Development of a new efficient method for cloning of microsatellite regions using magnetic beads and its application to the research on reproductive strategies of insects.

我们找到了一种新的有效方法，用于在吉普赛人、Lymantria dispar基因组中克隆微细胞区域。基因组DNA片段，其中含有限制性酶被添加到等离子体载体中，并被转化为大肠杆菌JM 109效力细胞。与助手相位M13 KO 7和单stranded DNA （ssDNA）一起感染的细胞是由标准程序准备的。Dynabeads M-280链霉菌素（DYNAL），与生物素-（GT）绑定D225链霉菌D2，与ssDNA混合以选择携带DNA片段（AC）n序列。有效的退火温度在70℃至75℃之间。间接地，双链DNA从那些选定的DNA中合成，并转化为E。C.M.109。我们找到了24个克隆人（GT/AC）n个序列，其中11个是独一无二的，使我们能够在吉普赛人身上完成完整性测试。每一个女人都可以连续地和两个男人约会。DNA片段来自每个女性、两个男性和前Larvae，在女性Laid的鸡蛋里。最后一个匹配的男性生殖器的比例， P2值，已计算。The P-D22-D2 value was variable between 0.22 and 1.00 and was negatively correlated with the interval between first and second mating of the female。这些结果建议说，如果人口密度很高，女性保护吉普赛人的行为有效地保护吉普赛人的生育率，这是因为女性似乎很快就会在这种条件下终止伴侣的终止。

We searched a new efficient method for cloning of the microsatellite regions in the genome of the gypsy moth, Lymantria dispar. The genomic DNA fragments which were obtained digestion with restriction enzyme were ligated into plasmai vector and transformed into Escherichia coli JM109 competent cells. The cells were infected with helper phage M13KO7 and single-stranded DNA (ssDNA) was prepared by standard procedures. Dynabeads M-280 Streptavidin (DYNAL), bonded with biotin-(GT)ィイD225ィエD2, were mixed with the ssDNA to select the DNA segments carrying (AC)n sequences. The efficient annealing temperature was from 70℃ to 75℃. Subsequently, double-strand DNA was synthesized from those selected ssDNA and transformed into E. coli JM109. We found 24 clones containing (GT/AC)n sequences and 11 of them were unique.Those microsatellite sequences enabled us to perform the paternity test in the gypsy moth. Each female was allowed to mate with two males sequentially. DNA fragments were obtained from each female, two males and pre-larvae in eggs that the female laid. The proportion of progeny sired by the last mated male, P2 value, was calculated. The PィイD22ィエD2 value was variable between 0.22 and 1.00 and was negatively correlated with the interval between first and second mating of the female. These results suggest that mate guarding behavior of gypsy moth males is efficient to ensure their paternity if population density is high, because females seem to remate soon after termination of copulation under such conditions.