Research of Improving Success Ratio of T-DNA Flanking Sequence Protocol

• Main Authors: CHO, KUAN-YU, 卓冠佑
• Other Authors: FAN, MING-JEN
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/578t7y

碩士 === 亞洲大學 === 生物科技學系 === 105 === Rice rice (Oryza sativa L.). Gramineae monocotyledons. As one of the important food. Recent studies on rice functional genes are mainly focus on Transfer-DNA (T-DNA) as a tag. To obtain T-DNA insertion mutations site, Flanking Sequence Tag (FST) is indispensable for reverse genetics studies. Therefore, to stablish T-DNA insertion mutations point of FST database, can accelerate rice functional genomics researches. The establishment of this database for rice functional gene research has a great help. At present the total number of more than 120,000 mutant plant lines. And entrusted the Asian University T-DNA Taged Rice Service Center(TTRSC) to deal with the rice T-DNA insert mutant seed preservation and sequencing flanking sequence of T-DNA insertion site . The team led by Yu Shu-mei of the Central Research Institute used the TN67 T-DNA insertion mutant to establish the Taiwan rice insert mutant population and related databases(Taiwan Rice Insertional Mutants Database,TRIM). For many years, it was believed that only the DNA between the repeats, the T-DNA, and not the external vector DNA is transferred to the plant cell. However, recent and more detailed characterization of the DNA inserts in transgenic plants demonstrate that very frequently also vector backbone sequences are integrated into the plant genome. Sequencing of the T-DNA flanking sequence need to face uncertainties in the PCR experiment and sequencing then blast rice genome and plasmid sequence database. Researcher can confirm the success of the FST in the way which is time-consuming and costly. The data used for this study is Taiwan Rice Insertional Mutants Database. We analysis data according to the results of PCR outcomes to explore the reasons for success and failure and comparing of the superior of PCR method. The successful rate of sequencing is 52%. A total of 27878 lines but 14774 lines is successfully sequenced. It doesn’t achieve the ideal situation. The determinants of failure include plasmid pollution and Backbone sequence. This study has confirmed that the Backbone sequence is one major determinants for the failure of the sequencing. Removing samples of Backbone before sequencing can cut costs and increase the successful rate of the experiment. This study aimed to use Taiwan rice insertional Mutagens (TRIM) to compare the Flanking Sequence inserted sequence which is found has duplication and compare the single nucleotide polymorphism (SNP) of both site. In this way, the different function of gene insertion site on rice genome can be compared. This researcher use Basic Local Alignment Search Tool (BLAST) combined Perl programming language to construction a program named the "rice T-DNA insertion mutants Flanking Sequence alignment automation system”. And use this system to distinguish the sequences obtain from PCR, such as of rice T-DNA sequence, plasmid sequence and rice gene sequences. This database is the foundation of rice FST laboratory backboned for analysis FST and BLAST sequence alignment. The research expected to find the same expectations with different insertion point chromosomes. In order to explore the effects of gene (Os11g0143600) insertion points on rice, this study will compare 200bp insertion point sequence to find high similarity of repeats and compare their differences. To analysis the insertion point and gene SNP, the results between the insertion point and a total of seven gene SNP C/T、G/C、G/A、G/C 、G/A 、A/G、T/C. Differential gene expression of a type known these SNP regard, need to do more field research. In this study, 20 DNA lines were subordinated by IRRI, A total of 18 lines of success order, the success rate increased to 80%, while the replacement cost from the original 28600 down to 17400, about 40% reduction in the cost of the order.