Fibroin heavy chain gene replacement with a highly ordered synthetic repeat sequence in Bombyx mori

IF 3.2 2区 农林科学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Yoko Takasu, Nobuto Yamada, Katsura Kojima, Masatoshi Iga, Fumiko Yukuhiro, Tetsuya Iizuka, Taiyo Yoshioka
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Abstract

The exceptional quality of silkworm silk is attributed to the amino acid sequence of its fibroin heavy chain (Fib-H) protein. The large central domain of Fib-H, which consists of glycine- and alanine-rich crystalline regions interspersed with amorphous motifs of approximately 30 amino acid residues, is considered crucial for fibrilization and determines the properties of the silk fiber. We established a technical platform to modify the Fib-H core region systematically using transcription activator-like effector nuclease-mediated homologous recombination through a somatic and germline gene knockin assay along with PCR-based screening. This efficient knockin system was used to generate a silkworm strain carrying a mutant Fib-H allele, in which the core region was replaced with a highly ordered synthetic repeat sequence of a length comparable with native Fib-H core. Heterozygous knockin mutants produced seemingly normal cocoons, whereas homozygotes did not and exhibited considerable degradation in their posterior silk glands (PSGs). Cross-sectional examination of the PSG lumen and tensile tests conducted on reeled silk threads indicated that the mutant Fib-H, which exhibited reduced stability in the PSG cells and lumen, affected the mechanical properties of the fiber. Thus, sequence manipulation of the Fib-H core domain was identified as a crucial step in successfully creating artificial silk using knockin technology.

Abstract Image

家蚕丝蛋白重链基因的高度有序合成重复序列替换
蚕丝的特殊品质归因于其纤维蛋白重链(Fib-H)蛋白的氨基酸序列。纤维- h的大中心结构域,由富含甘氨酸和丙氨酸的晶体区域组成,散布着大约30个氨基酸残基的无定形基序,被认为是纤维化的关键,并决定了丝纤维的性质。我们建立了一个技术平台,通过体细胞和种系基因敲入试验以及基于pcr的筛选,系统地使用转录激活子样效应核酸酶介导的同源重组来修饰Fib-H核心区域。这种高效的敲入系统被用于产生携带突变Fib-H等位基因的家蚕品系,其中核心区域被高度有序的合成重复序列取代,其长度与天然Fib-H核心相当。杂合子敲除蛋白突变体产生看似正常的茧,而纯合子则没有,并且在其后丝腺(psg)中表现出相当大的降解。PSG管腔的横断面检查和对纺丝进行的拉伸试验表明,突变纤维- h在PSG细胞和管腔中表现出稳定性降低,影响了纤维的机械性能。因此,Fib-H核心结构域的序列操作被认为是使用敲入技术成功制造人造丝绸的关键步骤。
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来源期刊
CiteScore
7.40
自引率
5.30%
发文量
105
审稿时长
40 days
期刊介绍: This international journal publishes original contributions and mini-reviews in the fields of insect biochemistry and insect molecular biology. Main areas of interest are neurochemistry, hormone and pheromone biochemistry, enzymes and metabolism, hormone action and gene regulation, gene characterization and structure, pharmacology, immunology and cell and tissue culture. Papers on the biochemistry and molecular biology of other groups of arthropods are published if of general interest to the readership. Technique papers will be considered for publication if they significantly advance the field of insect biochemistry and molecular biology in the opinion of the Editors and Editorial Board.
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