Secretory Mechanism of Fibroin,a Silk Protein,in the Posterior Silk Gland Cells of Bombyx mori

There are two microtubule-microfilament systems in the posterior silk gland cells of Bombyx mori. One is a radial microtubule system; the other is a circular microtubule-microfilament system. These two systems are presumably concerned with the intra-cellular transport of secretory granules of fibroin and the secretion of fibroin into the lumen, respectively. Conventional and scanning electron microscopic observations of the two microtubule-microfilament systems in the posterior silk gland cells are reported. Scanning electron micrographs showed that a number of parallel linear cytoplasmic processes ran circularly on the luminal surface of the posterior silk gland cells. These processes were assumed to correspond to the circular microtubule-microfilament systems. The effects of cytochalasin (B or D), a secretion stimulating agent of fibroin, on the intracellular recording of membrane potential from the posterior silk gland cells are also reported. Exposure to cytochalasin resulted in depolarization of the membrane potential of the gland cells. Possible functional roles of the two microtubule-microfilament systems in the secretory mechanism of fibroin are discussed with reference to the effects of antimitotic reagents and cytochalasin on these two systems.     

家蚕丝素蛋白轻链基因（fib-L）启动子序列的克隆及其活性分析

家蚕Bombyx mori丝素蛋白轻链（fibroin light-chain, fib-L）基因fib-L具有在后部丝腺组织专一性、高效性表达的特点。为了利用其启动子构建能够表达外源基因的丝腺生物工厂,本实验对fib-L启动子活性进行了研究。通过PCR法克隆了fib-L启动子元件,序列分析显示fib-L启动子由位于-33 ～ -25处的TATA盒元件和位于－128～－121处的特征性序列GTCAATTT共同组成。用fib-L启动子控制报告基因DsRed进行家蚕BmN细胞和蚕体内的瞬时表达研究,结果表明fib-L启动子可以驱动DsRed报告基因在BmN细胞和家蚕后部丝腺组织中瞬时表达。     

家蚕 Fhx/P25 基因的一种新的转录模式分析研究

家蚕 Fhx/P25 蛋白是丝素蛋白的主要成分之一,过去报道只在家蚕后部丝腺特异的转录表达 . 通过对大规模的家蚕 EST 序列分析发现, Fhx/P25 基因不仅在家蚕后部丝腺高效转录,而且在家蚕幼虫五龄第三天的卵巢组织及其他组织也有转录;分析还发现 Fhx/P25 基因在丝腺和卵巢组织中有不同的转录起始位点,在卵巢组织中的转录起始位点比在丝腺中的至少要提前 115 bp 左右 . 用 RT-PCR 和 FQ-PCR 进一步验证,以上分析结果均正确 . 分析还发现 Fhx/P25mRNA 存在选择性拼接 . 以上结果表明 Fhx/P25 基因并不是组织特异转录基因,它的转录表达存在复杂的调控机制,可能还有其他功能 .     

Ras1(CA) overexpression in the posterior silk gland improves silk yield

Sericulture has been greatly advanced by applying hybrid breeding techniques to the domesticated silkworm, Bombyx mori, but has reached a plateau during the last decades. For the first time, we report improved silk yield in a GAL4/UAS transgenic silkworm. Overexpression of the Ras1(CA) oncogene specifically in the posterior silk gland improved fibroin production and silk yield by 60%, while increasing food consumption by only 20%. Ras activation by Ras1(CA) overexpression in the posterior silk gland enhanced phosphorylation levels of Ras downstream effector proteins, up-regulated fibroin mRNA levels, increased total DNA content, and stimulated endoreplication. Moreover, Ras1 activation increased cell and nuclei sizes, enriched subcellular organelles related to protein synthesis, and stimulated ribosome biogenesis for mRNA translation. We conclude that Ras1 activation increases cell size and protein synthesis in the posterior silk gland, leading to silk yield improvement.     

Purification and characterization of an enhancer-binding protein of the fibroin gene. I. Complete purification of fibroin factor 1

An enhancer-binding protein of the fibroin gene, fibroin factor 1 (FF1), has been purified to homogeneity from crude nuclear extracts of posterior silk gland cells where this gene is transcribed specifically. There is a multiplicity of FF1; the FF1 activity was eluted as at least three major fractions on column chromatographies. FF1 is able to form a stable complex with the enhancer DNA sequence in the presence of another proteinous factor named FF2, which lacks ability to bind DNA molecules by itself. One of FF1 forms, FF1a, was purified with a combination of classical purification techniques without using a sequence-specific affinity column, and identified as a protein with molecular mass 125 kDa using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To obtain homogeneous protein of FF1a, purification of more than 26,000-fold from the starting nuclear extract was necessary.