将180 μL含有20倍稀释马铃薯葡萄糖液体培养基(5% PDB)的P. digitatum孢子悬浮液和20 μL抗菌肽溶液混合后立即加入无菌96孔微量板中,P. digitatum孢子的终浓度为1×104 CFU/mL,抗菌肽的终浓度分别为0、3.12、6.25、12.5、25、50、100 μmol/L;然后将96孔板置25℃恒温培养箱中培养,48 h时用酶标仪测定其OD600值,以48 h时依旧能完全抑制病原菌生长的最低浓度作为最低抑菌浓度(MIC)。实验重复3次。
3、不同pH对抗菌肽抑菌活性的影响
将抗菌肽溶于pH值分别为3.0、4.0、5.0、6.0、7.0、8.0、9.0、10.0的无菌水中,以不调节pH的无菌水为对照,室温静置1 h后,调节pH值为7.0,再用无菌水将抗菌肽溶液稀释,进行最低抑菌浓度测定试验。
4、金属离子对抗菌肽抑菌活性的影响
分别用经121℃灭菌20 min的不同浓度的KCl溶液(5、20、35、50 mmol/L)或NaCl溶液(50、100、150、200 mmol/L)溶解抗菌肽,以无菌水为对照,进行最低抑菌浓度测定试验。
三、实验结果
1、抗菌肽对柑橘果实采后绿霉病的控制作用
首先采用抗菌肽与病原菌混合后立即接种的方式,考察了七种抗菌肽(O3TR、C12O3TR、dhvar5、hLF(1-11)、LDP-NLS、Jelleine-I、Osmin)对柑橘果实采后绿霉病的控制作用。结果如图1和图2所示,七种抗菌肽对柑橘果实采后绿霉病的控制作用存在较大差异,其中,抗菌肽O3TR、C12O3TR和Jelleine-I能有效控制柑橘果实采后绿霉病的发生,而抗菌肽dhvar5、hLF(1-11)、LDP-NLS和Osmin对柑橘果实采后绿霉病无明显控制作用。
其次分别以抗菌肽与病原菌混合后立即接种和混合16 h后接种这两种接种方式,对比了抗菌肽O3TR、C12O3TR、Jelleine-I与文献报道的能有效控制柑橘果实采后绿霉病的抗菌肽PAF56、BP21在瞬时杀菌和长效杀菌两方面的能力。混合后立即接种的对比结果如图3A、3B和图4所示,抗菌肽O3TR、C12O3TR和Jelleine-I的控病效果都显著好于抗菌肽PAF56和BP21,其中经C12O3TR处理后,柑橘果实一直不发病,说明抗菌肽O3TR、C12O3TR和Jelleine-I的瞬时杀菌能力优于抗菌肽PAF56和BP21。混合16 h后接种的对比结果如图3C、3D和图4所示,抗菌肽PAF56的控病效果最差,抗菌肽BP21、O3TR、C12O3TR和Jelleine-I的控病效果无显著性差异(P < 0.05),说明抗菌肽O3TR、C12O3TR和Jelleine-I的长效杀菌能力优于抗菌肽PAF56。
2、不同温度对抗菌肽抑菌活性的影响
结果如图5所示,抗菌肽O3TR、C12O3TR和Jelleine-I对P. digitatum的生长抑制作用均不因抗菌肽所处温度变化而改变,说明这三种抗菌肽的抑菌活性具有热稳定性。抗菌肽O3TR、C12O3TR和Jelleine-I的MIC分别为12.5、6.25和6.25μmol/L。
3、不同pH对抗菌肽抑菌活性的影响
结果如图6所示,抗菌肽O3TR、C12O3TR和Jelleine-I在不同pH条件下均能抑制P. digitatum的生长且MIC值不发生变化,说明这三种抗菌肽的抑菌活性具有酸碱稳定性。
4、金属离子对抗菌肽抑菌活性的影响
结果如图7所示,在不同浓度的NaCl溶液中,抗菌肽C12O3TR和Jelleine-I对P. digitatum的生长抑制能力不发生改变,MIC值不变;抗菌肽O3TR在高浓度NaCl溶液(150、200 mmol/L)中抑菌能力下降,MIC值均为25μmol/L;在不同浓度的KCl溶液中,抗菌肽O3TR、C12O3TR和Jelleine-I均能保持对P. digitatum的生长抑制能力,MIC值不发生变化;说明抗菌肽C12O3TR和Jelleine-I的抑菌能力不受Na+、K+的影响。
最后说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管通过参照本发明的优选实施例已经对本发明进行了描述,但本领域的普通技术人员应当理解,可以在形式上和细节上对其作出各种各样的改变,而不偏离所附权利要求书所限定的本发明的精神和范围。
序列表
<110> 西南大学
<120> 抗菌肽O3TR和C12O3TR在制备防治柑橘果实采后绿霉病的药物中的应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> SITE
<222> (1,2)
<223> Xaa=Orn
<400> 1
Xaa Xaa Trp Trp
1
<210> 2
<211> 14
<212> PRT
<213> 智人(Homo sapiens)
<400> 2
Leu Leu Leu Phe Leu Leu Lys Lys Arg Lys Lys Arg Lys Tyr
1 5 10
<210> 3
<211> 11
<212> PRT
<213> 智人(Homo sapiens)
<400> 3
Gly Arg Arg Arg Arg Ser Val Gln Trp Cys Ala
1 5 10
<210> 4
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Lys Trp Arg Arg Lys Leu Lys Lys Leu Arg Pro Lys Lys Lys Arg Lys
1 5 10 15
Val
<210> 5
<211> 8
<212> PRT
<213> 意大利蜜蜂(Apis mellifera)
<400> 5
Pro Phe Lys Leu Ser Leu His Leu
1 5
<210> 6
<211> 17
<212> PRT
<213> 红壁蜂(Osmia rufa)
<400> 6
Gly Phe Leu Ser Ala Leu Lys Lys Tyr Leu Pro Ile Val Leu Lys His
1 5 10 15
Val
<210> 7
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Gly His Arg Lys Lys Trp Phe Trp
1 5
<210> 8
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Phe Lys Leu Phe Lys Lys Ile Leu Lys Val Leu
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Claims (2)
1.抗菌肽O3TR在制备防治柑橘果实采后绿霉病的药物中的应用;所述抗菌肽O3TR的氨基酸序列如SEQ ID No.1所示,其C端酰胺化修饰。
2.抗菌肽C12O3TR在制备防治柑橘果实采后绿霉病的药物中的应用;所述抗菌肽C12O3TR的氨基酸序列如SEQ ID No.1所示,其N端十二烷酰化修饰,C端酰胺化修饰。
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