Thesis or Dissertation Pooled RNA-seqを用いたアカショウジョウバエ低温耐性候補遺伝子の探索

中村, 遥

pp.1 - 57 , 2016-03-25
In a recent few decades, Drosophila albomicans has expanded their distribution from tropical zone to temperate zone. It has been reported that the current northernmost limit of D. albomicans distribution is west or central Japan. In previous studies, the variation of cold tolerance among D. albomicans strains was observed: the temperate zone population has a stronger cold temperature than the tropical population. This suggests that D. albomicans has expanded their distribution to temperate zone by adapting to the cooler climate. Therefore, I tried to identify genes responsible for the adaptation, using pooled RNA-Seq. The pooled RNA-Seq is a very challenging way by applying RNA Seq to a pool of many strains. Because it is very new, I should create pipeline for analyzing the data from a next generation sequencer. However, I could get information on gene expression and single nucleotide polymorphism (SNP) in exons at a population level in a cost-effective way. According to previous studies, it was found that cold tolerance of insect was enhanced by cold acclimation. It was reported that cold tolerance was enhanced by a cold acclimation at 20 ℃ for several days accompanied by changes in expression level of many genes in a strain of D. albomicans, but these changes have not been examined at a population level. In this study, the total RNA was extracted from cold-acclimated flies from three populations classified by location and time, i.e., from Southeast Asia (SEA), Japan in 1991 (J-1991) and Japan in 2011 (J-2011). Using the transcriptome data obtained by pooled RNA-Seq by Illmina HiSeq2000 sequencer, I compared differences in gene expression levels and genetic variations at a genomic level among the populations. I calculated Fst, which is a measure of population differentiation due to genetic structure. As the result, I found that the genetic structure was different between SEA and the J-1991 and J-2011 populations, whereas genetic structure has not been differentiated between J-1991 and J-2011, compared to those and SEA. This indicates that in the west Japan population the genetic structure has not been changed during the last 20 years, suggesting that west Japan population has not been under strong natural selection to change the genetic structure of population. In addition, using Tajima's D, I estimated that population size has been decreased during the distribution expansion to west Japan. I found that gene expression level was different in 22 genes between SEA and the west Japan population. Among these genes, three genes (Cyp12d1-1, CG13422 and CG11889) were included in the genes whose expression level was changed by cold acclimation according to Isobe (2014). So those three genes are candidates of the genes to be related to cold tolerance. To examine the effect of natural selection for the adaptation to cold environment, I computed nucleotide diversity, Waterson's theta, and Tajima's D for those candidate genes. All parameters indicate that J-1991 and J-2011 have lower genetic diversity than SEA, suggesting some effect of natural selection. However, the expected molecular functions of those genes are unlikely involved in cold tolerance directly and the observed expression changes of those genes are more likely attributed to cross-talk with a signaling pathway of other genes responding to cold stress.
首都大学東京, 2016-03-25, 修士(理学)

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