Sericin Ser3 Ectopic Expressed in Posterior Silk Gland Affects Hemolymph Immune Melanization Response via Reducing Melanin Synthesis in Silkworm.
Sericin Ser3 Ectopic Expressed in Posterior Silk Gland Affects Hemolymph Immune Melanization Response via Reducing Melanin Synthesis in Silkworm.
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家蚕具有显著的蛋白质合成能力，受到生物材料和生物医学研究者的青睐。蚕丝腺是蚕桑最受关注的目标组织。许多研究都集中在将外源功能纤维蛋白基因导入家蚕基因组中，并利用丝腺生物反应器获得了具有良好生物活性的生物医学蛋白和具有特殊性质的多功能丝纤维。然而，转基因家蚕的丝腺经常出现生命力低下、发育迟缓等问题，原因尚不清楚；转基因家蚕的生物安全性也是未知的。因此，我们研究了家蚕后部丝腺表达的重组Ser3异位基因对家蚕血淋巴黑化和代谢的影响。结果表明，虽然该突变体在正常环境下具有正常的生命力，但对血淋巴代谢和免疫功能有明显的影响。因此，研究结果对促进转基因生物的安全评估和发展具有积极意义。家蚕转基因是创新遗传资源和蚕丝功能的重要途径。然而，转基因家蚕的丝腺作为养蚕最受关注的靶组织，常出现生命力低下、发育迟缓等问题，其原因尚不清楚。本研究将中间丝腺(MSG)特异表达基因Ser3导入家蚕后丝腺(PSG)，并研究了突变纯系SER(Ser3+/+)血淋巴免疫黑化反应的变化。结果表明，该突变体虽然具有正常的生命力，但与体液免疫相关的血淋巴中黑色素含量和酚氧化酶(PO)活性显著降低，导致血液黑化明显减慢，杀菌能力减弱。机制研究表明，突变血淋巴中黑素合成途径中的苯丙氨酸羟基酶(PAH)、酪氨酸羟基酶(TH)和多巴胺脱羧酶(DDC)的mRNA水平和酶活性以及丝氨酸蛋白酶级联中PPAE、SP21和Serpins基因的转录水平均受到显著影响。此外，与血淋巴氧化还原代谢能力相关的总抗氧化能力、超氧阴离子抑制能力和过氧化氢酶(CAT)水平显著提高，而超氧化物歧化酶(SOD)和谷胱甘肽还原酶(GR)活性以及过氧化氢(H_2O_2)和谷胱甘肽(GSH)水平显著降低。综上所述，转PSG基因家蚕SER的血淋巴中黑素合成受到抑制，氧化应激的基本应答水平升高，血淋巴免疫黑化反应降低。这一结果将显著改善转基因生物的安全评估和开发。
Silkworms have remarkable protein synthesis ability, which is favored by biomaterials and biomedicine researchers. The silk gland is the most concerned target tissue of sericulture. Many studies have focused on introducing exogenous functional fibrin genes into the silkworm genome, and have obtained biomedical proteins with good biological activity and multifunctional silk fibers with special properties using the silk gland bioreactor. However, the silk gland of transgenic silkworms often suffers from low vitality, stunting and other problems, and the reasons are still unknown; the biosafety of transgenic silkworms is also unknown. Therefore, we studied the effect of the recombinant Ser3 ectopic gene expressed in the posterior silk gland on hemolymph melanization and metabolism in silkworms. The results showed that although the mutant had normal vitality in normal environments, hemolymph metabolism and immunity were significantly affected. Therefore, the results have positive significance to promote the safe assessment and development of genetically modified organisms. The transgenesis of silkworms is an important way to innovate genetic resources and silk function. However, the silk-gland (SG) of transgenic silkworms, which is the most concerned target tissue of sericulture, often suffers from low vitality, stunting and other problems, and the reasons are still unknown. This study trans engineered recombinant Ser3, a middle silk gland (MSG) specific expression gene, in the posterior silk gland (PSG) of the silkworm, and studied hemolymph immune melanization response changes in mutant pure line SER (Ser3+/+). The results showed that although the mutant had normal vitality, the melanin content and phenoloxidase (PO) activity in hemolymph related to humoral immunity were significantly reduced, and caused significantly slower blood melanization and weaker sterilization ability. The mechanism investigation showed that the mRNA levels and enzymatic activities of phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH) and dopamine decarboxylase (DDC) in the melanin synthesis pathway in mutant hemolymph, as well as the transcription levels of the PPAE, SP21 and serpins genes in the serine protease cascade were significantly affected. Moreover, the total antioxidant capacity, superoxide anion inhibition capacity and catalase (CAT) level related to the redox metabolic capacity of hemolymph were significantly increased, while the activities of superoxide dismutase (SOD) and glutathione reductase (GR), as well as the levels of hydrogen peroxide (H2O2) and glutathione (GSH), were significantly decreased. In conclusion, the anabolism of melanin in the hemolymph of PSG transgenic silkworm SER was inhibited, while the basic response level of oxidative stress was increased, and the hemolymph immune melanization response was decreased. The results will significantly improve the safe assessment and development of genetically modified organisms.
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