意大利蝗卵发育过程中血蓝蛋白基因表达分析

Expression profiling of hemocyanin gene in the developmental process of eggs of Calliptamus italicus (Orthoptera: Catantopidae)

【目的】意大利蝗Calliptamus italicus是新疆草原的主要优势危害种,以卵在土壤中越冬。呼吸代谢可反映蝗卵的生理状态,呼吸蛋白对于呼吸系统不完善的蝗卵尤为重要。本研究旨在明确意大利蝗卵发育过程中血蓝蛋白基因的表达情况。【方法】采用实时荧光定量PCR方法检测不同发育阶段的蝗卵以及1龄蝗蝻的血蓝蛋白2个亚基基因Hc1和Hc2的表达量。【结果】根据解剖形态观察,将意大利蝗越冬卵的整个发育过程分为10个阶段,包括9个卵发育阶段(C-Ⅰ-C-Ⅹ)和1龄蝗蝻阶段(C-Ⅹ)。Hc1和Hc2在越冬蝗卵各发育阶段以及1龄蝗蝻中均有表达。其中,在蝗卵早期发育阶段(C-Ⅰ,C-Ⅱ和C-Ⅲ),Hc1表达量逐渐增加,C-Ⅲ阶段表达量显著高于C-Ⅰ和C-Ⅱ阶段;滞育阶段(C-Ⅳ,C-Ⅴ和C-Ⅵ),胚胎发育停滞,Hc1表达量较C-Ⅲ,C-Ⅶ和C-Ⅷ阶段低;滞育后发育阶段(C-Ⅶ和C-Ⅷ),蝗卵解除滞育,快速发育,Hc1表达量较早期发育阶段和滞育阶段高,其中,C-Ⅷ阶段Hc1表达量最高(212.3156±10.5470),显著高于其他所有阶段;1龄蝗蝻(C-Ⅹ)的Hc1表达量最低,为0.4017±0.1010。Hc2表达量在C-Ⅴ阶段最高(679.7511±54.5719),显著高于其他所有阶段;除C-Ⅴ阶段外,其他各阶段之间Hc2表达量差异均不显著。Hc1在蝗卵滞育后阶段高表达,而Hc2在蝗卵滞育阶段高表达。【结论】血蓝蛋白亚基基因Hc1和Hc2在整个意大利蝗卵发育过程均有表达,且具有阶段特异性。Hc1与Hc2协同作用为蝗卵发育供氧,其中,Hc1主要负责蝗卵滞育后发育期间的氧气运载,而Hc2主要维持滞育期间的氧气运载,且载氧效率较低。研究结果可为进一步探讨意大利蝗卵的抗逆机制提供科学依据。

【Aim】 Calliptamus italicus is a major dominant pest in Xinjiang and overwinters in the soil with eggs.Respiratory metabolism can reflect the physiological state of locust eggs.Respiratory protein is especially important for C.italicus eggs,whose respiratory system is not well-developed.This study aims to clarify the expression profiles of hemocyanin gene in the developmental process of C.italicus eggs.【Methods】 The expression levels of hemocyanin subunit genes Hc 1 and Hc 2 in different developmental stages of eggs and the 1st instar nymph of C.italicus were analyzed by quantitative real-time PCR.【Results】 Based on anatomical observation,the developmental process of overwintering eggs of C.italicus is divided into ten stages,including nine egg stages (C-Ⅰ-C-Ⅸ) and the 1st instar nymph stage (C-Ⅹ).Hc 1 and Hc 2 were expressed in all the developmental stages of eggs and the 1st instar nymph of C.italicus.During the early stage of embryogenesis (C-Ⅰ,C-Ⅱ and C-Ⅲ),the expression level of Hc 1 increased gradually,and was significantly higher at the stage C-Ⅲ than at the stages C-Ⅰ and C-Ⅱ.During the diapause stage (C-Ⅳ,C-Ⅴ and C-Ⅵ),the embryonic development was stagnant,and the expression level of Hc 1 was lower than those at the stages C-Ⅲ,C-Ⅶ and C-Ⅷ.During the post-diapause developmental stage (C-Ⅶ and C-Ⅷ),the diapause of eggs were terminated and eggs developed rapidly,and the expression level of Hc 1 was higher than those at the early developmental stage and diapause stage.Especially,the expression level of Hc 1 in eggs at the stage C-Ⅷ was the highest (212.3156±10.5470),significantly higher than that in eggs at all of the other stages,and the expression level of Hc 1 in the 1st instar nymph was the lowest (0.4017±0.1010).The expression level of Hc 2 in eggs at the stage C-Ⅴ was the highest (679.7511±54.5719).The expression level of Hc 2 was not significantly different at various developmental stages of eggs except stage C-Ⅴ.Hc 1 was highly expressed during the post-diapause stage of eggs,while Hc 2 was highly expressed during the diapause stage of eggs.【Conclusion】 The hemocyanin subunit genes Hc 1 and Hc 2 are expressed throughout the developmental process of C.italicus eggs and show phase-specific expression.Hc1 and Hc2 synergistically provide oxygen for egg development.Hc1 is mainly responsible for oxygen transport during the post-diapause developmental stage of eggs,while Hc2 is mainly responsible for oxygen transport during the diapause stage of eggs with low oxygen carrying efficiency.The results can provide a scientific basis for further exploration of the stress resistance mechanism of C.italicus eggs.