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Fine Structure Mapping Of AST Gene In Arabidopsis

Posted on:2003-08-09Degree:DoctorType:Dissertation
Country:ChinaCandidate:A J MaoFull Text:PDF
GTID:1100360092966072Subject:Botany
Abstract/Summary:PDF Full Text Request
The ast (anthocyanin spotted testa) mutant,which was induced by carbon ion beam,was a single recessive gene mutant of Arabidopsis thaliana and involved in the anthocyanin biosynthesis. Due to the abnormal anthocyanin accumulation,the purple spotted pigmentation was observed in the immature seed coats. In the wide type plants,the normal immature seeds were pale green. A mapping analysis revealed that AST gene was located on chromosome I,and located between SSLP molecular marker nga280 and CAPS molecular marker PAB5. AST gene was closely linked to nga280 with a genetic distance of 3.2 cM. The genetic distance between AST gene and PAB5 was 21.6 cM.DDRT-PCR method was exploited to study the differential gene expression between immature siliques of Arabidopsis wide-type and ast mutant. The procedure of DDRT-PCR applicable for silver staining was optimized by adjusting the amount of several critical reagents,including total RNA,anchor primer,arbitrary primer,cDNA and dNTP. The PCR amplification products were separated on 6% vertical denaturing polyacrylamide gels. Numerous and distinct bands could be detected by silver staining. The minimum number of bands in one lane was 40,the maximum was 80 and the average was 60. The range of displayed PCR products extended from about 100 base to about 900 base. The sensitivity of silver staining was 5pg/mm2. This procedure was simple,time-saving,highly sensitive and reproducible. Based on the improved procedure,nearly 16,000 cDNA fragments were analyzed between the immature siliques of the wide type and ast mutant,and 28 differential cDNA fragments were screened. After the second PCR amplification,10 differential cDNA fragments were identified among which 6 fragments were wild type specific and 4 fragments were ast mutant specific. All of the 10 differential cDNA fragments were sequenced. BLASTN analysis showed that each of the 10 sequences had no homology with the structural genes or regulatory genes in the anthocyanin biosynthesis. It was suggested that there were some limitations to isolate the specific AST gene by DDRT-PCR.The map-based cloning strategy was used to clone the AST gene. A series of molecular markers were designed according to the SNPs (simple nucleotide polymophisms) and the insertion/deletion polymophisms in Arabidopsis database. With these molecular markers,fine-structure mapping of the AST gene was finished. The AST gene was located in BAC clone T13M11. It was suggested that the gene T13M11.8 could be the AST candidate gene. This gene was 1432bp long with 6 exons and 5 introns. The putative protein of T13M11.8 gene was highly homologous to dihydroflavonol 4-reductase (DFR),which was an important enzyme in the anthocyanin biosynthesis pathway. The functional complement experiments are in progress.
Keywords/Search Tags:Arabidopsis, ast mutant, AST gene, DDRT-PCR, silver staining, map-based cloning, molecular marker, fine structure mapping
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