Differential Proteomics Research During Juice Sacs Development In Citrus Grandis Cv. Guanximiyou

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.