Clonging And Characterization Of TY3/Gypsy Retrotransposon In Chilo Suppressalis

Chilo suppressalis Walker, the borer pest, is widely distributed in China's rice production areas. In recent years, C. suppressalis populations have significantly risen to a serious threat to rice production. The difficulty in control of the C. suppressalis populations is related to the genetic diversity of populations and adaptation mechanisms in different habitats. Retortransposons are the sequences of DNA that can move in the host. When they transpose and proliferate in the host, mutations have been accumulated and ultimately lead to population differentiation and species variation. Plenty of evidence has suggested that retrotransposons are a major source of genome evolution and the form of species diversity. They have become an important tool to research on the gene function and expression, biological diversity and the evolutionary mechanism. There are a lot of retrotransposon sequences in C. suppressalis populations. Therefore, we try to study the genetic diversity of C. suppressalis populations by the endogenous transposons.In this paper, a retrotransposon was cloned from C. suppressalis, named Csu-Ty3. The AP sequence in the Csu-Ty3 retrotransposon was analyzed. Then, the single-base variation characteristics of two types of transposons in C. suppressalis were also researched. In this study, on one hand, we can get a comprehensive understanding of the characteristics of Csu-Ty3 retrotransposon in C. suppressalis. On the other hand, the extensive study on the DNA mutation patterns as inferred from the two kinds of transposon in C. suppressalis was done. It is hoped that this study will add perspective, in effort to understand transposons' mutations. The occurrence and mutation of C. suppressalis could be combined with its endogenous molecular genetic factors. A further study could be maked of the relationship between the endogenous transposon and population genetic diversity. We can have a known of the molecular formation mechanism adapted to the population genetic diversity. Finally, providing the theoretical basis for elucidating the genetic adaptation mechanism to the population dynamics and outbreak of C. suppressalis.1. Cloning and characterization of TyS/gypsy retrotransposon in C. suppressalisBased on a retrotransposon sequence segment of Ty3/gypsy, using the Inverse-PCR and Genome walking methods, a novel member of Ty3/gypsy retrotransposons was cloned from Chilo suppressalis. This new member was named Csu-Ty3 ?GenBank accession No. KJ191261?. The sequence is 4 934 bp in length and integrated into the "AACGT" target site duplications ?TSDs? of the genome. There is a pair of non-completely identical long terminal repeats ?LTRs? in the Csu-Ty3 retrotransposon. The 5'LTR is 161 bp in length and the 3'LTR is 168 bp in length, with 93.5% similarities each other. There are three independent open reading frames ?ORFs? in Csu-Ty3 and encode the protein of GAG, AP and a polyprotein in turn. The southern hybridization indicated that there were many Csu-Ty3 copies in different C. suppressalis populations. At this locus, all the individuals have the Csu-Ty3 copy insertion except some individuals that tested in Deyang and Jiangjin populations.2. Molecular cloning and analysis of aspartic protease ?AP? in Ty3/gypsy retrotransposon in C. suppressalisThe AP gene ?GenBank:KF886014?, in Ty3/gypsy retrotransposon, was first cloned from C. suppressalis in this study. The AP gene has a single open reading frame ?ORF? of 528 bp encoding a protease of 175 amino acids. There is a conserved Asp_protease₂ domain in this AP protein. Multiple sequences alignment revealed that there were 46 single-base substitutions. These single-base substitutions consisted of 31 transitions and 15 transversions. In the set of substitution mutations, the most frequently occurring base changes are A?G ?15? and T?C ?11?. Other base change forms are few.69 of 70 copies have intact ORF and can code a complete protein.3. Single-base substitutions analysis of two types of transposons in C. suppressalisUsing the multiple sequence alignment, the DNA and RNA transposons' single-base variations in C. suppressalis were analyzed, respectively. These two kinds of transposon sequences show similar characteristics in the substitutions of single-base. The number of transitions in a single-base is significantly higher than transversions. In the single-base transitions, the number of A::T?G::C mutations is significantly higher than G::C?A::T, which is different from the other similar researches. Further, the single-base substitutions in ORF regions were analyzed. The results show that the probability of single-base substitutions is no significant difference at the three positions of the codons. The number of amino acid missense mutations is significantly higher than the other kinds of amino acid mutations. The base substitutions at the third position of codon lead to the highest number of amino acid synonymous mutation.