致病疫霉木瓜蛋白酶亚家族基因C1A-PH-SCH1的克隆与表达分析

刘霞, 张哲, 钱红洁, 张利杰, 杨艳丽

中国农学通报. 2021, 37(29): 13-19

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中国农学通报 ›› 2021, Vol. 37 ›› Issue (29) : 13-19. DOI: 10.11924/j.issn.1000-6850.casb2020-0693
生物科学

致病疫霉木瓜蛋白酶亚家族基因C1A-PH-SCH1的克隆与表达分析

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Cloning and Expression Analysis of the Papain-like Cysteine Proteases Subfamily Gene C1A-PH-SCH1 of Phytophthora Infestans

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摘要

为进一步明确引起马铃薯晚疫病的致病因子,通过克隆马铃薯晚疫病致病疫霉菌分泌蛋白中的半胱氨酸蛋白酶家族基因,构建该基因的原核表达载体,在大肠杆菌BL21中诱导表达。本试验采用TA克隆法,提取来自云南马铃薯产区的致病疫霉菌XH05-5-4的总RNA,反转录cDNA,PCR扩增后将该基因片段连接到载体,转化大肠杆菌BL21并诱导表达。克隆获得半胱氨酸蛋白酶家族基因全长为1176 bp,包含1个最大的开放阅读框1077 bp,编码387个氨基酸。推测蛋白相对分子量41.671 kD,理论等电点4.88;经IPTG诱导和SDS-PAGE检测,所构建的原核表达载体表达的蛋白与预期蛋白相符合。生物活性测定表明:马铃薯叶片出现坏死,类似于晚疫病症状。利用TA克隆技术从致病疫霉菌XH05-5-4中克隆得到了1个半胱氨酸蛋白酶家族基因(登录号:KP938956命名:C1A-PH-SCH1),通过同源性比对发现C1A-PH-SCH1为木瓜蛋白酶亚家族基因,成功构建了原核表达载体,并使其在大肠杆菌中得到了表达,对生物活性测定结果显示出现了类似于晚疫病症状的水渍坏死斑症状,推测C1A-PH-SCH1参与马铃薯晚疫病的发病过程。

Abstract

To further clarify pathogenic factor of potato late blight, we cloned the cysteine protease family gene in the secreted protein of Phytophthora infestans causing potato late blight and express in E. coli BL21. In this study, TA cloning method was used to extract total RNA of P. infestans XH05-5-4 of Yunnan, synthesize cDNA by reverse transcription and connect gene fragment to the vector after PCR amplification. Then we induced the expression of it in E. coli BL21. The full length of cysteine protease family gene is 1176 bp, containing 1077 bp open reading frame (ORF) encoding 387 amino acids. Predicted by bioinformatics analysis, its protein molecular weight is 41.671 KDa, isoelectric point is 4.88. After IPTG induced expression and SDS-PAGE identification, the protein expressed in the constructed prokaryotic expression vector is consistent with the expected protein. The results of biological activity examination show that potato leaves have necrosis and this symptom is similar to potato late blight. A cysteine protease family gene (GenBank Accession: KP938956, name: C1A-PH-SCH1) was cloned from P. infestans XH05-5-4 by TA cloning method. It is a papain enzyme subfamily gene by homology comparison, and the prokaryotic expression vector is successfully constructed and expressed in E. coli BL21. The symptom is similar to watery necrosis of potato late blight, therefore, it is speculated that C1A-PH-SCH1 is involved in the pathogenic process of potato late blight.

关键词

马铃薯 / 致病疫霉 / 木瓜蛋白酶 / C1A-PH-SCH1基因 / 克隆

Key words

potato / Phytophthora infestans / papain-like cysteine proteases / C1A-PH-SCH1 gene / clone

引用本文

导出引用
刘霞 , 张哲 , 钱红洁 , 张利杰 , 杨艳丽. 致病疫霉木瓜蛋白酶亚家族基因C1A-PH-SCH1的克隆与表达分析. 中国农学通报. 2021, 37(29): 13-19 https://doi.org/10.11924/j.issn.1000-6850.casb2020-0693
Liu Xia , Zhang Zhe , Qian Hongjie , Zhang Lijie , Yang Yanli. Cloning and Expression Analysis of the Papain-like Cysteine Proteases Subfamily Gene C1A-PH-SCH1 of Phytophthora Infestans. Chinese Agricultural Science Bulletin. 2021, 37(29): 13-19 https://doi.org/10.11924/j.issn.1000-6850.casb2020-0693

0 引言

马铃薯晚疫病是马铃薯生产的主要病害之一,每年都有不同程度的发生和流行,已成为制约马铃薯产业发展的因素[1]。近几年来,全球每年因致病疫霉菌引起的马铃薯晚疫病所造成的损失不可估量[2],而云南省是中国马铃薯主产地之一,其致病疫霉菌具有多样性、致病性强等特点。目前对马铃薯晚疫病菌的致病机理也未完全了解,基于此,探索由致病疫霉引起的马铃薯晚疫病致病机制,为以后防治晚疫病奠定基础。半胱氨酸蛋白酶是一类在酶的活性中心含有半胱氨酸并受巯基反应基不可逆抑制的蛋白水解酶,广泛存在于人类、哺乳动物、植物、原生动物、真菌、细菌、病毒等生物体内,共有49个家族[3]。植物中大部分属于木瓜蛋白酶亚家族和豆类天冬氨酸蛋白内切酶亚家族,还有天冬氨酸特异性的半胱氨酸蛋白酶亚家族和钙依赖半胱氨酸蛋白酶亚家族,之后还发现催化蛋白去泛素化的泛素类半胱氨酸蛋白酶和蛋白酶体[4,5]。半胱氨酸蛋白酶参与植物中某些细胞沉积和降解贮存蛋白,非生物胁迫和生物胁迫的响应,启动子中常含有胁迫响应元件,但在马铃薯(Solanum tuberosum)中发现tdi-65只受干旱诱导,而不受ABA诱导[6]。半胱氨酸蛋白酶是植物体重要的蛋白水解酶[4,7],广泛参与对生物和非生物胁迫的响应[8]、参与植物体的衰老和细胞程序性死亡[9,10]、贮存蛋白的沉积和降解[11,12]等生理过程。半胱氨酸蛋白酶也参与植物生物节律的调节,近年来发现可能具有转录因子的作用。木瓜蛋白酶(Papain-like cysteine proteases, PLCPs)是C1A家族蛋白酶,是其中最具代表性、研究最为广泛深入的一种蛋白酶[13]。最初是在木瓜的乳汁和果实中得到的4类木瓜蛋白酶,随后在病毒、细菌、原生动物、真菌、动物和植物中被发现。这类蛋白酶在酸性pH条件下发挥活性。在植物半胱氨酸蛋白酶中,papain蛋白酶研究较多。其中拟南芥发现有32个编码papain型的半胱氨酸蛋白酶,主要分为:aleurain型、KDEL型、类actinidain型、类菠萝蛋白酶型、末端序列型、类组织蛋白酶B型、受衰老和逆境诱导型[14]。在酶前体会形成α螺旋和β折叠结构域,结构域之间会构成裂隙,裂隙底部为催化活性位点[15],在保守区内其4个催化活性位点分别是Gln-Cys-His-Asn/Asp[16,17],这也是papain蛋白酶的结构特点。酶前体分为2种,一种有EX3RX3FX2NX3IX3N(ERFNIN)花式,另一种无花式,还有的成员N端酶前体序列中含有液泡靶向信号NPIR,papain型绕过高尔基体而与蛋白贮存液泡直接融合[18]。据不完全统计,目前已发表超过50个类papain植物蛋白酶,对甜荞半胱氨酸蛋白酶基因研究发现,该基因能被干旱、高盐、ABA和衰老胁迫诱导[19],但在致病疫霉中未见报道。实验室前期研究以云南省致病疫霉菌不同生理小种的3个菌种为试验材料,通过提取纯化3个致病疫霉菌的代谢产物,进行双向电泳检测,用液质联用质谱分析,得到了3个菌种共有的16个蛋白[20]。本试验对其中假定能引起毒害的登录号为gi|262108704的半胱氨酸蛋白酶家族基因进行克隆及基因功能分析,对原核表达产物活性进行测定,并明确原核表达蛋白能否引起马铃薯叶片的坏死,确定该蛋白是否参与马铃薯晚疫病的侵染。

1 材料与方法

1.1 试验材料

1.1.1 马铃薯品种 供试马铃薯品种为‘大西洋’,植株不抗晚疫病。
1.1.2 病菌样本 本研究使用的病菌样本为XH05-5-4,其生理小种为2.4.8.9.10,由云南农业大学马铃薯研究室保存提供。
1.1.3 分子生物学试剂 反转录试剂盒TransScript® II Frist-Stand cDNA Synthesis SuperMix、克隆载体pEASY®-T1 Cloning Kit、原核表达载体pEASY®-E1 Expression Kit均购自北京全式金生物公司,切胶回收试剂盒TARAKA MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0购自大连宝生物工程公司,高纯度质粒小提中量试剂盒购自天根生化科技有限公司,其他试剂均为国产分析纯。

1.2 试验方法

1.2.1 半胱氨酸蛋白酶家族 C1A基因克隆ZH05-5-4菌株总RNA按照Trizol试剂说明进行,反转录按照试剂盒说明书进行。以蛋白编号为gi|262108704(登录号NW_003303706)的核苷酸序列为模版,利用Primer5.0软件设计引物F(5’-TGTTCAAGGCTGAGGGTG-3’)、R(5’-AGTTGGCTACATTCCATTCG-3’),PCR反应条件为94℃变性7 min,94℃ 40 s,57.4℃ 40 s,72℃ 1 min,35个循环,72℃延伸10 min。PCR产物回收按照试剂盒进行,并连接到pEASY®-T1载体上,转入大肠杆菌Trans1-T1感受态细胞,经氨苄青霉素、X-gel筛选,随机选取独立的阳性克隆子,用通用引物做菌落PCR检测,并送上海华大基因公司测序鉴定,对样品进行备份。
1.2.2 序列分析 用ProtParam (http://www.expasy.org/tools/protparam.html)程序预测等电点及分子量;NetNGlyc Server (http://www.cbs.dtu.dk/services/NetNGlyc)工具预测氨基酸序列中可能存在的糖基化位点[20,21];ProtScale预测不同肽段的亲水性疏水性变化;NPS@( https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopm.html)对蛋白质二级结构进行预测[22];用TMHMM2.0(http://www.cbs.dtu.dk/services/TMHMM2.0)工具预测蛋白质跨膜结构,SingalP预测信号肽(http://www.cbs.dtu.dk/services/SignalP/)[20]
1.2.3 原核表达 将反转录cDNA的切胶回收产物和pEASY®-E1 Expression Kit进行连接,转化到感受态细胞Trans1-T1,经抗性筛选后挑取阳性单克隆子,接种至含Amp抗性的LB培养基中,培养至A600为0.5~0.8时提取质粒,导入BL21中,菌落PCR检测,取1 mL作为未诱导的阴性对照,同时建立空载体对照,向剩余培养液加入IPTG(终质量浓度为1 mg),37℃、220 r/min分别诱导2、4、6、12 h后各取菌液1 mL,离心10 min收集菌体,加入1 mL SDS凝胶加样缓冲液重悬,沸水浴,取上清液SDS-PAGE检测表达产物诱导情况,将诱导后的样品离心后重溶于PBS缓冲液,超声波破碎,再离心后留上清液备用。将菌液接种马铃薯品种‘大西洋’离体叶片,以无菌水和空载体作为对照组,对蛋白生物活性进行测定。

2 结果与分析

2.1 半胱氨酸蛋白酶家族C01A基因克隆

2.1.1 PCR扩增产物 用1%琼脂糖凝胶电泳检测,得到长约1000 bp的cDNA片段,切胶回收后连接克隆载体pEASY®-T1后进行菌落PCR检测,获得目的片段(图1)。片段长度为1176 bp,最大的开放阅读框1077 bp(图2),编码387个氨基酸。蛋白相对分子量41.671 kD,等电点4.88,说明蛋白呈酸性,该基因含Ser(S)最多,占10.0%,不含Asx(B)、Glx(Z)、Xaa(X)。总的带正电点残基(Arg+Lys)为29,负电残基(Arg+Glu)为41,其脂溶指数为75.77,不稳定系数41.91,为不稳定蛋白。
图1 PCR扩增图谱 M:DNA marker(DL2000);1:目的片段

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图2 半胱氨酸蛋白酶基因最大ORF核苷酸序列及其编码的氨基酸序列
绿色:起始密码子和终止密码子;蓝色:活性位点Cys,His,Asn和Gln;红色:N-糖基化位点

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2.1.2 同源性分析 经BLAST分析,半胱氨酸蛋白酶家族C01A基因核苷酸编码区序列与Genbank登录号为XP_002896821.1的核苷酸序列同源性为99%,与登录号为XM_008906598.1的核苷酸同源性为86%,与登录号为XM_004348520.1的核苷酸同源性为90%。对半胱氨酸蛋白酶家族C01A基因的氨基酸序列进行BlastP分析发现其在保守区域与其他半胱氨酸蛋白酶具有较高的相似性,其中与Phytophthora infestans T30-4 cysteine protease family C01A同源性最高,利用SMART在线工具对蛋白质的结构域进行预测,发现C1A-PH-SCH1编码的氨基酸在143~357位具有Pept-C1结构域,可推断C1A-PH-SCH1属于木瓜蛋白酶亚家族。

2.2 生物学信息学分析

2.2.1 信号肽预测 利用SingaIP进行信号肽预测显示,蛋白第1~18个氨基酸残基为其信号肽(图3),信号肽的裂解位点在第18位的丙氨酸。
图3 信号肽预测

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2.2.2 糖基化位点分析 利用NetNGlyc 1.0 Server预测其在第138、283位含有N-糖基化位点。
2.2.3 亲水性疏水性分析 利用ProtScale预测不同肽段的亲水性疏水性变化,结果表明:第5~23位的氨基酸均为疏水性,而第8、9位的亮氨酸(L)疏水性最强,第287位的Gln(Q)亲水性最强,但亲水性氨基酸多于疏水性氨基酸,整个蛋白可能表现亲水性(图4)。
图4 亲水性/疏水性预测结构

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2.2.4 跨膜结构预测 TMHMM2.0预测该蛋白有跨膜螺旋结构,具有跨膜结构(图5)。
图5 跨膜螺旋结构预测

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2.2.5 蛋白二级结构预测 通过NPS@对基因进行蛋白质二级结构预测,结果显示:α-螺旋(H)含量为28.21%;无规则卷曲(C)含量为41.62%;而β转角(T)含量仅为9.78%;延伸链€含量为20.39%(图6)。
图6 二级结构

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2.3 原核表达载体的检测

目的片段与表达载体pEASY®-E1连接后导入T1感受态中,经涂板挑取阳性菌导入BL21后,经终浓度为1 mmol/L IPTG,37℃下分别诱导2、4、6、12 h重组蛋白的表达,诱导完成后收集菌丝,对上清进行SDS-PAGE电泳检测(图7),以空载体和没有经IPTG诱导的菌液蛋白做对照,结果表明,在诱导6 h后,发现41.67 kDa有1条明显条带,对照组不明显,表明外源蛋白在大肠杆菌中得到表达,原核表达载体构建成功。
图7 IPTG诱导大肠杆菌表达C1A-PH-SCH1重组蛋白的SDS-PAGE电泳结果

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2.4 外源表达蛋白生物活性测定

将原核表达的蛋白稀释成3个浓度接种马铃薯品种‘大西洋’的离体叶片,1周后供试叶片出现与晚疫病症状类似的坏死斑,对照组正常(图8)。因此可以推测C1A-PH-SCH1参与马铃薯晚疫病的发病过程。
图8 生物活性测定

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3 讨论

木瓜蛋白酶参与了植物对逆境的反应[20,21,24],植物衰老[22,25]及植物细胞程序化死亡[23,26],Lee等发现OsCP1基因参与水稻雄性不育过程[24,27];zhang等[25,28]在烟草花药中反义抑制NtCP56的表达,导致烟草花粉粒败育;严秀蕊等从水稻中克隆了OsCP2基因[26,29];王高峰在马铃薯腐烂茎线虫中克隆了L型半胱氨酸蛋白酶[27,30];张晓梅等发现NtCP56在反义RNA介导的转基因烟草花药中绒毡层的降解延迟从而导致花粉发育异常[25,28];赵冬梅等报道PcFiSCR.1对马铃薯和烟草叶片有毒害作用,而PcFiSCR.1富含小的半胱氨酸的分泌蛋白,含有保守的半胱氨酸序列和N-末端信号肽[28,31],还有大量报道称半胱氨酸蛋白酶家族是植物的凋亡或编程性细胞死亡的效应器等,在植物中半胱氨酸蛋白酶能引起植物的坏死,但在致病疫霉菌中未见有相关报道。所以本试验对致病疫霉中的分泌蛋白进行了生物活性测定,重组蛋白诱导表达结果显示当pEASY-E1在BL21 DE3中表达,得到了很好的表达,在未诱导、诱导2、4 h均未看见有明显的条带,在6、8、12 h处蛋白出现大量的表达。本研究对木瓜蛋白酶进行生物活性测定的结果显示,叶片出现水渍状坏死斑的症状,而空载体、无菌水均未出现坏死症状,所以初步判断半胱氨酸蛋白酶家族对马铃薯有毒害作用,推测C1A-PH-SCH1参与了致病疫霉引起马铃薯晚疫病的过程。本试验采用原核表达系统,对于初步筛选蛋白毒性具有培养条件简单、基因操作容易、从构建到产物纯化周期短、成本低、产量高等特点。为后续对毒性蛋白进行基因定位,致病机理研究等工作奠定基础。

4 结论

本试验利用TA克隆技术从致病疫霉菌XH05-5-4中克隆得到了1个半胱氨酸蛋白酶家族基因(登录号:KP938956命名:C1A-PH-SCH1),编码387个氨基酸。通过同源性比对发现C1A-PH-SCH1为木瓜蛋白酶亚家族基因,成功构建了原核表达载体,并使其在大肠杆菌中得到了表达,对生物活性测定结果显示出现了类似于晚疫病症状的水渍坏死斑症状,推测C1A-PH-SCH1参与马铃薯晚疫病的发病过程。
生物学信息分析表明该蛋白为分泌蛋白,并且该蛋白存在一个N-糖基化位点,表明该蛋白翻译产生信号肽,当转译进行到第18位氨基酸之后,与信号肽识别体(SRP)识别并牵引至内质网进行糖基化修饰。通过进化树分析,发现C1A-PH-SCH1与致病疫霉菌半胱氨酸蛋白酶家族-木瓜蛋白酶亚家族在进化树上同源性最高,以及在SMART的蛋白功能结构域预测显示C1A-PH-SCH1拥有Pept-C1结构域,在保守区域内也具有木瓜蛋白酶亚家族共有的活性位点Gln-Cys-His-Asn/Asp。因此,可推断C1A-PH-SCH1属于木瓜蛋白酶亚家族。

参考文献

[1]
柳玲玲, 芶久兰, 秦松. 马铃薯晚疫病研究进展[J]. 耕作与栽培, 2016(2):73-75.
[2]
张贺兰, 王鑫, 张若晗, 等. 利用基因工程育种提高马铃薯晚疫病抗性的研究进展[J]. 分子植物育种, 2018(21):7038-7041.
[3]
刘海东, 肖军海, 李松. 半胱氨酸蛋白酶拟肽抑制剂设计新进展[J]. 生物技术通讯, 2006, 17(1):119-122.
[4]
Vierstra R D. The Ubiquitin/26S proteasome pathway, the complex last chapter in the life of many plant proteins[J]. Trends in Plant Science, 2003, 8(3):135-142.
Plants use a repertoire of methods to control the level and activity of their constituent proteins. One method, whose prominence is only now being appreciated, is selective protein breakdown by the ubiquitin/26S proteasome pathway. Remarkably, recent analyses of the near-complete Arabidopsis thaliana genome identified >1300 genes, or approximately 5% of the proteome, involved in the ubiquitin/26S proteasome pathway, making it one of the most elaborate regulatory mechanisms in plants. Molecular genetic analyses have also connected individual components to almost all aspects of plant biology, including the cell-cycle, embryogenesis, photomorphogenesis, circadian rhythms, hormone signaling, homeosis, disease resistance and senescence. Consequently, it appears that the ubiquitin/26S proteasome pathway rivals transcription complexes and protein kinase cascades as the main player in plant cell regulation.
[5]
Basset G, Raymond P, Malek L, et al. Changes in the expression and the enzymic properties of the 20S proteasome in sugar-starved Maize roots, evidence for an in vivo oxidation of the proteasome[J]. Plant Physiology, 2002, 128(3):1149-1162.
[6]
Harrak H, Azelmat S, Baker E N, et al. Isolation and characterization of a gene encoding a drought-induced cysteine protease in tomato (Lycopersicon esculentum)[J]. Genome, 2001, 44(3):368-374.
In a previous study, a 65 kDa protein, TDI-65, was found to be accumulated in the leaves of drought-stressed tomato (Lycopersicon esculentum cv. Starfire) plants. The protein level returns to control level when the drought-stressed plants are rewatered. Antibodies raised against the purified protein were used to elucidate the subcellular localization of the protein. The protein was found to be mainly localized in the nuclei and chloroplasts of drought-stressed leaf cells. To identify the nature of the protein, a cDNA library was constructed and screened by the purified anti-TDI-65 antibody. A cDNA clone designated tdi-65 was isolated and characterized. The deduced amino acid sequences of tdi-65 protein has extensive homology with known cysteine proteases such as actinidin and papain. Northern blot analysis revealed that tdi-65 mRNA is 10-fold higher in drought-stressed plants as compared to control and rewatered plants. Similar results were observed in the tomato cultivar Ailsa and its near isogenic abscisic acid (ABA)-deficient mutant line, flacca, suggesting that the gene does not require ABA for its expression under drought conditions. Based on the previous immunolocalization findings we suggest that tdi-65 encoded cysteine protease functions in relation to drought-induced senescence and programmed cell death.
[7]
闫龙凤, 杨青川, 韩建国, 等. 植物半胱氨酸蛋白酶研究进展[J]. 草业学报, 2006, 14(5):11-19.
[8]
Grudkowska M, Zagdanska B. Multifunctional role of plant cysteine proteinases[J]. Acta Biochimical Polonica, 2004, 51(3):609-624.
[9]
Hassan H, Souad A, Edward N B, et al. Isolation and characterization of a gene encoding a drought-induced cysteine protease in tomato (Lycopersicon esculentum)[J]. Genome, 2001, 44:368-374.
[10]
Rojo E, Martin R, Carter C, et al. VPE gamma exhibits a caspase-like activity that contributes to defense against pathogens[J]. Current Biology, 2004, 14(21):1897-1906.
[11]
Bhalerao R, Keskitalo J, Sterky F, et al. Gene expression in autumn leaves[J]. Plant Physiology, 2003, 131(2):430-442.
Two cDNA libraries were prepared, one from leaves of a field-grown aspen (Populus tremula) tree, harvested just before any visible sign of leaf senescence in the autumn, and one from young but fully expanded leaves of greenhouse-grown aspen (Populus tremula x tremuloides). Expressed sequence tags (ESTs; 5,128 and 4,841, respectively) were obtained from the two libraries. A semiautomatic method of annotation and functional classification of the ESTs, according to a modified Munich Institute of Protein Sequences classification scheme, was developed, utilizing information from three different databases. The patterns of gene expression in the two libraries were strikingly different. In the autumn leaf library, ESTs encoding metallothionein, early light-inducible proteins, and cysteine proteases were most abundant. Clones encoding other proteases and proteins involved in respiration and breakdown of lipids and pigments, as well as stress-related genes, were also well represented. We identified homologs to many known senescence-associated genes, as well as seven different genes encoding cysteine proteases, two encoding aspartic proteases, five encoding metallothioneins, and 35 additional genes that were up-regulated in autumn leaves. We also indirectly estimated the rate of plastid protein synthesis in the autumn leaves to be less that 10% of that in young leaves.
[12]
Kingston S A H, Bollard A L, Minchin F R. Stress-induced changes in protease composition are determined by nitrogen supply in non-nodulating white clover[J]. Journal of Experimental Botany, 2005, 56:745-753.
An inbreeding line of white clover has been identified which remains non-nodulated under appropriate physiological conditions and so the nitrogen concentration of the plant can be manipulated by altering the nitrate supply to the roots. Non-nodulating plants were used to test the hypothesis that acclimation to nitrogen limitation in white clover involves changes in protease activity and composition. These results indicate that acclimation to nitrogen limitation involves the realignment of constituent proteases without necessarily incurring significant changes in total protease activity. Plants grown at 2.5, 5.0, 7.5, and 10 mM nitrate showed a positive correlation between nitrate supply and foliar protein concentration. Protein profiles, revealed by Coomassie-stained SDS-PAGE, were unchanged between treatments for a given amount of protein. Serine, aspartate/metalloprotease, and two cysteine proteases were identified in the leaves. Although total protease activity per gram fresh weight was unchanged between treatments, the relative contributions of these four proteases was determined by nitrate supply. When plants were stressed further by withholding nitrate there was an increase in cysteine protease activity, but a senescence-related aspartate/metalloprotease was not visible. Hence, while protease expression in white clover leaves responded to the current and past nitrogen status of the plant, the proteases involved in remobilization during nutrient limitation were distinct from those involved during the main senescence period. It is suggested that nitrogen limitation induced an early, reversible stage of senescence in which perturbations in protease activity facilitated the degradation of non-essential proteins in order to increase the chances of plant survival or seed set.
[13]
姜军, 杨晓达, 王夔. 汞和金属离子及多硫化物对木瓜蛋白酶活性的抑制作用[J]. 中国药学:英文版, 2007, 1:1-8.
[14]
Simpson D J. Proteolytic degradation of cereal prolamins—the problem with proline[J]. Plant Science, 2001, 161(5):825-838.
[15]
Wiederanders B. Structure-function relationships in class CA1 cysteine peptidase propeptides[J]. Acta Biochemical Polonica, 2003, 50:691-713.
[16]
Guerrero C, de la Calle M, Reid M S, et al. Analysis of the expression of two thiol protease genes from daylily (Hemerocallis spp.) during flower senescence[J]. Plant molecular biology, 1998, 36(4):565-571.
A cDNA clone encoding a daylily (Hemerocallis spp.) thiolprotease (SEN11), whose expression is strongly upregulated in flower tepal senescence, has been isolated. The amino acid sequence, deduced from the nucleotide sequence, showed highest similarity to plant thiolproteases of Vigna mungo, Phaseolus vulgaris and Hemerocallis (SEN102), and contains a putative ER retention signal that has been described in Vigna mungo. SEN102 and SEN11 transcripts were not detectable in flower buds at the opening stage, but two peaks of transcripts were seen after 9 h and 19 h, in both petals and sepals, when wilting symptoms were apparent. The pattern of protease activity migrating on a 26.3 kDa protein was similar to the SEN102 and SEN11 transcript profiles. These two genes were also expressed in stamens and leaves, but their transcripts were undetectable in carpels and rhizomes. The expression of SEN102 was lower in the senescent leaf than in the green leaf. The pattern of expression of these genes suggests their involvement in the protein hydrolysis occurring in tepals at the late senescence stage, whereas in leaves they could be involved in the constitutive protein turnover machinery. Exogenous gibberellic acid application to cut flowers increased transcripts of both genes.
[17]
Eric P B, Bonnie J W, Zhao C S. Plant proteolytic enzymes: possible roles during programmed cell death[J]. Plant Molecular Biology, 2000, 44:399-415.
[18]
Grudkowska M, Zagdanska B. Multifunctional role of plant cysteine proteinases[J]. Acta Biochimica Polonica, 2004, 52(3):609-624.
[19]
方正武, 李来运, 李晓方, 等. 甜荞木瓜类半胱氨酸蛋白酶基因FeRD21的克隆与表达分析[J]. 西北植物学报, 2015, 35(3):459-464.
[20]
彭玉玲. 致病疫霉菌分泌蛋白anx-1的克隆、表达及pipme1基因载体构建[D]. 昆明:云南农业大学, 2014.
[21]
阳永学, 程维舜, 曾红霞, 等. 西瓜半胱氨酸蛋白酶基因ClCP1克隆及其生物信息学分析[J]. 安徽农业科学, 2013, 41(28):11286-11288.
[22]
李永生, 方永丰, 李玥, 等. 玉米逆境响应基因ZmGST23克隆和表达分析[J]. 农业生物技术学报, 2016(5):667-677.
[23]
Koizumi M, Yamaguchi-Shinozaki K, Tsuji H, et al. Structure and expression of two genes that encode distinct drought-inducible cysteine proteinases in Arabidopsis thaliana[J]. Gene, 1993, 129:175-182.
Among nine cDNA clones (named RD) corresponding to genes that are responsive to dehydration in Arabidopsis thaliana, two clones, RD19 and RD21, were analyzed further. Northern blot analysis revealed that both the RD19 and RD21 mRNAs were not induced by abscisic acid. Neither RD19 nor RD21 mRNA synthesis was responsive to cold or to heat stress. On the other hand, transcription of both the RD19 and RD21 mRNAs was strongly induced under high-salt conditions, which suggests that the genes corresponding to RD19 and RD21 may be induced by changes in the osmotic potential of plant cells. Putative proteins, RD19 and RD21, encoded by two of the RD cDNAs have amino acid (aa) sequences typical of the catalytic sites of cysteine proteinases (CysP). RD21 and RD19 appeared to contain signal peptides that function in protein secretion. RD21 contains an aa sequence similar to that of the C-terminal extension peptide. Phylogenetic tree analysis indicated that the putative RD21 and RD19 proteins are quite different types of CysP. Genomic Southern analysis revealed that each gene family contains at least two members, which do not cross-hybridize. The two genes corresponding to RD19 and RD21 (rd19A and rd21A, respectively) were cloned and their structural analysis revealed the presence of two and four introns, respectively. The numbers and sites of introns differ between the genes, supporting our hypothesis that rd19A and rd21A belong to different subfamilies of genes encoding CysP. The transcription start points were determined by primer extension. Two conserved sequences were found in the promoter regions of the two genes.
[24]
McLellan H, Gilroy H, Yun B W, et al. Functional redundancy in the Arabidopsis Cathepsin B gene family contributes to basal defence, the hypersensitive response and senescence[J]. New Phytologist, 2009, 183:408-418.
Cysteine proteases are required for programmed cell death (PCD) in animals. Recent work in Nicotiana benthamiana has implicated cathepsin B-like cysteine proteases in the hypersensitive response (HR) in plants, a form of PCD involved in disease resistance. Here, we investigate the function and regulation of Cathepsin B (CathB) genes in plant defence, and in both pathogen-inducible and developmental forms of PCD. Single, double and triple knockout mutants were isolated for the three Arabidopsis thaliana AtCathB genes. AtCathB genes were redundantly required for full basal resistance against the virulent bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. By contrast, AtCathB genes were not required for R gene-mediated resistance to Pst DC3000 expressing AvrB or AvrRps4. Neither did they contribute to PCD triggered by AvrRps4, although they were crucial for the full development of PCD during HR triggered by AvrB. Cathepsin B has also been proposed to play a positive regulatory role in senescence. Atcathb triple mutants showed a delay in senescence and a seven-fold decrease in accumulation of senescence marker gene SAG12. Our results demonstrate a redundant function for AtCathB genes in basal defence as well as a potential regulatory role in distinct forms of plant PCD.
[25]
Noh Y S, Amasino R M. Regulation of developmental senescence is conserved between Arabidopsis and Brassica napus[J]. Plant Molecular Biology, 1999, 41(2):195-206.
SAG12 is a developmentally controlled, senescence-specific gene from Arabidopsis which encodes a cysteine protease. Using SAG12 as a probe, we isolated two SAG12 homologues (BnSAG12-1 and BnSAG12-2) from Brassica napus. Structural comparisons and expression studies indicate that these two genes are orthologues of SAG12. The expression patterns of BnSAG12-1 and BnSAG12-2 in Arabidopsis demonstrate that the senescence-specific regulation of this class of cysteine proteases is conserved across these species. Gel-shift assays using the essential promoter regions of SAG12, BnSAG12-1, and BnSAG12-2 show that the extent of binding of a senescence-specific, DNA-binding protein from Arabidopsis is proportional to the expression levels of these genes in Arabidopsis. Therefore, the expression levels of these genes may reflect the affinities of the senescence-specific DNA-binding protein for the promoter element.
[26]
Solomon M, Belenghi B, Delledonne M, et al. The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plan[J]. Plant Cell, 1999, 11:431-444.
Programmed cell death (PCD) is a process by which cells in many organisms die. The basic morphological and biochemical features of PCD are conserved between the animal and plant kingdoms. Cysteine proteases have emerged as key enzymes in the regulation of animal PCD. Here, we show that in soybean cells, PCD-activating oxidative stress induced a set of cysteine proteases. The activation of one or more of the cysteine proteases was instrumental in the PCD of soybean cells. Inhibition of the cysteine proteases by ectopic expression of cystatin, an endogenous cysteine protease inhibitor gene, inhibited induced cysteine protease activity and blocked PCD triggered either by an avirulent strain of Pseudomonas syringae pv glycinea or directly by oxidative stress. Similar expression of serine protease inhibitors was ineffective. A glutathione S-transferase-cystatin fusion protein was used to purify and characterize the induced proteases. Taken together, our results suggest that plant PCD can be regulated by activity poised between the cysteine proteases and the cysteine protease inhibitors. We also propose a new role for proteinase inhibitor genes as modulators of PCD in plants.
[27]
Sanghyun L, Ki-Hong J, et al. Isolation and characterization of a rice cysteine protease gene, OsCP1, using T-DNA gene-trap system[J]. Plant Molecular Biology, 2004, 54:755-765.
The T-DNA gene-trap system has been efficiently used to elucidate gene functions in plants. We report here a functional analysis of a cysteine protease gene, OsCP1, isolated from a pool of T-DNA insertional rice. GUS assay with the T-DNA tagged line indicated that the OsCP1 promoter was highly active in the rice anther. Sequence analysis revealed that the deduced amino acid sequence of OsCP1 was homologous to those of papain family cysteine proteases containing the highly conserved interspersed amino acid motif, ERFNIN. This result suggested that the gene encodes a cysteine protease in rice. We also identified a suppressed mutant from T2 progeny of the T-DNA tagged line. The mutant showed a significant defect in pollen development. Taken together, the results demonstrated that OsCP1 is a cysteine protease gene that might play an important role in pollen development.
[28]
Zhang X M, Wang Y, Lv X M, et al. NtCP56, a new cysteine protease in Nicotiana tabacum L, involved in pollen grain development[J]. Journal of Experimental Botany, 2009, 60(6):1569-1577.
[29]
严秀蕊, 张大生, 梁婉琪, 等. 水稻半胱氨酸蛋白酶OsCP2的特征分析及其原核表达与纯化[J]. 上海交通大学学报:农业科学版, 2010, 28(2):140-146.
[30]
王高峰, 彭德良, 孙建华, 等. 马铃薯腐烂茎线虫L型半胱氨酸蛋白酶新基因(Dd-cpl-1)的克隆与序列分析[J]. 生物工程学报, 2011, 27(1):60-68.
[31]
赵冬梅, 徐进, 杨志辉, 等. 致病疫霉坏死基因PcF/SCR.1的克隆及功能分析[J]. 农业生物技术学报, 2014, 22(6):744-752.

基金

国家重点研发项目“西南区马铃薯化学肥料和化学农药减施技术模式集成与示范”(2018YFD0200808)

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