Molecular cloning, expression and oxidative stress response of the vitellogenin Gene (AccVg) from Apis cerana cerana

Apidologie, Apr 2017

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.

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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)


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Zhang, Weixing, Liu, Zhenguo, Zhu, Ming, Ma, Lanting, Wang, Ying, Wang, Hongfang, Guo, Xingqi, Xu, Baohua. Molecular cloning, expression and oxidative stress response of the vitellogenin Gene (AccVg) from Apis cerana cerana, Apidologie, 2017, pp. 599-611, Volume 48, Issue 5, DOI: 10.1007/s13592-017-0503-9