抑制类胰岛素肽BBX-B8基因的表达对家蚕抗逆性的影响

Effects of the Expression of Repressive Insulin-like Peptide Gene BBX-B8 on Stress Resistance of the Silkworm,Bombyx mori

家蚕类胰岛素肽(bombyxin)具有调节糖代谢、促进翅芽和造血器官细胞增殖、调控前胸腺分泌活性等功能。为探讨家蚕类胰岛素肽其它的生理功能,将Bombyxin B家族的BBX-B8基因克隆进pGEM-T载体,通过体外转录合成BBX-B8 dsRNA,研究RNAi介导的BBX-B8基因沉默对蚕体耐高温、低温和饥饿的能力及部分保护酶活性的影响。Western blotting检测显示,给家蚕5龄2d幼虫注射1μg/μLBBX-B8 dsRNA(10μL/头)3d后,脑组织中BBX-B8的表达水平下降30.80%。给家蚕5龄3d幼虫注射1μg/μLBBX-B8 dsRNA(10μL/头)2d后,血液中超氧化物歧化酶(SOD)活性、总抗氧化能力(T-AOC)分别提高17.81%、41.86%,而过氧化氢酶(CAT)活性与对照无显著差异。给家蚕5龄3d幼虫注射BBX-B8 dsRNA 2d后进行高温(40℃,3h)和低温(4℃,3h)冲击,化蛹率比对照提高12.76个百分点;注射BBX-B8 dsRNA后禁食处理,在饥饿状态下的化蛹率比对照低9.29个百分点。研究结果提示:在家蚕5龄幼虫期抑制BBX的表达,可刺激血液中SOD活性上升,增强蚕体抗氧化能力,从而产生一系列生理应激反应,导致其对高温和低温的应激能力提高;家蚕的代谢活动加剧,能量消耗增加,耐饥饿能力下降。

The insulin-like peptide bombyxin could regulate glycometabolism,promote cell proliferation of wing buds and hematopoietic organ,and regulate secretory activity of prothoracic gland etc.To understand other physiological functions of this insulin-like peptide,the BBX-B8 gene which belongs to the bombyxin B family was cloned into pGEM-T vector,and BBX-B8 dsRNA was synthesized through in vitro transcription and used to survey the effects of BBX-B8 gene silencing mediated by RNAi on silkworm larvae＇s tolerance to high-to-low temperature impact and starvation,and on the activity of some protective enzymes.Western blotting results showed that,at 3 days post injection （dpi） of 10 μg of BBX-B8 dsRNA into 2-day-old 5th instar larvae,the level of BBX-B8 in the brain was decreased by 30.80%.At 2 dpi of 10 μg of BBX-B8 dsRNA into 3-day-old 5th instar larvae,the levels of superoxide dismutase （SOD） and total antioxidant capacity （T-AOC） in the blood were increased by 17.81% and 41.86% respectively,while no significant difference was observed on the catalase （CAT） level between the RNAi group and the control group.The pupation rate of the larvae was increased by 12.76 percentage points after the larvae were injected with BBX-B8 dsRNA followed by high-to-low temperature impact as from 40 ℃ 3 h to 4 ℃ 3 h.The pupation rate of the larvae under starvation condition was decreased by 9.29 percentage points after injection with BBX-B8 dsRNA compared with that of the control group.The above results suggest that expression of the repressive BBX in the 5th instar larvae could stimulate SOD expression,enhance T-AOC,and resulted in a series of physiological stress response,thus improving the larvae＇s tolerance to high-to-low temperature stress.Besides,the larvae＇s metabolic activities and energy consumption were increased,while their tolerance to starvation was decreased.