目的:分析4例doublesex和mab-3相关转录因子1(doublesex and mab-3 related transcription factor 1, DMRT1)基因变异/单倍剂量不足所致的46，XY性发育异常(46，XY DSD)患儿的临床特征，以提高医生对此病的认识。方法:回顾性收集4例46，XY DSD患儿的病史、体格检查、内分泌功能评估、性腺病理及遗传学资料。结果:例1为DMRT1基因变异c.332G>T(P.Arg111Met)杂合错义新发变异，因"闭经"于15岁就诊，外生殖器男性化得分(external masculinization scores, EMS)为0分。DMRT1基因单倍剂量不足3例：9p末端依次存在1.2 Mb、5.1 Mb和6.0 Mb片段缺失，其中例3合并5p区域33.3 Mb重复。3例就诊年龄均在1岁以内，社会性别2例为女性，1例为男性，均因"外生殖器外观异常"就诊，EMS依次为1分、0分、5分。例1和例3内分泌评估提示原发性性腺功能减退，性腺病理均为完全性性腺发育不良，例2提示Leydig细胞功能良好、Sertoil细胞功能欠佳，性腺病理为混合性性腺发育不良。例3在18个月龄诊断为性腺母细胞瘤。例4患儿内分泌评估示睾丸功能正常，暂未同意活检。4例染色体核型均为46，XY。结论:在DMRT1基因变异/单倍剂量不足的46，XY DSD患者中，前者初诊年龄偏大，后者就诊年龄偏小，性腺功能可表现为从正常到完全性性腺发育不良。此类患者存在性腺母细胞瘤高风险，且发病年龄小，对性腺发育不良者应及早行性腺活检。

蚊属于典型的性二态昆虫,两性在行为上有着巨大差异,雄性成蚊主要依靠植物汁液获取碳源,而雌性只有吸血后卵巢才能够正常发育,这也是蚊传播多种人类疾病的重要基础.蚊的性二态由复杂的性别决定基因通路调控,虽然通路中两个关键因子双性基因(doublesex,dsx)与fruitless(fru)在结构和功能上与其他昆虫具有高度保守性,通过性别可变剪切最终调控下游性别表型与性行为相关基因,但是其性别决定初始信号即雄性决定因子(Male-determining factor,M-factor)无论与其他昆虫相比还是属种间均具有巨大的差异,而从M-factor至dsx的调控通路亦尚未明确.目前,利用已知的蚊性别决定基因对蚊进行基因改造,在实验室已经初步达到了性别逆转或种群抑制的效果.本文综述了蚊性别决定调控通路中初始信号雄性决定因子的进化和作用机制,其下游信号通路的响应模式,以及以性别决定基因为靶点的转基因蚊防制策略,为深入理解蚊性别决定机制与探讨蚊媒防制提供新思路.

[目的]RBP1是一种参与黑腹果蝇Drosophila melanogaster性别决定基因doublesex(dsx)pre-mRNA选择性剪接的重要剪接因子.本研究旨在克隆RBP1基因在西方蜜蜂Apis mellifera中的同源基因AmRBP1,分析其结构域及其在西方蜜蜂性别决定初期胚胎和幼虫不同发育阶段中的表达模式,以期为研究该基因是否参与蜜蜂性别决定奠定基础.[方法]基于NCBI中提交的西方蜜蜂基因数据库和转录组数据库,利用黑腹果蝇rbp1对数据库进行同源检索获得RBP1的转录组序列,并设计引物进行PCR扩增克隆AmRBP1基因,利用生物信息学软件对其进行核酸及氨基酸序列分析.采用半定量RT-PCR技术分析该基因在西方蜜蜂胚胎及幼虫期的表达模式.[结果]由序列比对分析可知,西方蜜蜂AmRBP1基因的pre-mRNA含有3种不同的选择性剪切形式.通过设计不同的引物,克隆得到3种成熟的mRNA序列,分别命名为AmRBP1-R1(GenBank登录号:KY404154)、AmRBP1-R2(GenBank登录号:KY404155)和AmRBP1-R3(GenBank登录号:KY404156);mRNA长度分别为696,790和771 nt,编码的蛋白大小分别为130,166和162 aa;结构域分析发现AmRBP1的3种选择性剪切形式具有相同的结构域,氨基端含有一个RRM结构域,羧基端含有一个RS结构域.同源性分析表明,西方蜜蜂AmRBP1与丽蝇蛹集金小蜂Nasonia vitripennis、红火蚁Solenopsis invicta、埃及伊蚊Aedes aegypti、黑腹果蝇D.melanogaster、家蚕Bombyx mori、家蝇Musca domestica和中华按蚊Anopheles sinensis RBP1蛋白氨基酸序列一致性分别为96.97％,94.20％,84.85％,81.48％,80.28％,78.15％和69.23％.半定量RT-PCR结果表明,AmRBP1基因的3种剪切形式(AmRBP1-R1-3)在西方蜜蜂胚胎和幼虫各时期均有表达,无雌特异性或雄特异性表达的剪切形式.[结论]西方蜜蜂AmRBP1可能是一种SR家族剪切因子,其有可能参与调控基因表达的特定剪切,但其是否参与doublesex(dsx)基因pre-mRNA的性别特异性剪切需要进一步研究.

为明确牙鲆雄性性别相关关键基因dmrt1 (doublesex and mab-3-related transcription factor 1)是否为pol-miR-26a、pol-miR-26b作用的靶基因,本研究利用PCR技术克隆了dmrt13'UTR区,并成功引入了特殊限制性内切酶PemⅠ和NotⅠ的识别位点;将得到的dmrt1 3'UTR区片段和psiCHECK-2载体进行双酶切、连接后得到野生型重组质粒;并且利用定点诱变法对dmrt1 3'UTR区进行体外定点诱变,将pol-miR-26a、pol-miR-26b识别的靶序列ACTGAA突变为TGACTT,形成突变型重组质粒.经琼脂糖凝胶电泳鉴定、双酶切及测序分析,成功获得了可用于pol-miR-26a、pol-miR-26b的靶基因dmrt1验证的野生型psiCHECK-dmrt1-3'UTR和突变型psiCHECK-mutated dmrt1-3'UTR的报告质粒,为进一步研究pol-miR-26a、pol-miR-26b的靶基因dmrt1鉴定和功能奠定了基础.

Fusion of the testis occurs in most Lepidoptera insects,including Spodoptera litura,an important polyphagous pest.Testicular fusion in S.litura is advantageous for male reproduction,and the molecular mechanism of fusion remains unknown.Doublesex influences the formation of genitalia,the behavior of courtship,and sexually dimorphic traits in fruit-fly and silkworm,and is essential for sexual differentiation.However,its purpose in the testis ofS.litura remains unknown.The doublesex gene ofS.litura (Sldsx) has male-specific SldsxM and female-specific SldsxF isoforms,and exhibits a higher expression level in the male testis.At the testicular fusion stage (L6D6),Sldsx attained the highest expression compared to the pre-fusion and post-fusion periods.Moreover,Sldsx had a higher expression in the peritoneal sheaths of testis than that of germ cells in the follicle.CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) was applied to S.litura to determine the role of Sldsx.A mixture of single guide RNA messenger RNA and Cas9 protein (300 ng/μL each) was injected into eggs within 2 h following oviposition.CRISPR/Cas9 successfully induced genomic mutagenesis of Sldsx at Go generation.The mutant males had smaller testis surrounded by less tracheae.Moreover,the mutant males had abnormal external genitalia and could not finish mating with wild-type females.Additionally,testes were fused for almost all mutant males.The results showed that Sldsx was not related to testicular fusion,and is required for both testis development and the formation and function of external genitalia in S.litura.The main roles of doublesex on the male are similar to other insects.

Sex determination has been studied in the model lepidopteran species Bombyx mori,but it remains poorly understood in lepidopteran pests.In the present study,we identified and characterized the Masculinizer (Masc) gene in a Noctuidae pest species,Agrotis ipsilon.Sequence analysis revealed that AiMasc encodes a protein of 658 amino acids that has two CCCH-type zinc finger domains and two conserved cysteine residues (Cys-277 and Cys-280).We assessed the masculinizing activity of AiMasc in BmN cells and found that AiMasc induced expression of the male-specific doublesex isoform.Disruption of Masc via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) in A.ipsilon caused abnormalities in abdominal segments and external genitalia,resulting in male-specific sterility.These results suggest that Masc participates in the process of sex determination in A.ipsilon.Successful identification of sex-determination gene in a pest species may enable the development of novel genetic approaches for pest control.

Dmrt (Doble-sex and mab-3 relatated transcription factor)基因的主要功能是决定性别、分化性腺和形成组织和器官.为研究其在三角帆蚌(Hyriopsis cumingii)性腺发育和分化过程中的作用,本实验通过比对三角帆蚌转录组序列和RACE扩增的方法,获得了三角帆蚌Dmrt1基因(HcDmrt1)全长cDNA,采用实时定量PCR的方法研究该基因在性腺发育和分化的表达情况.结果 显示,HcDmrt1基因全长为1561bp,ORF区(开放阅读框区域)1 161bp,编码386个氨基酸.编码蛋白具有DM (Doublesex/MAB-3) Domain family保守结构域.三角帆蚌2～10月龄个体性腺荧光定量结果显示该基因参与了早期性腺发育;组织荧光定量结果显示,HcDmrt1在各组织中均有表达,但是在雌雄性腺组织中表达差异最大,雄性中表达量明显高于雌性.性腺的原位杂交结果显示,该基因主要在卵母细胞的细胞膜上检测到表达信号,在雄性性腺中,主要在滤泡腔中的精细胞观察到信号.由此推测,Hc Dmrt1基因参与了三角帆蚌的性腺发育过程.

Recent identification of a Piwi-interacting RNA (piRNA)-initiated sex deter-mination cascade in the silkworm,Bombyx mori,provides novel insights into high di-versity of insect sex determination pathways.In this system,the W-chromosome-derived Fem piRNA is the primary sex determination signal.A CCCH-type zinc finger gene Mas-culinizer (Masc),which is targeted by Fem piRNA-PIWI complex in female animals,is indispensable for male-specific splicing of B.mori doublesex (Bmdsx).Although many genes involved in this cascade have been identified,the regulatory mechanisms of silk-worm sex determination remain to be elucidated.Here we show that another CCCH-type zinc finger gene,Bmznf-2,is a masculinization factor in B.mori.Bmznf-2 shows testis-abundant expression and loss of Bmznf-2 function via clustered regularly interspaced short palindromic repeats / single-guide RNA-mediated mutagenesis results in feminized dif-ferentiation and appearance of the female-specific splicing variants of Bmdsx transcripts in males.In contrast,there is no phenotypic consequence in mutant females.In mutant males,relative messenger RNA expression levels of female-dominant genes such as vitel-logenin and sex-specific storage protein 1 are significantly elevated while several male-dominant genes are significantly down-regulated.Furthermore,male mutants show de-layed developmental timing,smaller body sizes of larvae and malformation of moth wings.Our data thus reveal that Bmznf-2 plays an indispensable role in silkworm male sexual differentiation.

Sexual development in insects is regulated by a complicated hierarchical cas-cade of sex determination.The primary signals are diverse,whereas the central nexus dou-blesex (dsx) gene is relatively conserved within the pathway.Aedes (Stegomyia) albopictus is an important vector with an extensive worldwide distribution.We previously reported that Ae.albopictus dsx (Aalbdsx) yields one male-(AalbdsxM) and three female-specific isoforms (AalbdsxF1-3);however,the spatiotemporal expression profiles and mechanisms regulating sex-specific altemative splicing require further investigation.In this study,we demonstrated that the AalbdsxM messenger RNA (mRNA) represents the default pattern when analyzed in human foreskin fibroblasts and HeLa cells.We combined reverse tran-scription polymerase chain reaction with RNA immunoprecipitation using specific anti-bodies against tagged Ae.albopictus male-determining factor AalNix and confirmed that AalNix indirectly regulates dsx pre-mRNA and regulates its alternative splicing.During the early embryo stage (0-2 and 4-8 h),matemal dsxF and default splicing dsxM were detected in both sexes;the expression of dsxM then decreased until sufficient AalNix tran-scripts accumulated in male embryos at 20-24 h.These findings suggest that one or more potential dsx splicing enhancers can shift dsxM to dsxF in both sexes;however,the pres-ence of Nix influences the function of this unknown splicing enhancer and ultimately leads to the formation of dsxM in males.Finally,our results provide important insight into the regulatory mechanism of dsx alternative splicing in the mosquito.

Gender differences in behavior are prevalent among animal king-doms.The causal links among a particular gene,neural circuitry,and gendered behaviors have long been of particular interest in neurosci-ence.The fruit fly,Drosophila melanogaster,is a wonderful animal model with fruitful genetic tools to tackle these questions(Guo et al.,2019).It has been found that two sex determination genes,fruitless(fru)and doublesex(dsx),jointly govern the sex differences in the nervous system that regulate the male-specific courtship behavior(Burtis and Baker,1989;Ito et al.,1996;Ryner et al.,1996).The male-specific forms of fru(fruM)act in a subset of neurons to establish the potential for courtship behavior(Demir and Dickson,2005;Manoli et al.,2005;Stockinger et al.,2005).In contrast,dsx acts in subsets of both neural and nonneural tissues of males and fe-males to regulate sexual development and behaviors(Rideout et al.,2010;Robinett et al.,2010).