Proteomic Study On The Pathogen Of Mulberry Dwarf Disease And Its Responsive Proteins In Mulberry Leaves

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.