Construction Of Chinese Cabbage Expressed Sequence Tags Database And Analysis Of The Genes Expression Under Erwinia Carotovora Stress

Chinese cabbage (Brassica rapa subsp. pekinensis) originated from China, is one of Chinese people's favorite vegetable and widely planted in east Asia. Soft-rot disease caused by soft-rot pathogens Erwinia carotovora subsp.carotovora (Ecc), is one of the three important diseases of Chinese cabbage, and often causes product quality reduction and bad quality. Due to lacking available disease resistance materials and limitations in understanding the resistant mechanisms, Ecc-resistance breeding has been limited in Chinese cabbage for a long time. Studies on the melocular interaction between Chinese cabbage and Ecc will provide assistance for solving the above problem. In this study, sequenced large scale cDNA clones from Ecc-diseased Chinese cabbage SSH cDNA library, constructed a Chinese cabbage ESTs database, developed the operation softwares to store and analyze these sequences, and made cDNA microarrays and finally analyzed gene expression in Chinese cabbage infected by Ecc. In addition, lignin biosynthesis and its composition was studied for understanding the roles of lignin in resisting Ecc. The main results are as follows:1. Obtaining and analyzing the Chinese cabbage ESTs from SSH cDNA library.A single-run sequencing was performed on soft-rot diseased Chinese cabbage SSH cDNA library, by which 6, 282 ESTs were obtained. Assembly analysis was performed to remove redundant sequence using SeqmanII software in DNAStar6.0 package, and 2975 clusters (uniESTs) generated, including 1173 contigs and 1802 singletons.UniESTs were annotated by searching against the local Non-redundant database (download from NCBI site) using BLAST(x) arithmetic, then were grouped into known function and unknown function categories according to the annotated results. 1,978 UniESTs in known function category were classified into 11 groups,, in which genes in high proporation concern basic metabolism (426 genes, 14.33%), defence and stress(262 genes, 8.81%), inter/intra cellular transporter (247 genes,8.30%), energy metabolism(183 genes, 6.16%), signal transduction(179 genes, 6.02%), secondary metabolism (160 genes, 5.38%), etc. In unknown function group, 485(16.31%) genes without significant similarities compared to genes in other organisms (including 49 "no hits found") in GenBank, suggesting these may be new sequences. The other 512 have significant similarities to genes in other organisms, but their function is unknown now.Among 2, 975 uniESTs, the clones of 61 (2.05%) genes were sequenced at least for 10 times in SSH cDNA library, and mainly consisted of photosynthesis related genes and defence/stress related genes. The former included genes encoding the light-harvesting chlorophyll a/b binding protein, NADPH oxidoreductase, ribulose bisphosphate carboxylase, oxygen-evolving enhancer protein etc; the latter included genes encoding chitinase, catalase, senescence-associated family protein, defensin, PR4-type protein, heat shock protein HSP70-1 etc.2. Construction of the Chinese cabbage ESTs database and compiling related operation softwareIn order to improve data process, I used several bioinformatics softwares, including Phred and DNAstar, constructed the a Chinese cabbage ESTs database, developed Perl script, the database operating software Genemaster, online searching system and ESTs data converting softwere Convert in processing the ESTs data. The format of ESTs storaged in Chinese cabbage ESTs database was similar to those in the dbEST of GenBank (NCBI). These systems facilitated the procedure in searching, editing, analyzing and using the ESTs data.3. Construction of cDNA microarray and analysis of gene expression profiles in Chinese cabbage infected by EccIn order to monitor the gene expression profile, 4128 ESTs were selected from Ecc diseased Chinese cabbage SSH cDNA library (6, 282) and heading leaf cDNA library (2,372), and the inserts in representive clones corresponding to the ESTs were amplified by PCR to make cDNA microarrays. The cDNA microarrays were performed to monitor the gene expression in Chinese cabbage at 0.5, 2, 6, 9, 12 and 24 hours post inoculated (hpi) with Ecc pathogens, respectively.As a result, 913 (22.11%) differentially expressed genes were identified, including 488 up-regulated, 309 down-regulated and 116 up/down-regulated at different timepoints. The number of differentially expressed genes varied depended on time-points. The number of differentially expressed genes increased significantly from 0.5 to 2 hpi, then decreased gradually at 6 hours, and by 24 hpi, the number of up-regulated and down-regulated expressed genes at 24 hpi was 1/2 and 1/4 of those at 2 hpi, respectively. These suggested genes were regulated positively in the first 6 hours after inoculation, and it may be an important phase in the regulation of defensive reaction in Chinese cabbage in response to Ecc.By using Cluster analysis, the genes were clustered into four groups according to their expression pattern. Genes in first class, e.g., genes in photosynthesis pathway, were up-regulated at 2hpi while down-regulated or normally expressed at other time points, suggesting the decrease of photosynthesis rate may be related with the damage of ROS. Genes in second class, e.g., senescence-associated cysteine protease, chitinase, catalase genes etc, were up-regulated at 0.5hpi while down-regulated or normally expressed at other time points, suggesting these may be early defensive response genes in Chinese cabbage against the Ecc infection. Genes in third class, e.g., genes in basic metabolism and protein synthesis, were down-regulated at 0.5 and 2 hpi while up-regulated or normally expressed at other time points, suggesting expression of these genes were suppressed in Chinese cabbage at the early stage of Ecc infection. Genes in fourth class were mainly up-regulated in the majority of time points, including genes involving in signal recognization and transduction, synthesis of hormones, protein degradation, transcription, secondary metabolism, and pathogen/stress defensive response et al, suggesting these genes played important roles in Chinese cabbage in the defensive response to infection of Ecc. In addition, one-third of differentially expressed genes were unknown function and distributed in all the four class, suggested that these genes may be new genes regulated by the Ecc infection and their roles should bc further investigated.4,Role of lignin in Chinese cabbage subjected to Ecc.To obtain nutrition from plants, Ecc infection mainly depends on destructing the plant cell wall by the secretion of cell wall degradation enzymes, while the lignification of cell wall conduces to block the penetration and the diffusion of pathogens. Microarray results indicated that several genes associated with lignin biosynthesis pathway were up-regulated. For better understanding the role of lignin pathway in resisting Ecc, variation of lignin content and gene expression was investigated in Ecc infected Chinese cabbage. H₂O₂ accumulation and peroxidase activity were detected by 3, 3'-Dmethoxybenzidine staining at mocked and Ecc inoculated site of Chinese cabbage leafstalks as compared with those of untreated plants. Klason lignin content in inoculated plants was increased about 7.84%, 40.37%, and 43.13% more than that in mocked site at 12, 24 and 72 hour after inoculation, respectively, suggesting the biosynthesis of lignin was activated. The nitrobenzene oxidative products of cell wall residue from untreated, Ecc inoculated and mocked plants were detected by GC-MS, respectively. GC-MS results demonstrated that there were more p-coumaryl (H) and less coniferyl (G) and sinapyl (S) monolignins in leafstalks of Chinese cabbage, and all three monolignins increased in mock and Ecc inoculated plants while G and S monolignins increased significantly than H monolignin at 72dpi.There are ten genes in lignin biosynthesis pathway, including phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumarate coenzyme A ligase (4CL) in general phenylpropanoid metabolism; courmate 3-hydroxylase (C3H), caffeic acid O-methyltransferase (COMT), caffeoyl-CoA 3-O-methyltransferase (CCoAOMT), ferulate-5-hydroxylase (FSH), Hydroxycinnamoyl CoA: shikimate/ quinate hydroxycinnamoyltransferase (HCT) involving in biosynthesis of G and S monolignins and cinnamoyl-CoA reductase and cinnamyl-alcohol dehydrogenase involving in the synthesis of three monolignins. In this study, two lignin biosynthesis genes were cloned by 3'or 5'RACE method. One C4H gene have 1722bp in length with 1518 bp of CDS encoding a 57.63 KD protein; another gene CCoAOMT have 1106 bp in full-length cDNA with 777 bp of CDS encoding a 29.02 KD protein. Liguin contents are associated with the expression of genes in lignin biosynthesis, 12 ESTs from genes putatively encoding enzymes involved in lignin biosynthesis were selected to study their expression。RT-RCR results showed that all these genes could be induced by mock inoculation and Ecc infection and the expression pattern was consistent with the increase of lignin, among which expression of COMT and CCoAOMT was consistent with the increase of lignin. Our results indicated that Ecc induced "defence lignin" was different from developmental lignin in composition; G and S monolignins were positively regulated by plants in response to the soft rot Ecc. Thus, G and S monolignins may play important role in resisting Ecc in Chinese cabbage.