Functional Domains Of Tgf2 Transposase In Goldfish

Transposon is a special genome can be present from a site "jump" to another DNA fragment sites. The DNA fragment which can between non-homologous loci can be done to move. Transposon into two broad categories: retrotransposons, DNA-DNA ways transposons of transposition. Retrotransposons are found in all types of organisms, but often present in higher plants are more abundant. Another category to the way the presence of “DNA-DNA” transposons(class II TEs); the DNA is copied by shearing or direct access to mobile fragment, and then inserted into the DNA sequence. This transposon is divided into two categories, namely autonomous transposon and non-autonomous transposon. In this study, the main subjects Tgf2 transposon, it is class II transposon, autonomous transposon.Tgf2 transposons were first found in goldfish, belonging h AT superfamily, it is found in the second example of independent transposon which has activity autonomous transposon in vertebrates. The transposon full length 4687 bp, containing the four open reading frames can be transcribed up to 686 amino acid transposase protein. Transposon function requires the participation of two necessary conditions: asymmetrical end-specific repetitive sequences; and a transposase activity. Therefore Tgf2 transposons, transposons as a kind of independent activity, the transposase enzyme in the process of its functions have played a vital role, which is a key element catalytic DNA fragmentation and reassembly. In this paper, for the Tgf2 transposase functional structure launched a series of forecasting and research.(1)Firstly, SWISS-MODEL protein analysis, Phyre2 protein analysis system, Tgf2 transposase protein structure analysis forecasts use homology modeling and fold recognition modeling method. Through the whole sequence of its amino acid sequence and partial sequence alignment, can be obtained in Tgf2 transposase near the N-terminal portion having a zinc finger domains than BED(65-120aa),the structure of the template has C2H2 key sites, regulation of gene expression related to it; In the zinc finger structure downstream has a helix- turn- helix(HTH) structure(153-213aa), plays the role of the polymerization; and in downstream of HTH found a longer domain, RNase H-like catalytic domain(211-683aa). RNase H-like catalytic domain there is generally highly conserved active site, so the use of Clustal X 1.83 compared to the Tgf2 transposase enzyme family with this sequence have been three other conservative sites DDE(228aa, 295 aa, 648aa). Use cNLS mapper analysis Tgf2 transposase amino acid full length sequence predicted a single type nuclear localization signal,which length is 15 amino acids LLFSPKRARLDTNNF.(2) To study the localization of Tgf2 transposase protein, using the existing Tgf2 transposase full-length cDNA sequence Tgf2 TP, according to NLS general research methods, giving three types of deletion mutant sequences were deleted Tgf2 transposase enzyme BED N terminal zinc finger domains Tgf2TP△120N; delete the C-terminal 31 amino acids Tgf2TP△31C; and retain cNLS mapper predictable sequence Tgf2TP△16C, is removed only downstream of the remaining 16 amino acids. The DNA sequences of four lengths construct into a plasmid pEGFP-C1 with EGFP green fluorescence protein. The resulting amount of four plasmids were transfected by liposome transiently transfected into 293 T cells. The results showed that the complete Tgf2 transposase has a nuclear localization tendency, 39.9% of the proportion of full access to the nucleus; removal of the N-terminal Tgf2 transposase zinc finger BED domains had similar result, 38.3% protein full access to the nucleus, indicating that zinc finger BED structure does not affect protein localization structure; When transfected with pEGFP-C1-Tgf2TP△31C, cells can not completely separate the fluorescence expression in the nucleus alone, only 67.2% of its cell fluorescence dispersed throughout the cell, the fluorescent protein cannot explain fully positioned into the nucleus; however turn after transfection pEGFP-C1-Tgf2TP△16C retains the nuclear localization sequence predicted 40% of the cells express independently fluorescent cytoplasm no expression in the nucleus. We thus conclude that, Tgf2 transposase enzyme having the ability to enter the nucleus; cNLS mapper predicted sequence LLFSPKRARLDTNNF, can affect Tgf2 transposase protein localization conditions.(3) To explore whether activity central DDE of RNase-H-like catalytic domain of Tgf2 transposase enzyme will affect the efficiency of transposition system. In the experiment, the establishment of two control groups, positive control to the original Tgf2 transposase pCS2-Tgf2 T in vitro transcription of mRNA and donor plasmid pTgf2-EF1α-eGFP injected embryos; negative control injection only donor plasmid pTgf2-EF1α-eGFP. Test group: he point mutation method three key amino acid mutation D228 N, D295 N, E648Q; And giving three types of helper plasmid pCS2-Tgf2TPD228 N, pCS2-Tgf2TPD295 N, pCS2-Tgf2TPE648 Q, it’s in vitro transcription of mRNA; respectively donor plasmid DNA pTgf2-EF1α-eGFP with microinjection into 1-2 cell stage zebrafish embryos. Zebrafish expressing green fluorescence was observed under an inverted microscope, respectively, and pharyngula at 12 h 24h with the original mRNA pCS2-Tgf2 TP as control. In the observation can be found mutated mRNA of zebrafish EGFP expression occurs weakened, after hatching their body fluorescence expression can only vaguely little expression on muscles, which in sharp contrast to the original Tgf2 transposase enzyme mRNA expression in the whole body expression closer to the negative control group. The fish were reared to extract DNA, the use of the positive rate of PCR to detect EGFP marker gene. Three kinds of point mutations integration rate is consistent with the negative control group, the positive rate compared to the original transposase enzyme significantly different. These results can be explained through the point mutation treatment Tgf2 transposase protein function changes, and cause transposition efficiency significantly decreased; further validate active center DDE RNase-H catalytic domain for efficient transposition system transposase important.