Based On Kanamycin Resistance Construct System To Study The Structure And Function Of Group ⅠIntron And The Study Of The Rehoming Mechanism Of The Group Ⅱ Intron In T Erythraeum

Both groupⅠintron and groupⅡintron belong to self-splicing ribozymes, they are catalytic RNAs and could catalyze their own excision from its mRNA, tRNA, rRNA precursor with the ligation of their flanking exons. The typical second structure of a groupⅠintron has approximately ten paired elements. GroupⅡintron RNAs are characterized by a conserved secondary structure, which is organized into six domains, DI-DVI, radiating from a central "wheel", of which the DIV loop encodes the IEP with reverse transcriptase, maturase and endonuclease activities. These activities have been found to play important roles in splicing and mobility of groupⅡintrons. The difference of the self-splicing between them is that the groupⅠintrons' transesterification reaction using a guanosin molecule as a cofactor. And the groupⅡintrons use an internal bulged adenosine to initiate self-splicing.The first part of this study is to construct a system that based on kanamycin resistance screening to study the groupⅠintrons'structure and function in E.coli. As we known, groupⅠintron has become an ideal mode molecular to study the structure and function of RNAs, and around such molecules carried out base mutation testing is always the primary means of these studies. A rare groupⅠintron which is 383nt nucleotides long, has been found in the cyanobacterial ribonucleotide reductase gene of Nostoc punctiforme. In our research group I intron Npu RIR was inserted into kanamycin resistance (KanR) gene of the pDrive plasmid, the results plasmid was named pKR12. As the intron is within the heterologous gene and this insertion without its original exons, so it lost its intrinsic splicing activity and E.coli cells contain this plasmid could not grow up on the Kan resistance LB plate. Then we introduce random mutations into the intron using error-prone PCR and gain a large number of mutations successful. After the ligation into pDrive vector and transformation into E. coli (DH5a), We constructed a clone library and approximately about 1000 colonies grew up on the Amp&Kan double antibiotic resistance plates, Bacilli PCR and Enzyme digest test eliminate false-positive results and 34 plasmids with mutated introns in kanR gene were gained. Select one of the mutations pKR12m-3 and carry out RT-PCR identification, the results show that the mutation intron in the kanR gene has splicing activity. In this way, we set up a new system successfully to carry out base mutation testing to study intron RNA structures. Using this system we could select the group I introns with splicing activity easily and successfully by screening of KanR colonies on antibiotic plate. Due to the correlation of the RNAs'structure and function, then we can get more information about its structure.The second part of the study is the extension of the first one, since this system was constructed successfully, the next experiments involves senior structure study of the Npu RIR intron. We introduced random mutations at the site 2581～2585 in the P5 loop and at the site 2815～2818 in the P8 loop. Subsequent resistance plate controlled trials show that the mutations of these two sites lead to the intron lost its splicing activity, so these two sites are necessary to the Npu RIR introns sstructure. The next is designed to regain its splicing activity by directed evolution based on error-prone PCR, then may gain something more information about the senior structure, but there has no positive results gained and therefore not able to achieve the ultimate goal, so structural information could not be further advanced.The part III of this study explored the retrohoming mechanisms of groupⅡintrons come from marine cyanobacteria Trichodesmium erythraeum. Two groupⅡintrons have been found in theβ-subunit of DNA polymeraseⅢgene and IMP dehydrogenase gene of trichodesmiu erythraeum. The donor plasmid vector pDN1-C,pDIDD-C with intron contain group II intron DNA sequence and the recipient vector pAM1-H2 and pAMl-H3 which contain the maturase and the intron insert sites had been constructed in our lab. Notably, KanR gene was inserted in the donor expression plasmid while the recipient expression plasmid has a chloramphenical-resistance (CamR) gene on it. For co-expression experiment, two plasmid was introduced into the same E.coli cells and the specified clones was selected, then we got E. coli strains containing those two plasmid, this cell induced firstly by L-arabinose and then by IPTG, at this time the retrohoming reaction could be took place with low frequency and yield retrohoming plasmid. This retrohoming plamid contain the Cam R gene come form the recipient plasmid and the Kan R gene that the mobiled group II intron carried, so this plasmid can be selected on the LB plate with both kanamycin and chloramphenical. Extract the plasmids of the resulting colonies, respectively. Subsequent PCR were carried out using specified primers outside the intron insertion site. The result PCR products were then comfirmed through the agarose gel after electrophoresis. There are no bands on the agarose gel stained by EB under UV light after electrophoresis. May be the homing of this group II intron had occurred in a ratively low frequency, further research was required in this project. However, two strains of E.coli plasmid containing the double-successful, for the further study of marine cyanobacteria retrohoming mechanism of mobile even has provided preconditions. As the cyanobacteria is thought to be the ancestor of chloroplasts,so it is of great significance in evolutionary biology and this study could provided some evidence for the the origin and evolution of chloroplast.