Molecular cloning, expression and oxidative stress response of the vitellogenin Gene (AccVg) from Apis cerana cerana
Apidologie
Molecular cloning, expression and oxidative stress response of the vitellogenin Gene (AccVg ) from Apis cerana cerana
Weixing ZHANG 1
Zhenguo LIU 1
Ming ZHU 0
Lanting MA 1
Ying WANG 1
Hongfang WANG 1
Xingqi GUO 0
Baohua XU 1
0 College of Life Sciences, Shandong Agricultural University , Tai'an, Shandong , People's Republic of China
1 College of Animal Science and Technology, Shandong Agricultural University , Tai'an, Shandong , People's Republic of China
- Vitellogenin (Vg) is a yolk precursor protein in most oviparous females. However, Vg has not been studied in the Apis cerana cerana . In this work, the Vg gene of the A. cerana cerana has been cloned and sequenced. The gene codes for a protein consisting of 1770 amino acids in seven exons with a predicted molecular mass and isoelectric point of 200 kDa and 6.46, respectively. Additionally, an 807-bp 5′-flanking region was isolated, and potential transcription factor binding sites associated with development and stress response were identified. Quantitative real-time PCR analysis showed that AccVg is highly expressed in pupae during different developmental stages. In addition, the expression of AccVg could be induced by cold (4 and 16 °C), CdCl2, and pesticide treatment. Real-time quantitative PCR (RT-PCR) and western blot analyses indicated that AccVg transcription was induced by various abiotic stresses. Western blot was used to measure the expression levels of AccVg protein. Taken together, these results suggest that AccVg most likely plays essential roles in antioxidant defence and that it may be of critical importance to honey bee survival.
1. INTRODUCTION
In China, Apis cerana cerana is commonly
found and plays an important role in agricultural
economics and in the balance of regional
ecologies as a pollinator of flowering plants. In
honeybees, worker-worker division of labour is based
on a process of behavioural maturation; for the
first 2–3 weeks of adult life, workers perform
tasks inside the hive, such as brood care and food
storage, and as they become older, they progress
to tasks outside, including foraging for pollen and
nectar
(Winston, 1987)
.
Organisms are exposed to a series of
unfavourable environmental stressors on a nearly
constant basis, including temperature change,
heavy metals, pesticides, and ultraviolet (UV)
radiation, all of which are believed to induce the
formation of reactive oxygen species (ROS)
(Lushchak, 2011; Kottuparambil et al., 2012)
.
The generation of increased ROS and the
production of oxidative damage are associated with
induced thermal stress (Yang et al., 2010). ROS can
be generated by all oxygenic organisms during
aerobic metabolism
(Dröge, 2002; Balaban et al.,
2005)
. Therefore, understanding the antioxidant
system of the honeybee and its ROS defence
mechanisms has become a crucial issue for this
industry.
Large lipid transfer proteins (LLTP)2 have
multiple roles in animals, such as lipid
transporters, inflammation suppressors,
immunomodulators and blood coagulators. Vg is one of the
ancient forms of these proteins, with an
estimated 700 million-year history
(Hayward et al.,
2010)
.Vgs are mostly produced in the liver
(vertebrates), hepatopancreas (crustaceans) or fat
body (invertebrates); secreted into the blood
and taken up by targets via receptor-mediated
endocytosis
(Spieth et al., 1991; Tufail and
Takeda, 2008; Tseng et al., 2001)
. Honeybee
Vg, known as a conserved yolk precursor
protein, is a 180-kDa glycolipoprotein
synthesized in fat body cells and released to the
haemolymph (the insect blood)
(Wheeler and
Kawooya, 1990; Fleig, 1995)
. In egg-laying
queens, Vg constitutes over 50% of total
haemolymph proteins and is taken up by
developing oocytes (Hartfelder and Engels, 1998). In
the haemolymph of nurse bees, Vg comprise 30–
50% of total proteins and impacts both
antioxidant and immune function
(Amdam et al., 2004;
Seehuus et al., 2006; Corona et al., 2007)
.
Additionally, Vg is present in the haemolymph of
young worker bees, as well as in drones where it
may account for up to 5% of total haemolymph
proteins
(Trenczek et al., 1989)
. Vg has an
antioxidant function in workers
(Seehuus et al.,
2006)
. Knockdown of Vg expression in workers
causes decreased resistance to oxidative stress.
The Vg titre positively correlates with the
honeybee’s oxidative stress tolerance because the
antioxidant property of Vg shields other
haemolymph molecules from reactive oxygen
species (ROS)
(Seehuus et al., 2006)
.
Additionally, a causal link between honeybee vitellogenin
activity and lifespan is supported by the
zincbinding capacity of the protein product
(Amdam
et al., 2004)
, which also suggests an antioxidant
function
(Amdam and Omholt, 2002; Goto et al.,
1999; Quinlan et al., 2005)
.
The vitellogenins have been studied
extensively in a wide variety of animals, both vertebrates
and invertebrates, including insects. In the present
study, we isolated and characterised the Vg in (...truncated)