vasa基因在竹莖扁蚜及豌豆蚜的純化及定性
Isolation and characterization of vasa gene in bamboo stem aphids and pea aphids
Date Issued
2005
Date
2005
Author(s)
Lu, Shau-Lin
DOI
en-US
Abstract
Primordial germ cells (PGC) are segregated from somatic cells during early embryogenesis in most animals. The phenomenon is known by the observation of “nuage”, a group of electron-dense materials in the periphery of nuclear membrane. Vasa gene or its product has been known as a component of nuage in many species, showing that it is a conserved germline marker. In our study we cloned vasa-like genes in the bamboo stem aphid Pseudoregma bambucicola (bsavasa) and in the pea aphid Acyrthosiphon pisum (pavasa1). The bsavasa was used to discriminate the soldier and non-soldier nymphs, on the hypothesis that the expression of bsavasa is weaker in sterile soldiers; pavasa1 was used as a marker to study germline development in parthenogenetic aphids because we wanted to know its developmental expression from oogenesis to embryogenesis within an ovariole. RT-PCR shows that vasa mRNA was preferentially identified in the adult ovary, suggesting that it is a potential PGC marker in these two aphid species. However, expression of vasa was detected in soldier and non-soldier nymphs of bamboo stem aphids, implying that either bsavasa is expressed in soldiers’ non-degenerated germ cells or it is also expressed in somatic tissues at the 1st instar nymph. We identified a ~3 kb transcript of vasa with Northern blot in both species of aphids, implying that the length of vasa mRNA may be similar. Our effort to clone the 3’-end sequence of pavasa1 did not work, which was similar to the sequence provided by D. Stern (Princeton, USA). It might be caused by the abundant poly-A sequences scattering at the 3’-end area within the open reading frame, which consumed most of the poly-T primers in the 3’ RACE-PCR. In order to investigate whether pavasa1 is a single-copy gene, blots containing genomes digested respectively with HindIII, SpeI and EcoRI were probed with DIG-labeled vasa genes. Repeated results show that a single band was identified in each lane from different enzyme digestion (HindIII: ∼6kb; SpeI: ∼7 kb; EcoRI: ∼3.5 kb), suggesting that this vasa gene (pavasa1) is single copy. We also compared the deduced protein sequence encoded by pavasa1 with that encoded by pavasa2, a second vasa-like gene cloned in our laboratory. Sequence alignment shows that these two Vasa proteins only have 38% identity and N-terminal sequences appear divergent. It shows that there are at least two different vasa genes existing in the pea-aphid genome. In the last part, we will discuss whether pavasa1 is a germline marker according to the provided results of in situ hybridization and antibody staining.
Subjects
竹莖扁蚜
豌豆蚜
vasa
aphids
Type
thesis