Identification System Optimization Of The Rice Telomerase Rna And The Confirmation Of The Candidate Sequence

Telomere is a complex that formed with telomeric DNA and binding proteins, it can protect the ends of chromosomes that different from the general, and prevent recombining the chromosome ends. Telomerase is responsible for telomere synthesis, and consist of telomerase reverse transcriptase(TERT) and telomerase RNA(TER). TERT subunit of telomerase uses its TER subunit as a template to continuous replicate telomere DNA, thereby compensating for the end of the chromosome during replication process, to ensure the chromosome replication and structural stability.It has been reported that telomerase RNA had cloned from protozoans, yeast and human, also confirmed that the TER component plays an important role in maintaining the functionality of telomerase, but not had the length and sequence similarity between these species. And in addition to the conservation of telomerase RNA template sequence, the remaining regions are low, it is difficult to implement cloning based on homologous sequences. Currently, only Arabidopsis thaliana has been reported of cloning the telomerase RNA sequence in the plant. We also tried a number of occasions to clone telomerase RNA of rice(Oryza sativa L.) based on the RNA sequence of Arabidopsis, but were all unsuccessful. This study was designed to optimize a build rice telomerase RNA identification system, and to identify candidate sequences obtained with a laboratory rice TER features.This study aims at optimizing the established identification system of rice telomerase RNA, and identifying a candidate sequence with rice TER features that obtained from laboratory. Sequence alignment analysis through transcriptional regulatory regions of rice candidate sequences and Arabidopsis telomerase RNA sequence, it is found that they have similar transcriptional regulatory elements and related features. The mature embryos of Zhonghua 11 cultivar(Oryza sativa L. subsp. Japonica) were used as materials to induce callus. Through the site-directed mutagenesis T/A(5’-AAACCCTAA-3’) of the template sequence, non-mutant and mutant sequences were loaded into p MD18-T vectors for sequencing. Correct sequencing sequences were loaded into p Cambial1301 carriers to construct the non-mutant and mutant shuttle vectors, and using Agrobacterium-mediated method to introduce the two sequences into rice callus. By designing specific primers to verify the transgenic callus after extracting genomes and telomerase protein from resistant callus, then proceeding the subsequent induction of transgenic plant cultivation.Detection process of telomerase activity from rice callus showed that the telomeric repeat amplification protocol in the absence of d ATP, mutant telomerase activity was significantly higher than the no-mutant type. And sequencing the ss DNA extended from telomeres in vitro, the results showed that ten sequenced fragments all contained the sequences with characteristic features of mutant; then the telomeric sequences of the transgenic plants were identified, results showed that there were two containing the sequences with characteristic features of mutant out of seven sequenced telomeric sequences.In this study, comparison of the telomerase activity of mutant callus, as well as the identification of transgenic plants telomeres, further demonstrates the target sequence is the telomerase RNA gene of rice, but it still needs further comprehensive identification. Our study also verify the operability of the optimized identification system of telomerase RNA in rice, it establishes a reliable method and platform for further research.