不同致病力柑橘衰退病毒株系诱导甜橙表达差异蛋白质组学研究
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摘要
柑橘衰退病毒(Citrus tristeza virus, CTV)引起的柑橘衰退病是柑橘上一种具有经济重要性的病害,可危害甜橙和柚等品种,广泛分布于世界各柑橘产区。CTV主要通过感病接穗、苗和蚜虫传播,仅用无病毒苗木和药物不能完全控制该病的流行,应用弱毒株系交叉保护技术可在某些柑橘品种上防御部分强株系的侵入。目前对于CTV的致病机理和弱毒株系交叉保护机理仍然不够清楚。蛋白质是生物体功能的执行单元,从蛋白组学方面深入探讨CTV致病机理具有重要意义。因此,结合不同致病力株系开展CTV诱导甜橙表达差异蛋白质组学研究,对于了解CTV与寄主柑橘的互作,CTV弱毒株系交叉保护机理,进而为后期开展柑橘衰退病抗病育种和弱毒株系研发起到很好的推动作用。
本研究在柑橘病害实验室建立了甜橙叶蛋白质提取和双向凝胶电泳方法；利用普通双向电泳(2D-PAGE)、向荧光差异电泳(2D-DIGE)结合基质辅助激光解析二级质谱(MALDI-TOF/TOF MS/MS)的方法对CTV强毒株系TRL514和CT32、弱毒株系CT30和CT11A侵染甜橙叶片诱导表达的差异蛋白进行分离和鉴定；对差异蛋白表达量进行精确分析；利用KEGG数据库、Brite分类、Pathway通路和Module功能块、GO数据库检索,明确差异蛋白关联的细胞组分、分子功能和生物学进程信息；明确差异蛋白表达变化与CTV株系致病力的关系；采用RT-qPCR检测技术对来源于柑橘上的差异蛋白的相对表达量进行了初步验证。
主要研究结果如下：
1.甜橙叶蛋白双向凝胶电泳方法的建立
通过SDS-PAGE比较了酚提取法、三氯醋酸-丙酮沉淀法、三氯醋酸-酚结合法提取甜橙叶总蛋白效果,结果表明三氯醋酸-酚结合法的提取效果最好。以三氯醋酸-酚结合法提取的甜橙叶总蛋白进行双向电泳,得出：一般7cm胶条上样5-10μL,最高电压8000V条件聚焦30-100kVhr能够较好分离蛋白；13cm胶条上样10-15μL,最高电压8000V条件聚焦80-150kVhrR能够较好分离蛋白。
2.4个CTV株系诱导甜橙表达差异蛋白的2D分析
利用2D PAGE技术对4个CTV株系(强株系TRL514(?)CT32,弱株系CT30和CT11A)在锦橙叶上诱导表达的差异蛋白进行了比较分析,结果表明CTV诱导表达的差异蛋白主要分布在pH4-7范围内,利用pH4-7范围的13cm胶条更适合分离到最大量的差异蛋白。
3.CTV强弱株系诱导甜橙表达差异蛋白的2D-DIGE分析
利用2D-DIGE电泳从CTV强毒株系、弱毒株系和健康对照3组样本共10例样品的5张凝胶上获得了15幅不同荧光蛋白图谱。应用DeCyder2DTM软件进行蛋白图谱的胶内差异分析和凝胶间比较分析,在每块凝胶上获得超过3000个蛋白质点的信息。三组间one-way分析(0≤p≤0.001)结果显示存在82个显著表达差异蛋白,3Dgraph作图直观展示出差异蛋白在3组样本中的相对表达量高低。以p<0.05进行两组间差异蛋白t-test检验分析,在相对表达量差异1.2倍和1.5倍水平上,分别发现强毒株系与弱毒株系间存在差异表达蛋白点84个和32个,其中强毒株系相对于弱毒株系上调表达蛋白点分别为40个和10个,下调表达蛋白点分别为44个和22个。采用相同方式在1.2倍和1.5倍水平上,分别发现强毒株系与健康对照间的差异蛋白点139个和91个,其中强毒株系相对于对照上调蛋白点81个和56个,下调蛋白点58个和35个；分别发现弱毒株系与健康对照间的差异蛋白点116个和84个,其中弱毒株系相对于对照上调蛋白点79个和57个,下调蛋白点37个和27个。与健康对照比较,CTV强毒株系和弱毒株系样本中上调的差异蛋白点数均多于下调的差异蛋白点数。结果显示,CTV侵染诱导了甜橙更多蛋白的增量表达。
4.CTV诱导甜橙表达差异蛋白的质谱鉴定与功能分析
利用二级质谱分析和Mascot软件搜索,成功对123个差异蛋白点进行鉴定,共获得非重复蛋白质76个。其中,偏酸性蛋白点有82个,偏碱性蛋白点有41个,酸性蛋白点的等电点均在pH4-7的范围内,碱性蛋白点的等电点大部分在pH8-9的范围内：所鉴定蛋白质分子量绝大部分位于10kDa-55kDa之间。
利用KEGG工具对76个差异蛋白进行数据库搜索,表明54个差异蛋白质分属于27种KO分类号蛋白类型,22个差异蛋白质没有匹配的KO分类号。27种差异蛋白类型可按8种方式进行归类,其中超过20种属于同源功能蛋白类和酶类,显示出衰退病诱导表达的差异蛋白与多种生物学反应相关。其中6种属于光合作用蛋白类,各有1-2种差异蛋白类型属于肽酶类、蛋白酶体类、分子伴侣和折叠催化酶类、异戊烯基转移酶类或翻译因子类。同源功能蛋白涉及代谢、遗传信息处理、环境信息处理、细胞过程和人类疾病5个方面。通路功能聚类分析表明,组织中普遍含量较高的碳水化合物和能量代谢途径中找到了较多差异蛋白,神经组织退化疾病与折叠、分选和降解通路中也发现了较多差异蛋白。参与蛋白折叠、分选和降解通路的热激蛋白和蛋白酶体一般都与寄主抗病毒感染或病毒造成寄主致病的机制相关联,推测几种相关差异蛋白类型K13993、K02730、K02732、K01689、K00134和K04565有可能与衰退病诱导甜橙表达差异蛋白的关系密切。
特别发现了10种差异蛋白可能具有与衰退病侵染更为密切或某种特别的关系,它们是：3-磷酸甘油醛脱氢酶,在胞外域和共质体中有表达,影响了生物调控和定位进程,直接参与了疾病通路,可反映出衰退病与甜橙互作中信号传递与致病机制的某些信息；Cu/Zn超氧化物歧化酶,在胞外域中有表达,影响了生物调控进程,直接参与了疾病通路,可反映衰退病与甜橙的互作关系；HSP20家族热激蛋白和烯醇化酶,参与了遗传信息处理通路,可能反映衰退病毒通过干扰甜橙基础遗传信息加工、传递等实现对寄主多方面生物性状的影响；20S蛋白酶体α1亚基和β6亚基,在大分子复合物或胞外域中有表达,参与了两种免疫蛋白酶体功能块,影响了死亡进程、信号发送与免疫系统过程,可反映衰退病的致病机制信息和体现衰退病侵染后甜橙的免疫反应状况；异柠檬酸脱氢酶和I类果糖二磷酸醛缩酶,在胞外域或共质体中有表达,影响了生物调控或定位进程,参与了较多的通路和功能块,可反映出衰退病与甜橙互作中信号传递与致病机制的某些信息；F型氢离子运送ATP酶β亚基,在大分子复合物中有表达,影响定位进程,参与较多通路和功能块,是唯一具有转运子活性的差异蛋白,可能在衰退病影响甜橙反应中具有比较特别的功能作用；硫氧还蛋白H型5,是所有差异蛋白中唯一表现具有电子载体活性的蛋白质,值得关注是否在衰退病影响甜橙反应中具有某种特别的作用。
5.CTV强弱株系诱导甜橙表达差异蛋白的比较分析
鉴定到的123个差异蛋白中,匹配到强株系TRL514与健康对照Control(?)的差异点97个,其中61个上调；匹配到弱株系CT11A与健康对照Controll司的差异点89个,其中61个上调；匹配到强株系TRL514与弱株系CT11A间的差异点51个,其中24个上调。对10种比较重要的蛋白类型进行比较,匹配到差异蛋白点14个,CTV强弱株系侵染均诱导上调表达的差异蛋白为3-磷酸甘油醛脱氢酶、20S蛋白酶体α1亚基、20S蛋白酶体β6亚基、F型氢离子运送ATP酶β亚基、Cu/Zn超氧化物歧化酶和硫氧还蛋白H型5；CTV强弱株系侵染均诱导下调表达的蛋白为HSP20家族热激蛋白、I类果糖二磷酸醛缩酶和异柠檬酸脱氢酶。
6.CTV诱导差异蛋白表达的RT-qPCR检测
利用RT-qPCR方法,成功对差异蛋白NADP-异柠檬酸脱氢酶的相对表达进行验证。结果表明,衰退病强株系侵染时甜橙的JADP-异柠檬酸脱氢酶的表达被抑制,衰退病弱株系侵染时甜橙的NADP-异柠檬酸脱氢酶的表达被轻微抑制,暗示感病甜橙植株中NADP-异柠檬酸脱氢酶表达量的变化与衰退病毒侵染力密切关联。多数差异蛋白表达没有被成功验证,但在某种程度上揭示了衰退病诱导差异蛋白表达的不稳定性和复杂性。验证结果总体上仍然支持衰退病不同毒系在诱导甜橙差异表达蛋白的事实。
Citrus tristeza virus (CTV), a member of the genus Closterovirus within the family Closteroviridae, is widely distributed worldwide and is the causal agent of one of the most economically important diseases of citrus. CTV can cause great harm to sweet orange and pommulo trees. Multi-isolates have been found and cause a variety of symptoms such as decline and death of sweet orange and grapefruit on sour orange rootstock, stem pitting, seedling yellows, vein clearing etc.. CTV is dispersed to new areas by propagation of infection buds and then locally spread by aphids in a semipersistent manner. Simple application of Virus-free seedlings and chemical pesticides can't entirely control the spread of CTV. Mild strain cross protection (MSCP) has been proven to be one effective way in preventing sensitive citrus varieties from damaged by some severe CTV strains. So far, little knowledge has been known on the CTV pathogenesis and mechanism of MSCP. Proteins are, in effect, the main actioners in cells and in an entire organism, which play animportant role in phosphorylation and glycosylation processes etc. More and more, the method of differential proteomics has been adopted to seek the pathogen-related proteins efficiently and sweet orange has been paid more attention due to its economic importance. Hence, the research on differential proteomics of CTV infection on sweet orange will make contributions to enhangce the understanding of its pathogenesis., and then further promote carryout of the CTV-resistant citrus breeding and developing the mild CTV strains, and in the end service the whole citrus industry.
In this research, we established the suitable methods of total sweet orange(Citrus sinensis) leaf protein extraction and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).2D-PAGE and Two-dimensional fluorescence difference gel electrophoresis(2D-DIGE) combined with MALDI-TOF/TOF MS/MS techology were adopted to separate and analyse the differential expressed proteins induced by four CTV isolates, which included two severe isolates (TRL514and CT32), two mild isolates (CT30and CT11A). DeCyder software was used to accurately analyse the protein spots gained from2D-DIGE, which outputed the relative expression levels of protein spots, and Mascot software to search protein informations in NCBI database to blast the peptide sequences and gave out the matched protein accession IDs. KEGG databases were used to search out the corresponding KO number of each matched protein. We conducted three KEGG searches including Brite search, Pathway search and Module search to get the protein functional information. GO(gene ontology) analysis was adopted to find out gene products descriptions in terms of their associated biological processes, cellular components and molecular functions according to protein sequences. Then through integrated analysis based on above results, revealed the relationships between CTV isolates pathogenicity and induced protein expressions. In order to tentatively test the relations between CTV isolates and induced protein expressions, Realtime RT-PCR techology was adopted to detect differential genes expression in CTV-infected and healthy sweet orange leaves. Followed are the main results.
1. Establishment of2D method of sweet orange leaf protein
Three protein extraction methods were compared, including phenol method, trichloroacetic acid and acetone precipitation method, trichloroacetic acid and combined phenol method, and SDS-PAGE was run to test. Result showed that the trichloroacetic acid and combined phenol method was the most suitable protein extration method for sweet orange leaves. Isoelectrofocusing experiments showed that focusing parameters from gel instructions couldn't be adopted directly and should find out the suitable program for your samples. In our study, suggested conditions of isoelectrofocusing were:8000V and30-100kVhr for7cm gel;8000V and80-150kVhr for13cm. Combined2D-MALDI-TOF MS analysis vestified the validity of this2D method.
2.2D electrophoresis of differential expressed proteins in sweet oranges induced by four CTV isolates
Two sweet orange varieties including Jincheng and Symons, four CTV isolates including two severe isolates (TRL514and CT32) and two mild isolates(CT30and CT11A) were adopted in this experiment. Through2D method, we found that most differential expressed protein spots located within ph4-7area. To Jincheng seedling samples, seperated20differential expressed spots in pH3-10,18cm gels, wheras42spots in pH4-7,13cm gels. It indicated that more protein spots could be seperated in pH4-7,13cm gels. After that, we seperated35spots from Jincheng budling samples, and53spots from Symons budling samples.
3.2D-DIGE analysis of differential expressed proteins in sweet oranges induced by severe and mild CTV isolates.
2D-DIGE is the representive gel quantitative techology for differential expressed protein analysis. In this study, we used DIGE method to analysis the differential protein expression induced by one severe CTV isolate(TRL514) and one mild CTV isolate(CT11A) compared to healthy Control. The results showed that total82differential protein spots were detected with one-way analysis(0≤p≤0.001) between three groups. And when employing relative average volume ratio≥1.2or≥1.5statistical variance of two groups(t-test,P<0.05)respectively,84or32differential protein spots were detected between severe and mild isolate, and139or91spots between severe isolate and control, and116or84spots between mild isolate and control. Altogether up-expressed protein spot nubmers were more than down-expressed, indicating the reality of more protein expressions happening in CTV-infected sweet orange plants.
4. CTV-induced differential expressed protein analysis using MALDI-TOF/TOF MS/MS and protein function searches
The CTV-induced differential expressed proteins were identified by4800Plus MALDI TOF/TOFTM Analyzer mass spectrometer produced by ABI company. Results showed a total of76non-repeated proteins were identified successfully from140DIGE protein spots. Approximately two times of acid protein(all pI ranged between pH4-7) spot number to basic protein(most pI ranged between pH8-9). And most proteins ranged between10-55kDa.
KEGG analysis
Application of KEGG blast search indicated that54identified proteins belonged to27KO types. Further Brite search results showed that all27KO proteins could be classified into8classes. In which, over20KO proteins classified into protein ontology and enzymes, that indicated CTV-induced expressed proteins took part in many biological proccesses. Pathway clustering results showed quite a number of proteins affected two specific pathways, that were neurodegenerative diseases pathway and folding、sorting and degradation pathway. The related KO proteins contained K13993、K02730、K02732、K01689、K00134and K04565, which might play some key roles in affecting the host protein expression when diseased with CTV.
To learn how the27KO proteins behave in pathway, pathway and module searches were conducted. Results indicated all27protein types involving30pathways and21functional module, or having quite more functions.
GO analysis
Through Gene Ontology analysis of these76identified proteins, we found that CTV-induced expressed proteins mainly located in cell, cell part, organelle and membrane, which had catalytic activity and binding activity. There were a number of proteins located in extracellular region and macromolecular complex which had antioxidant activity, enzyme regulator activity and transporter activity, attending the processes of biological regulation, response to stimulus and localization. It indicated the referred proteins'close relation to CTV pathogenesis.
Combined analysis of KEGG and GO ontology suggested10differential expressed proteins may play the key roles in response to CTV infection, including glyceraldehyde3-phosphate dehydrogenase, Cu/Zn superoxide dismutase, HSP20family protein, enolase,20S proteasome subunit alpha1,20S proteasome subunit beta6, isocitrate dehydrogenase, F-type H+-transporting ATPase subunit beta, Thioredoxin H-type5.
5. Comparison of differential protein expressions in sweet orange induced by severe and mild CTV isolates
Based on above analyzations, we compared the protein expression differences in sweet orange induced by severe and mild CTV isolates. Results showed that there were97differential protein spots betweet TRL514isolate(severe) and Control(healthy) found in123identified protein spots, among which61differential protein spots were up-regulated compared to Control. And89spots between CT11A(mild) and Control, among which61up-regulated compared to Control. And51spots between TRL514and CT11A,among which24up-regulated compared to CT11A.
There were14differential protein spots corresponding to the10key protein types. And6kinds of proteins including glyceraldehyde3-phosphate dehydrogenase(K00134),20S proteasome subunit alpha1(K02730),20S proteasome subunit beta6(K02732), F-type H+-transporting ATPase subunit beta(kO2112), Cu/Zn superoxide dismutase(K04565) and thioredoxin H-type5(gi|357438577) were up-regulated in severe or mild CTV-infected sweet orange compared with healthy control, which usually play an important role in the immune system. And3kinds of proteins including HSP20family protein(K13993), fructose-bisphosphate aldolase class I(k01623) and isocitrate dehydrogenase (k00031) were down-regulated in severe or mild CTV-infected sweet orange compared with healthy control, which play a role in signal transduction.
6. Examination of mRNA expression induced by CTV using RT-qPCR techology
For the further examinaton of regarding protein expression relations, four differential protein genes expressions in TRL514-inoculated, CT11A-inoculated and healthy control Jincheng leaves were detected by RT-qPCR respectively. Tow citrus house-keeping genes were used imutaneously to balance the primary sample templates and adoption of relative expression level to compare the difference between samples. Results showed only one gene expression be verified the same orientation with that from DIGE. But results also supported great differences in protein expressions between severe and mild CTV isolates inductions.
本研究在柑橘病害实验室建立了甜橙叶蛋白质提取和双向凝胶电泳方法；利用普通双向电泳(2D-PAGE)、向荧光差异电泳(2D-DIGE)结合基质辅助激光解析二级质谱(MALDI-TOF/TOF MS/MS)的方法对CTV强毒株系TRL514和CT32、弱毒株系CT30和CT11A侵染甜橙叶片诱导表达的差异蛋白进行分离和鉴定；对差异蛋白表达量进行精确分析；利用KEGG数据库、Brite分类、Pathway通路和Module功能块、GO数据库检索,明确差异蛋白关联的细胞组分、分子功能和生物学进程信息；明确差异蛋白表达变化与CTV株系致病力的关系；采用RT-qPCR检测技术对来源于柑橘上的差异蛋白的相对表达量进行了初步验证。
主要研究结果如下：
1.甜橙叶蛋白双向凝胶电泳方法的建立
通过SDS-PAGE比较了酚提取法、三氯醋酸-丙酮沉淀法、三氯醋酸-酚结合法提取甜橙叶总蛋白效果,结果表明三氯醋酸-酚结合法的提取效果最好。以三氯醋酸-酚结合法提取的甜橙叶总蛋白进行双向电泳,得出：一般7cm胶条上样5-10μL,最高电压8000V条件聚焦30-100kVhr能够较好分离蛋白；13cm胶条上样10-15μL,最高电压8000V条件聚焦80-150kVhrR能够较好分离蛋白。
2.4个CTV株系诱导甜橙表达差异蛋白的2D分析
利用2D PAGE技术对4个CTV株系(强株系TRL514(?)CT32,弱株系CT30和CT11A)在锦橙叶上诱导表达的差异蛋白进行了比较分析,结果表明CTV诱导表达的差异蛋白主要分布在pH4-7范围内,利用pH4-7范围的13cm胶条更适合分离到最大量的差异蛋白。
3.CTV强弱株系诱导甜橙表达差异蛋白的2D-DIGE分析
利用2D-DIGE电泳从CTV强毒株系、弱毒株系和健康对照3组样本共10例样品的5张凝胶上获得了15幅不同荧光蛋白图谱。应用DeCyder2DTM软件进行蛋白图谱的胶内差异分析和凝胶间比较分析,在每块凝胶上获得超过3000个蛋白质点的信息。三组间one-way分析(0≤p≤0.001)结果显示存在82个显著表达差异蛋白,3Dgraph作图直观展示出差异蛋白在3组样本中的相对表达量高低。以p<0.05进行两组间差异蛋白t-test检验分析,在相对表达量差异1.2倍和1.5倍水平上,分别发现强毒株系与弱毒株系间存在差异表达蛋白点84个和32个,其中强毒株系相对于弱毒株系上调表达蛋白点分别为40个和10个,下调表达蛋白点分别为44个和22个。采用相同方式在1.2倍和1.5倍水平上,分别发现强毒株系与健康对照间的差异蛋白点139个和91个,其中强毒株系相对于对照上调蛋白点81个和56个,下调蛋白点58个和35个；分别发现弱毒株系与健康对照间的差异蛋白点116个和84个,其中弱毒株系相对于对照上调蛋白点79个和57个,下调蛋白点37个和27个。与健康对照比较,CTV强毒株系和弱毒株系样本中上调的差异蛋白点数均多于下调的差异蛋白点数。结果显示,CTV侵染诱导了甜橙更多蛋白的增量表达。
4.CTV诱导甜橙表达差异蛋白的质谱鉴定与功能分析
利用二级质谱分析和Mascot软件搜索,成功对123个差异蛋白点进行鉴定,共获得非重复蛋白质76个。其中,偏酸性蛋白点有82个,偏碱性蛋白点有41个,酸性蛋白点的等电点均在pH4-7的范围内,碱性蛋白点的等电点大部分在pH8-9的范围内：所鉴定蛋白质分子量绝大部分位于10kDa-55kDa之间。
利用KEGG工具对76个差异蛋白进行数据库搜索,表明54个差异蛋白质分属于27种KO分类号蛋白类型,22个差异蛋白质没有匹配的KO分类号。27种差异蛋白类型可按8种方式进行归类,其中超过20种属于同源功能蛋白类和酶类,显示出衰退病诱导表达的差异蛋白与多种生物学反应相关。其中6种属于光合作用蛋白类,各有1-2种差异蛋白类型属于肽酶类、蛋白酶体类、分子伴侣和折叠催化酶类、异戊烯基转移酶类或翻译因子类。同源功能蛋白涉及代谢、遗传信息处理、环境信息处理、细胞过程和人类疾病5个方面。通路功能聚类分析表明,组织中普遍含量较高的碳水化合物和能量代谢途径中找到了较多差异蛋白,神经组织退化疾病与折叠、分选和降解通路中也发现了较多差异蛋白。参与蛋白折叠、分选和降解通路的热激蛋白和蛋白酶体一般都与寄主抗病毒感染或病毒造成寄主致病的机制相关联,推测几种相关差异蛋白类型K13993、K02730、K02732、K01689、K00134和K04565有可能与衰退病诱导甜橙表达差异蛋白的关系密切。
特别发现了10种差异蛋白可能具有与衰退病侵染更为密切或某种特别的关系,它们是：3-磷酸甘油醛脱氢酶,在胞外域和共质体中有表达,影响了生物调控和定位进程,直接参与了疾病通路,可反映出衰退病与甜橙互作中信号传递与致病机制的某些信息；Cu/Zn超氧化物歧化酶,在胞外域中有表达,影响了生物调控进程,直接参与了疾病通路,可反映衰退病与甜橙的互作关系；HSP20家族热激蛋白和烯醇化酶,参与了遗传信息处理通路,可能反映衰退病毒通过干扰甜橙基础遗传信息加工、传递等实现对寄主多方面生物性状的影响；20S蛋白酶体α1亚基和β6亚基,在大分子复合物或胞外域中有表达,参与了两种免疫蛋白酶体功能块,影响了死亡进程、信号发送与免疫系统过程,可反映衰退病的致病机制信息和体现衰退病侵染后甜橙的免疫反应状况；异柠檬酸脱氢酶和I类果糖二磷酸醛缩酶,在胞外域或共质体中有表达,影响了生物调控或定位进程,参与了较多的通路和功能块,可反映出衰退病与甜橙互作中信号传递与致病机制的某些信息；F型氢离子运送ATP酶β亚基,在大分子复合物中有表达,影响定位进程,参与较多通路和功能块,是唯一具有转运子活性的差异蛋白,可能在衰退病影响甜橙反应中具有比较特别的功能作用；硫氧还蛋白H型5,是所有差异蛋白中唯一表现具有电子载体活性的蛋白质,值得关注是否在衰退病影响甜橙反应中具有某种特别的作用。
5.CTV强弱株系诱导甜橙表达差异蛋白的比较分析
鉴定到的123个差异蛋白中,匹配到强株系TRL514与健康对照Control(?)的差异点97个,其中61个上调；匹配到弱株系CT11A与健康对照Controll司的差异点89个,其中61个上调；匹配到强株系TRL514与弱株系CT11A间的差异点51个,其中24个上调。对10种比较重要的蛋白类型进行比较,匹配到差异蛋白点14个,CTV强弱株系侵染均诱导上调表达的差异蛋白为3-磷酸甘油醛脱氢酶、20S蛋白酶体α1亚基、20S蛋白酶体β6亚基、F型氢离子运送ATP酶β亚基、Cu/Zn超氧化物歧化酶和硫氧还蛋白H型5；CTV强弱株系侵染均诱导下调表达的蛋白为HSP20家族热激蛋白、I类果糖二磷酸醛缩酶和异柠檬酸脱氢酶。
6.CTV诱导差异蛋白表达的RT-qPCR检测
利用RT-qPCR方法,成功对差异蛋白NADP-异柠檬酸脱氢酶的相对表达进行验证。结果表明,衰退病强株系侵染时甜橙的JADP-异柠檬酸脱氢酶的表达被抑制,衰退病弱株系侵染时甜橙的NADP-异柠檬酸脱氢酶的表达被轻微抑制,暗示感病甜橙植株中NADP-异柠檬酸脱氢酶表达量的变化与衰退病毒侵染力密切关联。多数差异蛋白表达没有被成功验证,但在某种程度上揭示了衰退病诱导差异蛋白表达的不稳定性和复杂性。验证结果总体上仍然支持衰退病不同毒系在诱导甜橙差异表达蛋白的事实。
Citrus tristeza virus (CTV), a member of the genus Closterovirus within the family Closteroviridae, is widely distributed worldwide and is the causal agent of one of the most economically important diseases of citrus. CTV can cause great harm to sweet orange and pommulo trees. Multi-isolates have been found and cause a variety of symptoms such as decline and death of sweet orange and grapefruit on sour orange rootstock, stem pitting, seedling yellows, vein clearing etc.. CTV is dispersed to new areas by propagation of infection buds and then locally spread by aphids in a semipersistent manner. Simple application of Virus-free seedlings and chemical pesticides can't entirely control the spread of CTV. Mild strain cross protection (MSCP) has been proven to be one effective way in preventing sensitive citrus varieties from damaged by some severe CTV strains. So far, little knowledge has been known on the CTV pathogenesis and mechanism of MSCP. Proteins are, in effect, the main actioners in cells and in an entire organism, which play animportant role in phosphorylation and glycosylation processes etc. More and more, the method of differential proteomics has been adopted to seek the pathogen-related proteins efficiently and sweet orange has been paid more attention due to its economic importance. Hence, the research on differential proteomics of CTV infection on sweet orange will make contributions to enhangce the understanding of its pathogenesis., and then further promote carryout of the CTV-resistant citrus breeding and developing the mild CTV strains, and in the end service the whole citrus industry.
In this research, we established the suitable methods of total sweet orange(Citrus sinensis) leaf protein extraction and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).2D-PAGE and Two-dimensional fluorescence difference gel electrophoresis(2D-DIGE) combined with MALDI-TOF/TOF MS/MS techology were adopted to separate and analyse the differential expressed proteins induced by four CTV isolates, which included two severe isolates (TRL514and CT32), two mild isolates (CT30and CT11A). DeCyder software was used to accurately analyse the protein spots gained from2D-DIGE, which outputed the relative expression levels of protein spots, and Mascot software to search protein informations in NCBI database to blast the peptide sequences and gave out the matched protein accession IDs. KEGG databases were used to search out the corresponding KO number of each matched protein. We conducted three KEGG searches including Brite search, Pathway search and Module search to get the protein functional information. GO(gene ontology) analysis was adopted to find out gene products descriptions in terms of their associated biological processes, cellular components and molecular functions according to protein sequences. Then through integrated analysis based on above results, revealed the relationships between CTV isolates pathogenicity and induced protein expressions. In order to tentatively test the relations between CTV isolates and induced protein expressions, Realtime RT-PCR techology was adopted to detect differential genes expression in CTV-infected and healthy sweet orange leaves. Followed are the main results.
1. Establishment of2D method of sweet orange leaf protein
Three protein extraction methods were compared, including phenol method, trichloroacetic acid and acetone precipitation method, trichloroacetic acid and combined phenol method, and SDS-PAGE was run to test. Result showed that the trichloroacetic acid and combined phenol method was the most suitable protein extration method for sweet orange leaves. Isoelectrofocusing experiments showed that focusing parameters from gel instructions couldn't be adopted directly and should find out the suitable program for your samples. In our study, suggested conditions of isoelectrofocusing were:8000V and30-100kVhr for7cm gel;8000V and80-150kVhr for13cm. Combined2D-MALDI-TOF MS analysis vestified the validity of this2D method.
2.2D electrophoresis of differential expressed proteins in sweet oranges induced by four CTV isolates
Two sweet orange varieties including Jincheng and Symons, four CTV isolates including two severe isolates (TRL514and CT32) and two mild isolates(CT30and CT11A) were adopted in this experiment. Through2D method, we found that most differential expressed protein spots located within ph4-7area. To Jincheng seedling samples, seperated20differential expressed spots in pH3-10,18cm gels, wheras42spots in pH4-7,13cm gels. It indicated that more protein spots could be seperated in pH4-7,13cm gels. After that, we seperated35spots from Jincheng budling samples, and53spots from Symons budling samples.
3.2D-DIGE analysis of differential expressed proteins in sweet oranges induced by severe and mild CTV isolates.
2D-DIGE is the representive gel quantitative techology for differential expressed protein analysis. In this study, we used DIGE method to analysis the differential protein expression induced by one severe CTV isolate(TRL514) and one mild CTV isolate(CT11A) compared to healthy Control. The results showed that total82differential protein spots were detected with one-way analysis(0≤p≤0.001) between three groups. And when employing relative average volume ratio≥1.2or≥1.5statistical variance of two groups(t-test,P<0.05)respectively,84or32differential protein spots were detected between severe and mild isolate, and139or91spots between severe isolate and control, and116or84spots between mild isolate and control. Altogether up-expressed protein spot nubmers were more than down-expressed, indicating the reality of more protein expressions happening in CTV-infected sweet orange plants.
4. CTV-induced differential expressed protein analysis using MALDI-TOF/TOF MS/MS and protein function searches
The CTV-induced differential expressed proteins were identified by4800Plus MALDI TOF/TOFTM Analyzer mass spectrometer produced by ABI company. Results showed a total of76non-repeated proteins were identified successfully from140DIGE protein spots. Approximately two times of acid protein(all pI ranged between pH4-7) spot number to basic protein(most pI ranged between pH8-9). And most proteins ranged between10-55kDa.
KEGG analysis
Application of KEGG blast search indicated that54identified proteins belonged to27KO types. Further Brite search results showed that all27KO proteins could be classified into8classes. In which, over20KO proteins classified into protein ontology and enzymes, that indicated CTV-induced expressed proteins took part in many biological proccesses. Pathway clustering results showed quite a number of proteins affected two specific pathways, that were neurodegenerative diseases pathway and folding、sorting and degradation pathway. The related KO proteins contained K13993、K02730、K02732、K01689、K00134and K04565, which might play some key roles in affecting the host protein expression when diseased with CTV.
To learn how the27KO proteins behave in pathway, pathway and module searches were conducted. Results indicated all27protein types involving30pathways and21functional module, or having quite more functions.
GO analysis
Through Gene Ontology analysis of these76identified proteins, we found that CTV-induced expressed proteins mainly located in cell, cell part, organelle and membrane, which had catalytic activity and binding activity. There were a number of proteins located in extracellular region and macromolecular complex which had antioxidant activity, enzyme regulator activity and transporter activity, attending the processes of biological regulation, response to stimulus and localization. It indicated the referred proteins'close relation to CTV pathogenesis.
Combined analysis of KEGG and GO ontology suggested10differential expressed proteins may play the key roles in response to CTV infection, including glyceraldehyde3-phosphate dehydrogenase, Cu/Zn superoxide dismutase, HSP20family protein, enolase,20S proteasome subunit alpha1,20S proteasome subunit beta6, isocitrate dehydrogenase, F-type H+-transporting ATPase subunit beta, Thioredoxin H-type5.
5. Comparison of differential protein expressions in sweet orange induced by severe and mild CTV isolates
Based on above analyzations, we compared the protein expression differences in sweet orange induced by severe and mild CTV isolates. Results showed that there were97differential protein spots betweet TRL514isolate(severe) and Control(healthy) found in123identified protein spots, among which61differential protein spots were up-regulated compared to Control. And89spots between CT11A(mild) and Control, among which61up-regulated compared to Control. And51spots between TRL514and CT11A,among which24up-regulated compared to CT11A.
There were14differential protein spots corresponding to the10key protein types. And6kinds of proteins including glyceraldehyde3-phosphate dehydrogenase(K00134),20S proteasome subunit alpha1(K02730),20S proteasome subunit beta6(K02732), F-type H+-transporting ATPase subunit beta(kO2112), Cu/Zn superoxide dismutase(K04565) and thioredoxin H-type5(gi|357438577) were up-regulated in severe or mild CTV-infected sweet orange compared with healthy control, which usually play an important role in the immune system. And3kinds of proteins including HSP20family protein(K13993), fructose-bisphosphate aldolase class I(k01623) and isocitrate dehydrogenase (k00031) were down-regulated in severe or mild CTV-infected sweet orange compared with healthy control, which play a role in signal transduction.
6. Examination of mRNA expression induced by CTV using RT-qPCR techology
For the further examinaton of regarding protein expression relations, four differential protein genes expressions in TRL514-inoculated, CT11A-inoculated and healthy control Jincheng leaves were detected by RT-qPCR respectively. Tow citrus house-keeping genes were used imutaneously to balance the primary sample templates and adoption of relative expression level to compare the difference between samples. Results showed only one gene expression be verified the same orientation with that from DIGE. But results also supported great differences in protein expressions between severe and mild CTV isolates inductions.
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