Abstract：
Animal silk is economically important, while silk secretion is a complex and subtle mechanism regulated by many genes. We identified the poly (ADP-ribose) polymerase (PARP1) gene of the silkworm and successfully cloned its coding sequence (CDS) sequence. Using clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) technology, we screened single guide RNA (sgRNA) with high knockout efficiency by cellular experiments and obtained PARP1 mutants by knocking out the PARP1 gene of the silkworm at the individual level. We found that the mutants mainly exhibited phenotypes such as smaller cocoon size and reduced cocoon shell rate than the wild type. We also detected the expression of silk protein genes in the mutant by quantitative real-time PCR (qPCR) and found that the expression of some silk protein genes was slightly down-regulated. Meanwhile, together with the results of transcriptomic analysis, we hypothesized that PARP1 may affect the synthesis of silk proteins, resulting in their failure to function properly. Our study may provide an important reference for future in-depth refinement of the molecular mechanism of silk protein expression in silk-producing animals, as well as a potential idea for future development of molecular breeding lines of silkworms to improve silk production.
摘要：
动物丝在经济上很重要，而丝分泌是由许多基因调控的复杂而微妙的机制。我们鉴定了家蚕的聚（ADP-核糖）聚合酶（PARP1）基因，并成功克隆了其编码序列（CDS）序列。使用成簇的规则间隔短回文重复(CRISPR/Cas9)技术，我们通过细胞实验筛选了具有高敲除效率的单向导RNA（sgRNA），并通过在个体水平上敲除家蚕的PARP1基因获得了PARP1突变体。我们发现突变体主要表现出表型，例如比野生型更小的茧大小和降低的茧壳率。我们还通过定量实时PCR（qPCR）检测了突变体中丝蛋白基因的表达，发现某些丝蛋白基因的表达略有下调。同时,连同转录组学分析的结果，我们假设PARP1可能会影响丝蛋白的合成，导致他们无法正常运作。本研究可为今后深入完善产丝动物丝蛋白表达的分子机制提供重要参考,以及未来发展家蚕分子育种系提高蚕丝产量的潜在思路。
