Abstract：
The silk glands are the specialized tissue where silk protein synthesis, secretion, and conformational transitions take place, with pH playing a critical role in both silk protein synthesis and fiber formation. In the present study, we have identified erythrocyte carbonic anhydrase (BmeryCA) belonging to the α-CA class in the silk gland, which is a Zn2+ dependent metalloenzyme capable of efficiently and reversibly catalyzing the hydrated reaction of CO2 to HCO3-, thus participating in the regulation of acid-base balance. Multiple sequence alignments revealed that the active site of BmeryCA was highly conserved. Tissue expression profiling showed that BmeryCA had relatively high expression levels in hemolymph and epidermis but is barely expressed in the posterior silk gland (PSG). By specifically overexpressing BmeryCA in the PSG, we generated transgenic silkworms. Ion-selective microelectrode (ISM) measurements demonstrated that specifically overexpression of BmeryCA in the PSG led to a shift in pH from weakly alkaline to slightly neutral conditions. Moreover, the resultant PSG-specific BmeryCA overexpression mutant strain displayed a significant increase in both silk yield and silk fiber mechanical properties. Our research provided new insights into enhancing silk yield and improving the mechanical properties of silk fibers.
摘要：
丝腺是丝蛋白合成的专门组织，分泌，发生构象转变，pH在丝蛋白合成和纤维形成中起关键作用。在本研究中,我们已经鉴定了丝腺中属于α-CA类的红细胞碳酸酐酶（BmeryCA），它是一种依赖Zn2+的金属酶，能够有效和可逆地催化CO2到HCO3-的水合反应，从而参与酸碱平衡的调节。多序列比对显示BmeryCA的活性位点是高度保守的。组织表达谱显示BmeryCA在血淋巴和表皮中具有相对较高的表达水平，但在后丝腺（PSG）中几乎没有表达。通过在PSG中过度表达BmeryCA，我们产生了转基因蚕。离子选择性微电极（ISM）测量表明，PSG中BmeryCA的特异性过表达导致pH从弱碱性条件向弱中性条件转变。此外，所得的PSG特异性BmeryCA过表达突变菌株显示出丝产量和丝纤维机械性能均显着提高。我们的研究为提高丝产量和改善丝纤维的机械性能提供了新的见解。
