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Expressed Sequence Tage Analysis And Gene Expression Profile Of Heading Leaf Of Chinese Cabbage (Brassica Rapa L. Ssp. Pekinensis)

Posted on:2005-09-28Degree:DoctorType:Dissertation
Country:ChinaCandidate:D P DaiFull Text:PDF
GTID:1103360122993370Subject:Botany
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China is an origin center of Chinese cabbage {Brassica rapa L. ssp. pekinensis) which is one of the most important vegetables in Far East. Under the co-effects of natural evolution and artificial selection the leaf head serving as the nutrition deposited organ has been formed. Studying the mechanism of heading process is very important in practice, especially for the aim of increasing the yield and quality of Chinese cabbage. Unfortunately, the pace for studying the mechanism of heading formation is just at the begining so far. A A ZipLox mediated cDNA library using the leaves of Chinese cabbage at the early heading stage had been constructed, in order to explore the gene expression pattern of heading leaf from the genomics level. Our work was to achieve the designed aims by analyzing that library further. Several main results and conclusions were following:After transfecting Escherichia coli DH10B with the A ZipLox cDNA library we finally got 2697 plasmid clones and exacted all the plasmid among them. Totally 2802 sequences with better results were acquired after sequencing with DNA sequencer (Beckman & Coulter CEQ8000 genetic analysis system) or with the help of two biological company Shenggong and Dingguo Co.. We also studied the factors influencing the sequencing reaction and found that high purity and appropriate amount of plasmid template were the vital factors for obtaining good sequencing results.The sequencing results indicated that 268 clones had inserts shorter than 150bp or only polyA sequences and 162 clones had no cDNA inserts. The sequences of remaining 2372 clones, occupied 84.6 percent of all the sequencing results, were used for further analysis. Total length of all the usable sequences was 1088467 bp, and the average length per EST was 459 bp, which was longer than that of other researchers reported.The SeqMan II module of DNAStar software was used for contig analysis of all the 2372 ESTs, and a total of 1641 different contigs were obtained. Total length of all the contigs' consensus sequence was 782616 bp, and each contig had 477 bases for average. Further analysis revealed that 1247 contigs belonged to singletons which included only one EST in one contig. Combined with the later blast align results we predicted that the real number of non-redundant sequences was around 1499, so the redundant sequence occupied about 36.8%.After being compared with the non-redundant protein (nr) databases of NCBI using blastx program, 1232 contigs which included 1810 ESTs shared significant similarity with sequences registered in the database. For the sequences with lower score than 80 or even having no homologous in the nr database, biastn program was carried out for finding homological ESTs in the estothers database of NCBI. Searching results showed that 295 contigs which included 432 ESTs could find homologous, and the remaining 114 contigs accounting for 131 ESTs still could not find any homological ESTs. These new ESTs might be the sequences discovered firstly in the world.By analyzing the homological proteins' sources of the organisms, it was found that majority of the functionally known protein came from Arabidopsis thaliana; however, at the nucleotide level, only 51% (1205/2372) of the total ESTs showing highest homology to ESTs coming from Arabidopsis thaliana. This result suggested that gene expression profiles of Chinese cabbage differ from that of Arabidopsis thaliana. Sequences with lower similarity to the Arabidopsis nucleotide could be specially used to study the unique developmental process and metabolic pathway in Chinese cabbage, and those sequences were also important for the genetic map drawing in the future.For the ESTs whose homological protein had elucidatory functions, functional assignment was carried out based on the method used in the Arabidopsis thaliana genome sequencing initiative. The results revealed that ESTs of the genes involved in the process of protein synthesis had the highest proportion which took up 22.5 percent of all the analyzed ESTs, then in turn were the ESTs...
Keywords/Search Tags:Expressed sequence tags (ESTs), Expression profile, Functional category, PolyAPCR method, Dot-blotting, Heading leaf, Rosette leaf, Chinese cabbage (Brassica rapa L. ssp. pekinensis)
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