BmMITE-2 Induced Positive Selection And Affected Neighby Gene Expression In Bombyx Mori

Archaeological evidence suggested that the silkworm was domesticated from wild silkworm about 5,000 years ago. In ancient China, silkworm was regarded as a national treasure. In order to protect the secret of silk production, silkworm or silkworm eggs were forbidden smuggling to other countries. With the Silk Road, however, silk production spread to Japan, the Middle East and some European countries. As a representative of Lepidoptera, so far the silkworm is the only known economic insect fully domesticated and a good material for genetic research. According to statistics, there are about 1000 lines of silkworm in the worldwide. Based on geographical location, silkworm can be divided into four groups:Chinese, Japanese, European and tropical populations. At the same time there are abundant wild silkworm resources, which provides favorable conditions for us to study the genetic difference between domesticated silkworm (Bombyx mori) and wild silkworm (Bombyx mandarina). In China, Institute of Sericulture and Systems Biology of Southwest University maintained one of the largest silkworm gene pools in the world. These strains provide abundant genetic materials for studying the adaptive evolution of silkworm.With the large-scale genome sequencing development, it was found that eukaryotic genomes contain a large of repeat sequences. In order to study the origin and evolution of the eukaryotic genomes, it is necessary to study the effect of transposable elements (TEs) on the host genome. TE is a repeated DNA fragment which is able to move randomly in the genome and can change the structure and function of host genome. When the TE jumps and then inserts into a functional gene, it may trigger mutations. In general, this kind of mutations may be deterious. However, these mutations also provide abundant genetic variation for hosts to fit diverse environments, which accelerates evolution of host species.The proportion of TEs (40%) in the silkworm genome is lower than the corn, barley, human, mouse and so on, but much higher than the insects like Drosophila and Anopheles. TEs in the silkworm genome mainly consist of short interspersed nuclear elements (SINEs) and Non-long terminal repeats (Non-LTRs). In addition, the miniature inverted transposable elements (MITEs) in the silkworm can be divided into 17 families. Of the families, BmMITE-2 family has some features:large copy number, high homology and the possible recently burst expansion. In this study, 1 investigated the molecular population genetics of BmMITE-2 insertion. The main results are as follows:1. The insertion and deletion polymorphism of BmMITE-2 family in the silkwormNucleotide database of NCBI and SilkDB were Blastn searched using BmMITE-2 sequence as query. We found that BmMITE-2 inserted in the five genes. They were ecdysteroid-regulated 16 kDa protein precursor(5.16 kDa), Capa-a protein (Capa-a), methyltransferase, ubiquinone biosynthesis protein COQ7-like protein (COQ7-L) and glucose-6-pheshate isomerase (Ig6p). Except COQ7-L whose insertion site is in the 5'UTR region, the insertion points of the remaining four genes are in the 3'UTR region. The primers were designed in the flanking region of the insertion site. Through PCR, we could investigate the insertion and deletion polymorphism of the five genes in the four different geographic populations. And genetic relationship of host population was analyzed on the basis of these polymorphism data. The results showed that in 5'UTR region of COQ7-L,11 of 116 silkworm strains (9.48%) used in this study have BmMITE-2 insertion, and these lines mainly belong to Chinese and tropical populations. The proportions of insertion for remaining genes were: 68.6%, 71.95%, 60.33%. and 55.32% respectively. Then we selected 16 wild silkworm strains to analyze their insertion and deletion polymorphism. and we found in these wild strains, BmMITE-2 did not insert into the 5'UTR of COQ7-L, while did insert into the 3'UTR of other genes.According to population polymorphism of five insertion sites of BmMITE-2 family, we found that genotypes of BmMITE-2 were consistent with Hardy-Weinberg equilibrium (P> 0.05). except for 1g6p and COQ7-L. The genetic differentiation of different populations is mainly intra-population, and degree of differentiation varies among different insertion sites. At the same time, genetic distance among four geographical populations and their phylogeny showed that the genetic relationship among silkworm population was in accord with the geographic distribution.2. BmMITE-2 induced adaptive insertionFor COQ7-L, 10 insertion strains and 13 deletion strains were selected. Then, we cloned and sequenced for the insertion site and 2kb,5kb flanking region of this site in the 23 silkworm strains, and found that polymorphism of strains with BmMITE-2 insertion was lower than strains without insertion. Phylogenetic analysis showed that insertion and deletion strains grouped into different clusters, respectively. This result might be caused by the insertion of BmMITE-2, or probably caused by the closer relationship of these strains which have BmMITE-2 insertion. To clarify this, we determined phylogenetic relationship using CR region of mitochondrial sequence from partial strains, and the result rejected the second possibility.By RT-PCR method, we checked the period and tissue expression of COQ7-L in the insertion and deletion strains. The results showed that the period expression of this gene showed no significant difference between the insertion and deletion strains. But in tissue expression, the expression had great difference between insertion and deletion strains. This gene had no expression in the ovary, silk gland, fat body, midgut and trachea in the deletion strains. However, it had expression in these tissues in insertion strains.We investigated the population polymorphism and adaptive insertion of TEs in the silkworm for the first time using bioinformatics and molecular biology methods. In addition, our results emphasized the role of TEs played in gene regulation. This study not only helps us understand population polymorphism, but also provides some new insights into the role of TEs in increasing gene regulatory networks.