【作者】 钟凤林；

【导师】 潘东明；

【作者基本信息】 福建农林大学 ， 果树学， 2009， 博士

【摘要】 蛋白质组学是后基因组学的主要内容之一,近年来得到了迅速发展,在生物分子的分析方面广泛应用。本研究以琯溪蜜柚(Citrus grandis cv.Guanximiyou)汁胞为材料,利用双向电泳、质谱技术,对琯溪蜜柚汁胞不同发育时期进行了差异蛋白质组学的分析。建立了适合琯溪蜜柚汁胞蛋白质组学研究的技术平台。获得与汁胞发育、成熟和衰老相关的差异蛋白质,进行了差异蛋白质组分析和生物信息学的研究,并对其中的部分差异蛋白质进行cDNA克隆及原核表达分析。主要研究结果如下:1.建立了适合琯溪蜜柚汁胞蛋白质分离的、稳定性好、分辨率高的双向电泳技术体系,对汁胞发育不同时期和不同粒化程度的蛋白质组差异进行比较。汁胞共检测到1037±60个蛋白质点,其中有100个蛋白质点在不同发育时期上调表达, 217个蛋白质点下调表达,完全特异的蛋白质点有85~511个;有108个蛋白质点在不同粒化程度中上调表达,107个蛋白质下调表达,约400个完全特异的蛋白质点。2.选取琯溪蜜柚不同发育时期的汁胞50个差异蛋白质点进行MALDI-TOF/TOF质谱分析,48个获得了完整的肽指纹图谱,将肽段数据导入NCBInr Viridiplantae蛋白质数据库,用MASCOT搜索鉴定出24个蛋白质点;通过生物信息学分析,根据蛋白质的功能可以把他们划分为调控蛋白,转录因子,能量代谢,细胞骨架,防卫反应,代谢酶,以及未知功能的蛋白质。另外有10个只能在柑橘EST中得到鉴定。蛋白、核酸和EST序列数据库的综合应用提高了蛋白质鉴定的成功率。还有一些差异表达没有鉴定成功可能与现在的蛋白质数据库不够全面有关。选取琯溪蜜柚正常与粒化汁胞中的30个差异蛋白质点进行MALDI-TOF/TOF质谱分析,其中27个获得了完整的肽指纹图谱,将肽段数据导入NCBInr Viridiplantae和NCBI CitrusEST数据库进行检索,鉴定出19个蛋白质点,其中有8个只能在柑橘EST中得到鉴定,通过生物信息学分析是关于转录活性因子蛋白、GTP结合蛋白、Hsp70、APX、温度诱导脂质运载蛋白和Actin。3.鉴定出的差异蛋白质点G6能够与序列号gi|188431972的Citrus EST序列匹配上,通过RACE技术进行G6的cDNA克隆,得到一个902bp片段的序列。通过Blast和DNAMAN同源性比较分析,推测其是一个Germin-like protein。Germin蛋白的多糖部分为HS-GGAX,是细胞壁半纤维素的直接前体,与植物细胞壁伸展性的增加密切相关,同时,Germin具有草酸氧化酶活性。推测Germin通过控制细胞壁的伸展性在琯溪蜜柚汁胞的粒化中起作用。4.质谱鉴定结果中有3个和APX匹配的差异蛋白质点,表明APX与汁胞发育(粒化)是密切相关的。为此,对APX在汁胞发育过程和不同粒化程度中的活性动态和分子克隆方面作了进一步的研究。(1)选取不同发育时期的琯溪蜜柚汁胞进行它们的APX活性测定及同工酶分析,随着成熟度的增加,APX活性成一个上升趋势,到果实成熟时趋向稳定。取不同粒化程度的汁胞进行它们的APX活性测定及同工酶分析。APX与汁胞发育是密切相关的,同时发现粒化样品的APX活性随着粒化指数的提高而变大。(2)根据已克隆的其他植物的APX基因的保守序列设计引物,采用同源克隆的方法从琯溪蜜柚汁胞中获得了APX1 cDNA片段,根据获得的cDNA片段的序列设计RACE引物,通过RACE法获得了全长为1118 bp的琯溪蜜柚汁胞APX1 cDNA全序列,包含750bp的开放读码框,编码250个通读的氨基酸序列。具有过氧化物酶活性位点信号和亚铁血红素结合基信号。琯溪蜜柚APX1的氨基酸序列与已报道的拟南芥、烟草、番茄、水稻等植物APX有很高的同源性。其核酸序列与龙眼的APX同源性高达87%,与荔枝的同源性86%,与胡杨的同源性85%,与葡萄的同源性84%,与番茄的同源性81%。目前APX1cDNA序列已在GenBank上注册,登录号为gb|FJ595794.1。通过构建重组质粒p32-APX1,转化至Trans Rosetta(DE3)中,当诱导温度为37℃,IPTG浓度为1mM,诱导时间为4h时,其在大肠杆菌宿主中获得了表达。(3)根据鉴定出的蛋白质氨基酸序列反推出碱基序列,设计特异的APX引物,通过RT-PCR克隆的方法从琯溪蜜柚汁胞中获得了APX2cDNA片段,根据获得的cDNA片段的序列设计RACE引物,通过RACE法获得了全长为1061 bp琯溪蜜柚汁胞APX2的cDNA全序列,包含753 bp的开放读码框,编码250个通读的氨基酸序列。对已报道的拟南芥、烟草、番茄、水稻等植物APX与琯溪蜜柚APX 2的氨基酸序列分析,与可可的APX同源性高达82%,与橡胶、茶树、桉树的同源性81%,与甘薯的同源性80%,与葡萄、胡杨的同源性79%,与花生、大豆的同源性78%。同时,进行2个APX cDNA编码的氨基酸产物同源性分析,发现它们的同源性为81.67%,都含有APX基因家族的保守结构域。与差异蛋白质点的肽指纹数据分析,推测APX2是差异蛋白质点Aa。目前APX2 cDNA已在GenBank上注册,登录号为gb|FJ595795。通过构建重组质粒p28-APX2,转化至BL21(DE3)中,当诱导温度为37℃,IPTG浓度为1mM,诱导时间为4h时,其在大肠杆菌宿主中获得了表达。5.根据鉴定出的差异蛋白质ACTIN和HSP的氨基酸序列反推出碱基序列,设计ACTIN和HSP的特异引物,通过RT-PCR克隆的方法从琯溪蜜柚汁胞中获得了ACTIN cDNA的开放读码框和一个1879bp的HSP cDNA片段序列。ACTIN cDNA ORF共有1131个碱基组成,编码377个氨基酸。更多还原

【Abstract】 Progresses have been made enormously in proteomics in recent years. Means of proteomics have been widely applied in many research fields of plant science. In this thesis, the juice sacs of pomelo (Citrus grandis cv. Guanximiyou) were utilized as material, and the techniques of two dimensional electrophoresis(2-DE), biology mass spectrometry(MS), molecular cloning etc. were performed to investigate the mechanism of fruit development and granulation(sclerification) in citrus. The main results showed as follows:1. A system of 2-DE involving the preparation of protein sample, the electrophoresis and staining for the proteomic extracted from juice sac was developed. And, the changes of proteomic were studied in the process of fruit development and juice sac granulation in pomelo. The silver stained gel images were analyzed with Image MasterTM 2D Platinum software and about 1037±60 protein spots were determined. There are 100 protein spots up-regulated expression and 217 protein spots down-regulated expression, 85~511 specific protein spots in the juice sacs during the fruit development. There are 100 protein spots up-regulated expression and 217 protein spots down-regulated expression, about 400 specific protein spots in juice sac granulation.2. 50 spots of differential proteins on the juice sacs development were selected and identified through MALDI-TOF/TOF MS analysis, and fragment fingerprint (FFP) alignment followed by peptide mass fingerprint (PMF) MASCOT and ProFound engine respectively. Using complementary protein, nucleotide, and EST sequence libraries, we were able to achieve a protein identification success rate of 70% for our representative protein dataset. 24 protein spots were successfully identified, 12 protein spots were identified exclusively by searching Citrus EST database, and they falls into several functional categories including regulatory protein, transcription factor, energy metabolism, cytoskeleton, defense response, metabolic enzymes and others. 30 spots of differential proteins on the juice sacs granulation were selected and identified through MALDI-TOF/TOF MS analysis, and fragment fingerprint (FFP) alignment followed by peptide mass fingerprint (PMF) MASCOT and ProFound engine respectively. Using complementary protein, nucleotide, and EST sequence libraries. 19 protein spots were successfully identified, 8 protein spots were identified exclusively by searching Citrus EST database.3. The differential protein spot G6 was matched to the deduced amino acid sequence encoded by Citrus EST of No. gi|188431972. And a 902 bp of cDNA fragment encoding G6 was obtained by RACE technique. Homology analysis showed that the G6 can be classified into a germin-like protein. The polysaccharide part of germin protein was HS-GGAX, which was the direct precursor of hemicellulose and closely related to the extensibility and lignification of plant cell wall. Meanwhile, germin protein exhibited activity of oxalic acid oxidase. The results indicated that the germin might play a role in the granulation of pomelo by regulating the lignifcation of juice sac.4. Among the total of 80 differential protein spots analyzed by MALDI-TOF MS, three distinct protein spots were identified as ascorbate peroxidase (APX), suggested that APX might play a role in the development and/or granulation of juice sac. Hence, the fluctuation of APX and molecular cloning of cDNA encoding APX in juice sac were performed in the subsequent studies.(1) The changes of APX activity and zymogram in the process of pomelo juice sac development and granulation were studied. The results showed that the activity of APX increased gradually with the development of juice sacs, and then it kept constant relatively when the fruit tended to maturation. A new isozyme band (Rf 0.66) located in juice sacs was visualized at Sep 18th, and it’s staining intensity increased in the process of fruit development. Compared with normal juice sac, granulated juice sac exhibited higher activity of APX.(2) Total RNA of pomelo juice sacs was extracted, and conserved region of APX cDNA was obtained by degenerated oligonucleotide primed RT-PCR. After that, the rapid amplification of cDNA ends (RACE) was performed to the 3’ and 5’ direction, and full length cDNA encoding APX1 was obtained from pomelo juice sacs. The full sequence is 1118bp in length, containing a 753 bp-nucleotide of open reading frame (ORF), encoding a putative protein of 250 amino acid residues, which were 87%, 86%, 85%, 84% and 81% homology to that of Longan, Litchi, Populus, Vitis and Lycopersicon, respectively. When aligned with primary structure of APX by scanning Prosite, the active site and heme binding site of APX were found in the deduced amino acid sequence. APX1 cDNA was submitted to GenBank, the accession No. is FJ595794.1. The coding region of the cDNA was ligated into pET-32a vector to build a recombinant vector p32-APX1, which was expressed in Escherichia Coli Trans Rosetta (DE3) strain when induced with 1mM IPTG and grown for 4 hours at 37℃.(3) The nucleotide sequence was obtained from identified protein amino acid sequence. Based on the nucleotide sequence, the specific APX primers were designed. The APX2 gene segment in pomelo juice was amplified by RT-PCR. According to APX2 sequence, the RACE primers were designed and the full length cDNA sequence of APX2 cDNA was amplified by RACE method. The APX2 full length cDNA sequence is 1061bp, containing a 753bp ORF coding 250 amino sequence. The APX2 sequence of pomelo was compared with APX reported in Arabidopsis thaliana, Nicotiana tabacum, Solanum lycopersicum and Oryza sativa. The results show that it has a 82%, 81%, 81%, 81%, 80%, 79%, 79% 78%, 78% homology with theobroma cacao, Hevea brasiliensis, Camellia Sinensis, eucalyptus, sweet potato, vitis, Populus euphratica, Arachis Hypogaea, soybean respectively. The APX2 sequence of pomelo was compared with APX1. The results show that it has a 81.67% homology. Based on the nucleotide sequence homology analysis showed that the APX2 can be the differential protein spot Aa. APX2 cDNA was submitted to GenBank, the accession No. is FJ595795. The coding region of the cDNA was ligated into pET-28a vector to build a recombinant vector p28-APX2, which was expressed in Escherichia Coli BL21 (DE3) strain when induced with 1mM IPTG and grown for 4 hours at 37℃.5. Specific primers for PCR amplifying ACTIN ORF and HSP fragment from juicy sac of pomelo were designed from the reversely translated nucleotide sequences of differential proteins ACTIN and HSP. ACTIN ORF was composed of 1131 bases coding for 377 amino acids, while the HSP fragment was 1879 bases in length.更多还原