【作者】 冀宪领；

【导师】 牟志美；

【作者基本信息】 山东农业大学 ， 森林培育， 2008， 博士

【摘要】 植原体病害是世界性植物病害,全球范围内均有不同程度的发生,给农林业生产造成巨大损失。桑黄化型萎缩病是桑树上的重大病害,常导致大片桑树毁灭,严重制约了蚕业生产的发展。由于植原体难以培养,对于植原体的研究进展缓慢,关于植原体诱导植物发病的分子机制知之甚少。自从植原体基因组序列测定完成后,基因组学的研究重心便从揭示植原体的所有遗传信息转移到从整体水平上对植原体的生物过程进行研究,特别是对基因组注解的验证性研究,而蛋白质组学研究便是其中的一项重要的内容。近年来对于桑树黄化型萎缩病发生分子机制的研究并没有取得实质性的进展,桑树萎缩病病原响应蛋白的研究在国内外尚未见报道。本研究通过对植原体蛋白质组学及桑树萎缩病病原响应蛋白的研究,以期为从分子水平上揭示桑树萎缩病的发生机理提供理论基础。本研究利用Shotgun策略对植原体表达的蛋白质组进行了分析。结合SDS-PAGE电泳与毛细管液相色谱-串联质谱技术,准确地鉴定了242种植原体蛋白质,其中包括参与氨基酸合成、细胞膜、中间代谢、细胞过程、能量代谢、不饱和脂肪酸和磷脂的代谢、核苷及核苷酸代谢、复制、转录、翻译、运输和结合蛋白和其它功能的相关蛋白质。除了上述已知功能的蛋白外,还鉴定了76种假定蛋白或保守的假定蛋白。所鉴定的蛋白质总量占预测的植原体蛋白质组的35%。如此大通量地对植原体蛋白质组进行鉴定国内外还未见报道,该研究不仅为植原体蛋白质组学研究提供了技术参考,同时提供了一个具有参考价值的植原体蛋白质数据库,为更好地理解植原体的生物过程的功能和机制奠定了基础。本研究结合蛋白质双向凝胶电泳和质谱技术,利用差异蛋白质组学策略研究了桑树受植原体侵染后叶片蛋白表达谱的变化。利用ImageMasterTM 2D Platinum软件分析,共检测到500多个叶片可溶性蛋白,发现有37个蛋白点差异表达,其中18个被下调,19个被上调。质谱分析共鉴定出18个蛋白点,代表了15种不同的蛋白。被鉴定的蛋白包括Rubisco大亚基、景天庚酮糖-1,7-二磷酸酶、Rubisco活化酶、防御相关蛋白、蛋白酪氨酸磷酸化酶、NUDIX/mutT类水解酶家族蛋白、成熟酶K、Kunitz型蛋白酶抑制剂-1、20S蛋白酶体亚基、放氧复合体的33 kDa前体蛋白、苹果酸脱氢酶、甲硫氨酸亚砜还原酶、Gm-ck32857、F-box蛋白、未知蛋白。这些蛋白涉及到光合作用、氨基酸代谢、核苷酸代谢、信号传导及调控、防御应答、转录等多个生理过程。本研究为从分子水平上揭示桑树萎缩病的发生机理提供了理论基础。景天庚酮糖-1,7-二磷酸酶(SBPase)是卡尔文循环过程中的关键酶。本研究利用RACE技术得到桑树景天庚酮糖-1,7-二磷酸酶基因全长cDNA,命名为MSBPase (GenBank登录号:DQ995346)。MSBPase全长为1 527 bp,该序列含有一个1 179 bp的完整开放读码框,编码393个氨基酸,蛋白质理论分子量约为42.6 kDa,等电点为5.85,其氨基酸序列与其它植物中已分离的SBPase有很高的同源性。对MSBPase编码的蛋白质(命名为MSBPase)进行结构预测分析表明,该蛋白富含无规卷曲(Coil),高达64.29%,其次是α-螺旋(Helix),为22.19 %,而β-折叠(Strand)只有13.52 %。将MSBPase编码区插入原核表达载体pET30a (+),并转化到大肠杆菌菌株BL21中,经过IPTG诱导,MSBPase融合蛋白在BL21菌株中成功表达。将得到的MSBPase编码区插入植物表达载体pBI121中,构建了MSBPase植物表达载体pBI121-SBP。将植物表达载体pBI121-SBP,采用农杆菌介导的方法,转化拟南芥,经卡那霉素筛选获得若干再生植株。经Northern和Western杂交分析,证明MSBPase在转基因拟南芥中已成功得到表达。MSBPase在拟南芥中超表达可以提高拟南芥叶片的SBPase活性和净光合速率,叶片淀粉和可溶性糖含量增加,植物生长旺盛,干物质积累增加,开花提前。本研究为桑树基因工程提供了有效的候选基因,为深入研究SBPase的分子调控机制奠定了基础。1,5-二磷酸核酮糖羧化酶活化酶(RCA)广泛存在于光合生物中,它是一种由核基因编码的叶绿体蛋白,具有调节Rubisco活性的功能。本研究根据RCA的保守区域设计一对兼并引物,通过PCR扩增,获得RCA的基因功能区的中间片段,利用RACE技术获得RCA的基因cDNA的3’端片段。对获得的基因片段所编码的氨基酸进行BLAST分析,结果表明,其与GenBank中报道的其它植物来源的RCA有较高的同源性。RCA的部分编码区插入原核表达载体pET30a(+),并转化到大肠杆菌菌株BL21中。经过IPTG诱导,RCA的部分编码区在BL21菌株中成功表达。将得到的RCA基因片段反向插入植物表达载体,构建了RCA基因反义表达载体pBI121-RCA。本研究为深入研究光合作用的机理以及阐明RCA与1,5-二磷酸核酮糖羧化酶相互作用和调控关系奠定了基础。更多还原

【Abstract】 Phytoplasmas are wide spread pathogens responsible for a broad range of plant diseases and has caused huge losses to agriculture and forestry. The mulberry dwarf disease is one of the most destructive diseases of mulberry and has caused many mulberry trees in large areas destroyed. It is a serious restricting to the development of sericulture. No research laboratory is currently able to cultivate phytoplasmas in cell-free medium, making progress in their study slow. Little is known about the underlying molecular mechanisms for the symptoms evoked in the host plants. With the availability of the complete genome sequence of phytoplasma, attention is now shifting to the components specified by such genomes. In particular, it is becoming increasingly important to confirm that the predicted genes encode bona fide proteins. Proteomics is becoming one of the most important researches in the post-genomic era. Little is known about the underlying molecular mechanisms for mulberry dwarf disease and these is no reporter about the mulberry dwarf disease phytoplasma responsive proteins in mulberry leaves to my knowledge. In this research, the proteome of mulberry dwarf disease phytoplasmas and its responsive proteins in mulberry leaves were studied to provide a better understanding about the underlying molecular mechanisms for mulberry dwarf disease.In this study,the expressed proteome of phytoplasma was surveyed by using shotgun strategy. A combination of one-dimensional SDS-PAGE with capillary liquid chromatography-tandem mass spectrometry allowed a total of 242 phytoplasma proteins to be unambiguously assigned, including amino acid biosynthesis, cell envelope, central intermediary metabolism, cellular processes, energy metabolism, fatty acid and phospholipid metabolism, nucleosides and nucleotides metabolism, replication, transcription, translation, transport and binding proteins and some other function proteins. In addition to those known function proteins, 76 proteins previously was annotated as hypothetical or conserved hypothetical. Taken together, 35% of the predicted proteome for phytoplasma has been experimentally verified, representing the most extensive survey of any phytoplasma proteome to date. This research not only provides a technique to study phytoplasma proteome, but also a valuable dataset of phytoplasma proteins, thus providing better understanding of the functional mechanisms of phytoplasma in many biological processes.In this study, differential proteomic analysis was conducted to characterize the proteins in the mulberry leaf that were differently expressed in responsive to mulberry dwarf phytoplasmas with 2-DE, MS and MS/MS. The gels were analyzed by ImageMasterTM 2D Platinum software. About five hundred reproducible protein spots were detected, among which 37 protein spots displayed differential expression. There were 19 up-regulated and 18 down-regulated protein spots in the diseased leaf. MALDI-TOF MS and MALDI-TOF-TOF MS analysis followed by database searching helped to identify 18 spots representing 15 different proteins. The identified proteins include Rubisco activase, Sedoheptulose-1,7-bisphosphatase, RuBisCOLSU, Defense protein-related, Protein tyrosine phosphatase, NUDIX/mutT hydrolase family protein, Maturase K, Kunitz proteinase inhibitor-1, 20S proteasome subunit, 33 kDa precursor protein of oxygen-evolving complex, Malate dehydrogenase, Methionine sulfoxide reductase, Gm-ck32857, F-box protein and unknown protein. These proteins could be grouped into the categorizations such as photosynthesis, amino acid biosynthesis, nucleotides metabolism, transcription, defense response, signal transduction and regulation. This research provides a better understanding for the molecular mechanisms of mulberry dwarf disease.Sedoheptulose-1,7-bisphosphatase (SBPase) is a key enzyme in the regenerative phase of Calvin cycle. A full-length cDNA encoding SBPase (designated as MSBPase, GenBank accession No. DQ995346) was cloned from mulberry by rapid amplification of cDNA ends. The cDNA was 1 527 bp containing a 1 179 bp open reading frame which was deduced to encode a peptide of 393 amino acids whose predicted molecular mass was 42.6 kDa and isoelectric point was 5.85. Sequence comparison analysis showed that the SBPase from mulberry (MSBPase) had highest homology to SBPases from other plants. It was predicted that the structure of MSBPase was rich in coils and helixes, and was poor in strands. The coding region of the MSBPase was inserted into an expression vector, pET30a (+), and transformed into Escherichia coli BL2l. The fusion protein was successfully expressed with IPTG induction. The plant expression vector with this fragment under the control of 35S promoter was constructed and transformed into Arabidopsis thaliana plants. Northern blot and Western blot analysis indicated that the MSBPase was expressed successfully in the Arabidopsis thaliana plants. Activity of SBPase was increased by overexpression of MSBPase in Arabidopsis thaliana plants. In plants with increased SBPase activity, photosynthetic rates were increased, higher levels of soluble sugars and starch were accumulated and an increase in dry weight was also evident. Compared to wild-type plants, the onset of flowering was advanced. The results of this study may be useful in the mulberry gene engineering and the results may be helpful to study the regulation of SBPase.Rubisco activase (RCA) is a soluble chloroplast protein, coded in nucleus, and has the activation of Rubisco in photosynthetic autotrophs. The degenerate primers designed based on the conserved sequences among the known RCA were used to amplify the RCA fragment by PCR using the first strand of cDNA as templates. Amino acid sequence analysis indicated that the sequence deduced from the cloned cDNA fragment showed highly homology to other plant RCAs. The fragment of the RCA coding region was inserted into an expression vector (pET30a) and then was transformed into the Escherichia coli BL2l. The coding protein was successfully expressed in the Escherichia coli BL2l with IPTG induction. The antisense expression vector with the same fragment under the control of 35S promoter was constructed. The results of this study may be helpful to study the photosynthetic mechanism, the relationship and regulation between RCA and Rubisco.更多还原