Font Size: a A A

Study On Cloning Of Gene Related To The Leafy Head Formation From Pac-choi And Transgenic Plants With Exogous BcpLH Gene

Posted on:2002-10-01Degree:DoctorType:Dissertation
Country:ChinaCandidate:W X XueFull Text:PDF
GTID:1100360062475702Subject:Vegetable science
Abstract/Summary:PDF Full Text Request
The genomic fragment, designated as BccLH, homologous with the BcpLH gene of Chinese cabbage was isolated from the total genomic DNA of Pac-choi by PCR method, with a pair of primer designed according to the sequence of BcpLH. BccLH gene was compared to BcpLH gene in the aspects of genomic DNA sequence, cDNA sequence, as well as amino acid sequence and secondary structure of protein. BccLH gene is 1544bp in length and has 96.1% homology with BcpLH gene. Southern blotting indicated that there is only one copy of BccLffgene in the genomic DNA of Pac-choi. 2 introns of 485bp and 197bp respectively and 3 extrons are contained in BccLffgene, the same to BcpLH gene. It is the third extron in which exists the main difference between the BcpLH and BccLffgene. Many substitution and deletion of bases occur in the third extron of BcpLH gene compared with BccLH. Especially in the site from 1427-1448bp in the downstream to the TAG, BcpLH gene has deletion of 21 successive bases. There are 28 substitutions of bases all together in the third extron of BccLffand BcpLH. Little difference exists in the other sites of BcpLH and BccLH gene. It is suggested that the leafy head formation of Chinese cabbage may arise from the sequence variance of the third extron of BcpLH gene, compared with BccLH gene.The complete cDNA of BccLH contains 852bp in its read frame, 27bp more than that of BcpLH and 93. 1% homologous with BcpLH. The cDNA of BccLH encodes 283 amino acids, which shares 92. 9% homology with BcpLH, and 9 amino acids more than that of BcpLH. Compared with BccLH, there exists successive deletions of 7 amino acids, th^t is - P-E-N-A-E-T-M-, which begins from the 278th amino acid to N-terminal in sequence of amino acid of BcpLH gene. No published gene sequence has been found in the gene database, which shares high homology with BccLH gene except BcpLH and BoLH gene. The same to BcpLH, two fragments in BccLH gene are high homologous with the domains of double-stranded RNA binding motif(DRBM), but only one amino acid difference in the second fragment. Two DRBMs of BccLH have 51% and 59% homology respectively compared with the corresponding domains of RNA binding protein TRBP of human' . The secondary structure of protein of BcpLH and BccLffhas no difference prior to the 250th amino acid, but little difference exists after that site. This may be one of the important explanations that the Chinese cabbage and Pac-choi have different fashions of development during their growing period.According to the cDNA sequence homologous with BcpLH , the evolution tree of 8 homologous genes from 6 different species was drawn, which include Chinese cabbage,cabbage, rape, arabidopsis, rice and Pac-choi. Evolutionary relationships of 6 species, which were revealed by the evolution tree and plant category respectively, are identical.The function of BcpLH gene on morphogenesis of leafy head of Chinese cabbage was studied by non-tissue culture method, with which sense BcpLH gene and antisense BcpLH gene were transferred into Pac-choi and Chinese cabbage respectively. The transformation frequency is up to 0. 63% by using the method of vacuum infiltration on Chinese cabbage. While the transformation frequency of oval injection is 1.8% on Pac-choi. The kanamycine-resistent seedlings screened by tissue culture can root very well when subcultured on the MS medium supplemented with 0. 5mg/L NAA after cut at their bases. The survival rate of the kanamycine-resistant seedling with so many roots was increased obviously. Comparison of different screening methods indicated that seeds germinating in the plate containing kanamycine solution with the sponge and filter paper as supporter is the best method, in which a secondary screening by transplanting is needed. This method, designated as water culture method, is not only convenient to operate but also has a high screening efficiency, by which more seeds can be screened in one test. Appropriate concentration of kanamycine screening by water culture method is 50ug/ml on Chinese cabbage and 40 ug/ml on Pac-ch...
Keywords/Search Tags:BcpLH gene, BccLH gene, leafy head formation, transgenic plants, vcacuum infiltration, oven injection, Silwet L-77, gene transformation evolution tree, double-stranded RNA binding motif, copy number, Chinese cabbage, Pac-choi
PDF Full Text Request
Related items