Cloning And Molecular Evolutional Analysis Of The Nucleotide Sequence Of The A+T-rich Region Of Mitochondrial DNA From Silkmoth Insects

The domesticated silkworm, Bombyx mori is an important model of Lepidoptera insects. Researches on the origin and evolution of B. mori have been attached great importance by the sericulture scientific community. With the development of science and technology and the progress of archaeological discoveries, great progresses have been obtained in this field. The viewpoint that China is the origin of B. mori has became the consensus in the academic community. However, there are two different theories about the regional origin and the differentiation of voltinism of B. mori. One is that the Yellow River valley is the only origin of B. mori and the other is geographical region of multi-center theory.Mitochondrial DNAs (mtDNA) of animals are small in molecular weight with fast speed in DNA variation and maternal inheritance and easy to operate. They are therefore widely used in the study of mass genetics and molecular evolution of species. The control region of mtDNA is called A + T-rich region because of its high content of adenine and thymine. It is the most variant area in the mtDNA both in sequence and in length. Some researches have been done on the molecular phylogeny and evolution of silkmoths including B. mori and Philosamia cynthia ricini by using conservative protein coding genes or rRNA genes. But it was very seldom seen researches on the evolution of silkmoth, such as B. mori by using sequence of the A + T-rich region of mtDNA.Twelve silkworm strains of B. mori, Chinese B. mandarina Zhenjiang strain, P. cynthia ricini, Philosamia cynthia cynthia and Actias selene ningdoana were chosen as the materials for the study. And their mtDNAs were extracted from pupae separately. The B. mori strains are Sansi, Daisou, Handanzhong, Mysore, Dazao, Sanisi, Yuhang bivoltine, Yanhe 1, Xinchang 12, Ankang 4, Gansuzhong and Lan 5. There are monovoltinism, bivoltinisms and multivoltinism strains including Chinese, Japanese, European and Tropical races. Their cocoons are in white, jacinth, light jacinth, golden yellow and pistachio respectively. A pair of primers were designed according to the A + T-rich regions and flanking sequences of B. mori and B. mandarina published in the GenBank. The A + T-rich regions and flanking sequences of the silkmoth insects were cloned by polymerase chain reaction (PCR) method and sequenced.The result of sequence analysis showed the cloned fragments of 12 silkworm strains were about 1.1 kb in length, that of B. mandarina Zhenjiang strain was 1085 bp, and those of P. cynthia ricini, P. cynthia cynthia and A. selene ningdoana were 951 bp, 952 bp and 930 bp, respectively. The order of genes and their arrangement were identical to that of C108, viz. 3′-end portion of 12S rRNA, A+T-rich region, tRNA-Met, tRNA-Ile, tRNA-Gln and 5′-end portion of ND2 successively. There was a non-coding region between tRNA-Gln and ND2. The homology of nucleotide sequence of the cloned fragments among 12 silkworm strains tested, C108 and Aojuku was above 99%.Those of Lan5, C108 and Aojuku were completely the same. Comparing Yanhe1 and Dazao with C108, there were only one or two base differences which occurred in the thymine-stretch and adenine-stretch of the A+T-rich region. Among 14 B. mori strains, there were 19 base substitutions (including 15 bases transition,4 bases transversion) and 6 base absences which occurred mainly in the A+T-rich region.To study the origin of silkworm B. mori and the evolutional relationship between B. mori and the Saturniidae insects, a N-J phylogenetic tree based on the A + T-rich regions and flanking sequences of 14 silkworm strains and 4 strains of B. mandarina and a M-L phylogenetic tree based on the A + T-rich regions of 4 Saturniidae species including two strains of B. mori, one strain of A. pernyi, P. cynthia ricini, P. cynthia cynthia and A. selene ningdoana were constructed, respectively, by using Phylip 3.66 software package with Drosophila melanogaster mtDNA as the out-group. In the N-J tree, B. mori strains and two Chinese B. mandarina strains clustered in one subgroup, two Japanese B. mandarina strains clustered in the other subgroup, indicating that the genetic relationship between B. mori and Chinese B. mandarina was more nearer than that between B. mori and Japanese B. mandarina. Moreover, the genetic relationship of Gansuzhong was evolved earlier than that of the other 13 tested B. mori strains. The M-L tree showed that P. cynthia ricini P. cynthia cynthia and A. selene ningdoana species was genetically closer with each other, and they were a little far to A. pernyi and remote to B. mori. In addition, P. cynthia ricini and P. cynthia cynthia showed the closest genetic relationship among all insect species in the study.These results have provided new evidences in mitochondrial molecular level to the theories both of B. mori originated from the ancient Chinese B. mandarina and the Yellow River valley was one of the origins of B. mori.