Silkworm genetic sexing through W chromosome-linked, targeted gene integration.
Silkworm genetic sexing through W chromosome-linked, targeted gene integration.
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通过 W 染色体连锁的靶向基因整合来确定家蚕的遗传性别
DOI：
10.1073/pnas.1810945115
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发表时间：
2018-08-28
影响因子：
11.1
通讯作者：
Huang Y
中科院分区：
文献类型：
--
作者：
Zhang Z;Niu B;Ji D;Li M;Li K;James AA;Tan A;Huang Y
意义昆虫的性别是由遗传决定的，并在不同物种之间显示出高度的多样性。家蚕具有在鳞翅目昆虫中发现的WZ/ZZ性染色体系统，在该系统中雌性家蚕是异性恋的。性别分离方法对养蚕的商业方面至关重要，遗传性别鉴定系统也可以作为适应害虫鳞翅目昆虫不育技术的基础。在这里，我们描述了一个基于W染色体的遗传性别鉴定系统，该系统结合了转录激活物样效应核酸酶和CRISPR/Cas9技术。利用无处不在的雌性特异荧光家蚕进行遗传分选或完全雌性特异胚胎致死的家蚕品系的开发，为利用基因组编辑工具定位昆虫性染色体提供了一个成功的范例，这将有助于未来害虫不育昆虫技术的发展。在商业昆虫生产和不育昆虫技术中，性别分离方法是遗传性别鉴定系统的关键。将可选择的标记基因整合到性染色体中，对于具有异性配对性别决定系统的昆虫特别有用。在这里，我们描述了利用转录激活物样效应核酸酶(TALEN)介导的基因组编辑，将荧光标记表达盒整合到鳞翅目模式昆虫W染色体上随机扩增的多态DNA(RAPD)标记区的靶基因。这种家蚕品系从胚胎到成虫都表现出无处不在的雌性特有的红色或绿色荧光。此外，我们开发了一个二元的、雌性特有的胚胎致死系统，该系统结合了TALEN和聚集的规则间隔短回文重复序列(CRISPR)/CRISPR相关蛋白9(Cas9)技术。该系统包括一个由家蚕生殖细胞特异性Nanos(Nos)启动子驱动的、由TALEN介导的W-特异性Cas9表达的菌株，以及另一个针对桑蚕胚胎发育所必需的基因--变压器2(Tra2)的U6来源的单引导RNA(SgRNA)表达的菌株。子代1(F1)杂交种在胚胎期表现出完全的雌性特异性致死。本研究为家蚕遗传性别鉴定提供了一种有前景的方法，并为其他昆虫物种，特别是具有WZ/ZZ性染色体系统的鳞翅目害虫的不育昆虫技术的发展提供了参考。
Significance Insect sex is determined genetically and shows high diversity among different species. The silkworm, Bombyx mori, has a WZ/ZZ sex chromosome system seen in lepidopteran insects in which the females are heterogametic. Sex separation methods are critical for commercial aspects of rearing silkworms, and genetic-sexing systems also could serve as the basis for adaptation to sterile insect techniques for pest lepidopteran species. Here, we describe a W chromosome-based, genetic-sexing system combining transcriptional activator-like effector nucleases and CRISPR/Cas9 technologies in B. mori. Development of silkworm strains with ubiquitous female-specific fluorescence for convenient genetic sorting or complete female-specific embryonic lethality for male-only rearing provides a successful example of targeting an insect sex chromosome with genome editing tools, which should assist future sterile insect technique development for pest insects. Sex separation methods are critical for genetic sexing systems in commercial insect production and sterile insect techniques. Integration of selectable marker genes into a sex chromosome is particularly useful in insects with a heterogametic sex determination system. Here, we describe targeted gene integration of fluorescent marker expression cassettes into a randomly amplified polymorphic DNA (RAPD) marker region in the W chromosome of the lepidopteran model insect Bombyx mori using transcriptional activator-like effector nuclease (TALEN)–mediated genome editing. This silkworm strain shows ubiquitous female-specific red or green fluorescence from the embryonic to adult stages. Furthermore, we developed a binary, female-specific, embryonic lethality system combining the TALEN and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology. This system includes one strain with TALEN-mediated, W-specific Cas9 expression driven by the silkworm germ cell-specific nanos (nos) promoter and another strain with U6-derived single-guide RNA (sgRNA) expression targeting transformer 2 (tra2), an essential gene for silkworm embryonic development. Filial 1 (F1) hybrids exhibit complete female-specific lethality during embryonic stages. Our study provides a promising approach for B. mori genetic sexing and sheds light on developing sterile insect techniques in other insect species, especially in lepidopteran pests with WZ/ZZ sex chromosome systems.
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影响因子：
3.8
作者：
Ma, Sanyuan;Wang, Xiaogang;Xia, Qingyou
通讯作者：
Xia, Qingyou
影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
通讯作者：
Zhang F
影响因子：
1.7
作者：
Abe, H;Mita, K;Shimada, T
通讯作者：
Shimada, T
基于转基因的雌性特异性致死系统，用于家蚕 Bombyx mori 的遗传性别鉴定
DOI：
10.1073/pnas.1221700110
发表时间：
2013-04-23
影响因子：
11.1
作者：
Tan, Anjiang;Fu, Guoliang;Huang, Yongping
通讯作者：
Huang, Yongping
DOI：
10.1073/pnas.0500954102
发表时间：
2005-08-16
影响因子：
11.1
作者：
Tan, A;Tanaka, H;Shiotsuki, T
通讯作者：
Shiotsuki, T