Construction Of Non-Heading Chinese Cabbage Genome Database And Microrna, EST-SSR Data Mining And Analysis Of Brassica Crops

Non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) is one of the most important vegetable crops in the Brassica genus. Non-heading Chinese cabbage, known as Pak Choi, originated in China, is one of the most popular vegetables crops in eastern Asia like China, Korea and Japan and is now common in Europe and America. The molecular biology of non-heading Chinese cabbage was lagged behind other model plants due to the absence of genomic sequence information. It is important to construct the non-heading Chinese cabbage genome database. It will be help to characterize the gene function and promote the accession improvement and germplasm enhancement of non-heading Chinese cabbage. It is biological and genetic base for deeply knowing the origin, evolution, natural variation of non-heading Chinese cabbage. In this present study, non-heading Chinese cabbage genome database was constructed. And miRNA and EST-SSR of Brassica genus was searched by comparative genome method, and some miRNAs were validated by qRT-PCR experiment. The results are as follows:1. Construction of non-heading Chinese cabbage genome databaseRecently the whole genome sequence of non-heading Chinese cabbage has been obtained. But the database platform of bioinformatics is not used, so construction of non-heading Chinese cabbage genome database is of great significance. In this study, genome sequencing data of non-heading Chinese cabbage has been processed by bioinformatic technology, a server running environment and website has been set up in IBM high performance computing server. Non-heading Chinese cabbage genome database has been constructed by Linux, Apache, MySQL and PHP softwares, providing a variety of functions such as whole genome browsing, sequence alignment and search, physiological and morphological data query, photo data of numerous cultivars browsing and data download service for researchers. 2. Identification of conserved microRNAs and their targets in Chinese cabbage (Brassica rapa subsp. pekinensis)In the present study,168potential miRNAs, derived from22EST and119GSS sequences in Chinese cabbage were identified and classified into38miRNA families by well-defined computational analysis, in which most belonged to the miRNA1533, miRNA156, and miRNA2911families. Totally, there are129identified miRNAs potentially targeting1,386Chinese cabbage EST genes, which play roles in multiple biological and metabolic processes including metabolism, cell growth, signal transduction, stress response, and plant development. Gene ontology analysis, based on these target proteins, showed that688,532, and287genes were involved in molecular functions, biological processes, and cellular components, respectively. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis demonstrated that these miRNAs participated in214metabolism pathways, including of plant-pathogen interaction, fatty acid metabolism, amino acid metabolism, nitrogen metabolism, plant hormone signal transduction.3. Expression analysis of miRNA in different tissues and involved in cold response of non-heading Chinese cabbageThe objective of this study was to determine expression pattern of miRNA in different tissues including root, stem, leaf and flower during cold response of non-heading Chinese cabbage inbred line’Suzhouqing’by using qRT-PCR (quantitative real-time polymerase chain reaction). All miRNAs were expressed in four tissues with different level. Bra-miR390, bra-miR529and bra-miR845were up-regulated in flowers, and down-regulated in root. Expressions of non-haeding Chinese cabbage miRNAs under cold treatment were validated by qRT-PCR. The result showed that9miRNAs including of bra-miR390, bra-miR156, bra-miR158, bra-miR162were up-regulated and bra-miR529, bra-miR167were down-regulated under cold treatment (4℃) in leaf tissue. Other miRNAs were expressed without differential level.4. Identification and characterization of microRNAs and their target genes in Brassica oleraceaBy computational analysis and comparative genomics,193potential miRNA candidates were identified from17ESTs and152GSSs in B. oleracea. These miRNA candidates were classified into70families using a well-defined comparative genome-based computational analysis. Most miRNAs belong to the miRNA169, miR5021, miR156and miR158families. Of these,36miRNA families are firstly found in Brassica species. Total of1,393B. oleracea genes were predicted as candidate targets of175miRNAs. The mutual relationship between miRNAs and the candidate target genes was verified by checking differentially expression levels using qRT-PCR and RLM5’RACE analyses. These target genes participate in multiple biological and metabolic processes, including signal transduction, stress response, and plant development. Gene Ontology analysis shows that the818,514, and265target genes were involved in molecular functions, biological processes, and cellular component respectively. KEGG Pathway enrichment analysis suggests that these miRNAs might regulate186metabolic pathways, including those of lipid, energy, starch and sucrose, fatty acid and nitrogen.5. In silico detection of EST-SSRs in Brassica species and application in Brassica rapa25,631EST-SSRs from1,017,392Brassica ESTs are identified. These SSR-motifs average25bp in length, and tri-nucleotide type of SSRs are the most abundant. Among them, AGG/CCT is the predominant motif, accounting for25%. A total of20,683primer pairs are successfully designed, and some of the primer pairs from B.napus and B.oleracea can be successfully transferred to B. rapa by comparative analysis and PCR amplication.