CN113957077B - 一种剑麻半胱氨酸蛋白酶抑制剂基因及应用 - Google Patents
一种剑麻半胱氨酸蛋白酶抑制剂基因及应用 Download PDFInfo
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Abstract
本发明公开了一种剑麻半胱氨酸蛋白酶抑制剂基因及应用，涉及到生物工程领域，所述剑麻半胱氨酸蛋白酶抑制剂基因的核苷酸序列如SEQ No.1所示，由上述基因编码的蛋白的氨基酸序列如SEQ No.2所示。本发明获得的剑麻半胱氨酸蛋白酶抑制剂蛋白具有抗菌生物活性，可用于制备抗菌类产品，应用于作物病害防控，可抑制烟草疫霉、芒果炭疽病菌和西瓜枯萎病菌生长。
Description
技术领域
本发明涉及生物工程领域，特别涉及一种剑麻半胱氨酸蛋白酶抑制剂基因及应用。
背景技术
剑麻是一种硬质叶片纤维作物，在热带亚热带地区广泛种植。其用途较为广泛，主要涉及渔业、航海、航天、工矿、运输、油田、纺织等行业。剑麻在生长过程中常见病虫害较多，主要有剑麻斑马纹病、紫色卷叶病、茎腐病、红蜘蛛、新菠萝灰粉蚧等，病虫害爆发导致的剑麻纤维减产对剑麻产业影响较大。化学防治目前仍是剑麻病虫害防治的主要手段，但其对环境生态影响较大，因此，探索绿色高效的病虫害防治技术对剑麻产业可持续发展至关重要。
半胱氨酸蛋白酶抑制剂(Cysteine proteinase inhibitor，CPI)，也被称为巯基蛋白酶抑制剂，可特异性结合半胱氨酸蛋白酶，通过抑制酶活性保护蛋白质中的二硫键免遭破坏，进而保持相应蛋白功能完整性以调节生命进程。植物半胱氨酸蛋白酶抑制剂基因已在多种植物中被报道，例如水稻、大麦、小麦、玉米、拟南芥、豌豆和番茄等。CPI基因的表达受植物生长调节剂、机械损伤、逆境胁迫等因素影响。
目前已证实CPI基因在提高植物抗病性方面具有重要作用，国内外对于植物半胱氨酸蛋白酶抑制剂基因研究报道较多，但剑麻中尚未见报道。因此，发明一种剑麻半胱氨酸蛋白酶抑制剂基因及应用来解决上述问题很有必要。
发明内容
本发明的目的在于提供一种剑麻半胱氨酸蛋白酶抑制剂基因及应用，以解决上述背景技术中提出的问题。
为实现上述目的，本发明提供如下技术方案：
一种剑麻半胱氨酸蛋白酶抑制剂基因，核苷酸序列如SEQ No.1所示，具体如下：
ATGAAGGCTAGTTCTCTTGTTCTTCTTCTTCTTGCTTCAACTTTCATGCTTGCCAATTTATGCTCTGCTTCAAGAGGCTCTGGTCCAATGGTTGGGGGATGGAGCACAATCAAGAACATGAGTGACCCACATATTGCAGAGATTGGGGAGTTTGCAATCTCTGAGCACAACAAGGAGACCAACTCCAGGCTTGCATTCAACAGAGTGATCAAGGGTAAGATCCAAGTTGTGGCTGGTTTCAATTACAAGCTTGTTATTGAGTCCAAGGATGGGAATAAAGTTAGGAAGTATGAGGCAGTTGTTTGGGAGAAAGTTTGGGAGAATTTCTTGAAGCTTACTTCCTTCAAGCCTCTTAAGATCTGA
一种剑麻半胱氨酸蛋白酶抑制剂蛋白，氨基酸序列如SEQ No.2所示，具体如下：
MKASSLVLLLLASTFMLANLCSASRGSGPMVGGWSTIKNMSDPHIAEIGEFAISEHNKETNSRLAFNRVIKGKIQVVAGFNYKLVIESKDGNKVRKYEAVVWEKVWENFLKLTSFKPLKI
进一步的，所述一种剑麻半胱氨酸蛋白酶抑制剂蛋白，由所述SEQ No.1序列导入原核表达载体，通过原核表达获得剑麻半胱氨酸蛋白酶抑制剂蛋白。
进一步的，所述剑麻半胱氨酸蛋白酶抑制剂基因或所述剑麻半胱氨酸蛋白酶抑制剂蛋白应用于抗真菌类产品。
进一步的，所述剑麻半胱氨酸蛋白酶抑制剂基因或所述剑麻半胱氨酸蛋白酶抑制剂蛋白应用于作物真菌病害防控。
进一步的，所述剑麻半胱氨酸蛋白酶抑制剂基因或所述剑麻半胱氨酸蛋白酶抑制剂蛋白应用于抑制烟草疫霉、芒果炭疽病菌和西瓜枯萎病菌生长。
进一步的，一种抗真菌类产品或用于作物真菌病害防控产品或用于抑制烟草疫霉、芒果炭疽病菌和西瓜枯萎病菌生长产品，含有所述的剑麻半胱氨酸蛋白酶抑制剂蛋白。
本发明还提供了上述技术方案所述的蛋白在制备抗真菌类产品上的应用，所述抗真菌类产品包括含剑麻半胱氨酸蛋白酶抑制剂蛋白的平衡缓冲液。
本发明的技术效果和优点：
本发明涉及的基因及蛋白质序列与已公布的植物半胱氨酸蛋白酶抑制剂存在明显差异，其核苷酸序列长度为363bp，氨基酸序列长度为120。
本发明获得的剑麻半胱氨酸蛋白酶抑制剂蛋白具有抗菌生物活性，可抑制烟草疫霉、芒果炭疽病菌和西瓜枯萎病菌生长，可用于制备抗菌类产品，应用于作物病害防控。
附图说明
图1为剑麻半胱氨酸蛋白酶抑制剂基因原核表达载体琼脂糖凝胶电泳验证结果示意图；
图2为抑菌实验验证剑麻半胱氨酸蛋白酶抑制剂蛋白对烟草疫霉抑制效果示意图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明提供了如图1-2所示的一种剑麻半胱氨酸蛋白酶抑制剂基因，该基因通过对剑麻转录组数据库进行序列比对而获得，剑麻半胱氨酸蛋白酶抑制剂基因的核苷酸序列如SEQ No.1所示，具体如下：
ATGAAGGCTAGTTCTCTTGTTCTTCTTCTTCTTGCTTCAACTTTCATGCTTGCCAATTTATGCTCTGCTTCAAGAGGCTCTGGTCCAATGGTTGGGGGATGGAGCACAATCAAGAACATGAGTGACCCACATATTGCAGAGATTGGGGAGTTTGCAATCTCTGAGCACAACAAGGAGACCAACTCCAGGCTTGCATTCAACAGAGTGATCAAGGGTAAGATCCAAGTTGTGGCTGGTTTCAATTACAAGCTTGTTATTGAGTCCAAGGATGGGAATAAAGTTAGGAAGTATGAGGCAGTTGTTTGGGAGAAAGTTTGGGAGAATTTCTTGAAGCTTACTTCCTTCAAGCCTCTTAAGATCTGA
本发明还提供了上述技术方案的剑麻半胱氨酸蛋白酶抑制剂基因编码的蛋白，该蛋白通过将SEQ No.1序列导入原核表达载体，以大肠杆菌为宿主细胞，通过原核表达而获得，蛋白的氨基酸序列如SEQ No.2所示，具体如下：
MKASSLVLLLLASTFMLANLCSASRGSGPMVGGWSTIKNMSDPHIAEIGEFAISEHNKETNSRLAFNRVIKGKIQVVAGFNYKLVIESKDGNKVRKYEAVVWEKVWENFLKLTSFKPLKI
本发明还提供了上述技术方案的蛋白在制备抗真菌类产品上的应用，抗真菌类产品包括含剑麻半胱氨酸蛋白酶抑制剂蛋白的平衡缓冲液。
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例一
制备剑麻半胱氨酸蛋白酶抑制剂蛋白
本发明涉及的剑麻半胱氨酸蛋白酶抑制剂原核表达，包括以下步骤：
步骤一：将已公布的芦笋半胱氨酸蛋白酶抑制剂基因CPI1(NCBI序列号：XM_020388810.1)作为参考序列，在剑麻转录组数据库(SRA accession：PRJNA432160)中进行Blast比对，获得如SEQ No.1所示DNA片段，以SEQ No.1所示DNA片段为模板设计引物，通过实时定量PCR验证其表达模式，引物序列如下：
正向引物：5’-ATGACTGTGTTTATTTCTTGT-3’；
反向引物：5’-TCAACAAGGTCTAGTGCAGAA-3’。
采用刺伤接种法接种剑麻斑马纹病病原烟草疫霉，对照处理仅刺伤不接种病原，接种5d后取样。通过天根植物总RNA提取试剂盒(天根生化科技,中国北京)提取样品RNA，并通过GoScript Reverse Transcription System(Promega,美国)将其反转录为cDNA。通过QuantStudio 6实时荧光定量PCR系统(赛默飞,美国)对剑麻半胱氨酸蛋白酶抑制剂基因进行实时定量PCR验证。
扩增体系为20μL,其中包括0.5μL正向引物、0.5μL反向引物、1μLcDNA模板、10μLTransStart Tip Green qPCR Supermix(全式金,中国)、0.4μLPassive Ref-erence Dye(全式金,中国)和7.6μL双蒸水。
反应程序为94℃30s；94℃3min,94℃10s,58℃30s,共40个循环；溶解曲线循环。
以剑麻PP2A基因(protein phosphatase 2A)为内参基因,每个样品重复3次作为技术重复,结果用2-ΔΔCt法进行相对定量分析，结果显示剑麻半胱氨酸蛋白酶抑制剂基因在烟草疫霉侵染后显著上调，表明其参与了剑麻抗病应答。
步骤二：然后进行剑麻半胱氨酸蛋白酶抑制剂基因全基因合成，并将其导入
-Blunt E1 Expression Vector原核表达载体，采用热激法将原核表达载体导入BL21(DE3)Chemically Competent Cell感受态细胞，LB固体培养基平板孵育12小时候后挑取单克隆用于PCR检测及LB液体培养基培养。
步骤三：通过PCR检测其是否插入到原核表达载体，引物序列与步骤一相同。
20μl反应体系包括：10μl
PCR SuperMix(全式金，中国)、10pmol正向反向特异引物各1μl、含原核表达载体的菌液1μl、7μl ddH2O。
反应程序为：首先95℃变性5min，然后以“95℃变性15s、60℃变性15s、72℃变性15s”为条件循环扩增30次，最后72℃变性2min。
如图1所示，琼脂糖凝胶电泳检测显示PCR产物长度约为360bp，符合预期，对PCR产物进行Sanger测序，测序结果与SEQ No.1所示序列一致。
步骤三：将含有原核表达载体的BL21感受态细胞在LB液体培养基培养24小时，超声波破碎后通过
Ni-NTA Resin蛋白质纯化试剂盒(全式金，中国)提取剑麻半胱氨酸蛋白酶抑制剂蛋白。
实施例二
剑麻半胱氨酸蛋白酶抑制剂蛋白在制备抗真菌类产品上的应用
将烟草疫霉、芒果炭疽病菌和西瓜枯萎病菌分别接种在PDA培养基上，将菌块接种于培养皿正中央，同时将牛津杯置于菌块四周，将平衡缓冲液(300mM NaCl、50mM NaH2PO4、10mM imidazole、10mM Tris base，pH8.0)和含剑麻半胱氨酸蛋白酶抑制剂蛋白(100μg/mL)的平衡缓冲液分别加入到接种了三种真菌培养皿的牛津杯中，28℃培养4天后观察菌落生长情况。
结果如图2所示，平衡缓冲液(图2左)对烟草疫霉无抑制作用，含剑麻半胱氨酸蛋白酶抑制剂蛋白的平衡缓冲液使烟草疫霉菌落在牛津杯边缘产生弧状抑制区，表明剑麻半胱氨酸蛋白酶抑制剂蛋白对烟草疫霉具抑制作用。对剑麻半胱氨酸蛋白酶抑制剂蛋白的抑菌作用进行量化统计，结果显示其对烟草疫霉、芒果炭疽病菌和西瓜枯萎病菌的抑菌率分别为61.75％、50.65％和56.05％，表明其对三种真菌具抑制作用。
综上所述，剑麻半胱氨酸蛋白酶抑制剂蛋白可用于制备抗菌类产品，应用于作物病害防控。
最后应说明的是：以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换,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。
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Claims (7)
1.一种剑麻半胱氨酸蛋白酶抑制剂基因，其特征在于：所述剑麻半胱氨酸蛋白酶抑制剂基因的核苷酸序列如SEQ No.1所示。
2.一种剑麻半胱氨酸蛋白酶抑制剂蛋白，其特征在于：所述剑麻半胱氨酸蛋白酶抑制剂蛋白的氨基酸序列如SEQ No.2所示。
3.根据权利要求2所述一种剑麻半胱氨酸蛋白酶抑制剂蛋白，其特征在于：由权利要求1所述SEQ No.1序列导入原核表达载体，通过原核表达获得剑麻半胱氨酸蛋白酶抑制剂蛋白。
4.根据权利要求1所述剑麻半胱氨酸蛋白酶抑制剂基因或权利要求2所述剑麻半胱氨酸蛋白酶抑制剂蛋白应用于抗真菌类产品。
5.根据权利要求1所述剑麻半胱氨酸蛋白酶抑制剂基因或权利要求2所述剑麻半胱氨酸蛋白酶抑制剂蛋白应用于作物真菌病害防控。
6.根据权利要求1所述剑麻半胱氨酸蛋白酶抑制剂基因或权利要求2所述剑麻半胱氨酸蛋白酶抑制剂蛋白应用于抑制烟草疫霉、芒果炭疽病菌和西瓜枯萎病菌生长。
7.一种抗真菌类产品或用于作物真菌病害防控产品或用于抑制烟草疫霉、芒果炭疽病菌和西瓜枯萎病菌生长产品，其特征在于：含有权利要求2所述的剑麻半胱氨酸蛋白酶抑制剂蛋白。
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| WO1997014797A2 (en) * | 1995-10-20 | 1997-04-24 | Dana-Farber Cancer Institute | Cystatin m, a novel cysteine proteinase inhibitor |
| CN101186916A (zh) * | 2007-11-20 | 2008-05-28 | 青岛大学 | 一种编码双齿围沙蚕半胱氨酸蛋白酶抑制剂的基因序列及其氨基酸序列与应用 |
| CN106636122A (zh) * | 2017-01-03 | 2017-05-10 | 内蒙古农业大学 | 斯氏副柔线虫半胱氨酸蛋白酶抑制剂Pj_CPI基因克隆、重组表达方法及应用 |
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2021
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| WO1997014797A2 (en) * | 1995-10-20 | 1997-04-24 | Dana-Farber Cancer Institute | Cystatin m, a novel cysteine proteinase inhibitor |
| CN101186916A (zh) * | 2007-11-20 | 2008-05-28 | 青岛大学 | 一种编码双齿围沙蚕半胱氨酸蛋白酶抑制剂的基因序列及其氨基酸序列与应用 |
| CN106636122A (zh) * | 2017-01-03 | 2017-05-10 | 内蒙古农业大学 | 斯氏副柔线虫半胱氨酸蛋白酶抑制剂Pj_CPI基因克隆、重组表达方法及应用 |
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