The mechanism of sex-specific splicing at the doublesex gene is different between Drosophila melanogaster and Bombyx mori

We have previously reported that Bmdsx, a homologue of the sex-determining gene, doublesex (dsx), was found to be sex-specifically expressed in various tissues at larval, pupal, and adult stages in the silkworm, Bombyx mori, and was alternatively spliced to yield male- and female-specific mRNAs. To reveal sex-specific differences in splicing patterns of Bmdsx pre-mRNA, the genomic sequence was determined and compared with male- and female-specific Bmdsx cDNA sequences. The open reading frame (ORF) consisted of five exons. Exons 3 and 4 were specifically incorporated into the female type of Bmdsx mRNA. On the other hand, exon 2 was spliced to exon 5 to produce the male type mRNA of Bmdsx. As in the case of Drosophila dsx, the OD2 domain was separated by a female-specific intron into sex-independent and sex-dependent regions. Sex-specific splicing occurred in equivalent positions in the Drosophila dsx gene. However, unlike Drosophila dsx, the female-specific introns showed no weak 3′ splice sites, and the TRA/TRA-2 binding site related sequences were not found in the female-specific exon, nor even in any other regions of the Bmdsx gene. Moreover, an in vitro splicing reaction consisting of HeLa cell nuclear extracts showed that the female-type of Bmdsx mRNA represented the default splicing. These findings suggest that the structural features of the sex-specific splicing patterns of Bmdsx pre-mRNA are similar to those of Drosophila dsx but the regulation of sex-specific alternative splicing of Bmdsx pre-mRNA is different.     

小桐子YABBY全基因组家族成员的鉴定、表达和可变剪接分析

为发掘能源植物小桐子(Jatropha curcas)的YABBY转录因子,以最新公布的小桐子基因组序列为参考,在全基因组层面鉴定出5个亚家族的7个YABBY基因,同一亚家族的成员具有相似的氨基酸序列、基因结构和保守基序组成。YAB2和FIL/YAB3亚家族的2个旁系同源基因对(JcYAB2A/JcYAB2B、JcYAB1/JcYAB3)具有良好的共线性关系,表明片段复制或全基因组复制是小桐子YABBY家族扩张的主要方式。纯化选择是进化的主要动力,而YAB2亚家族成员可能在进化中经历了更明显的功能分化。基因表达模式和蛋白互作预测分析表明JcYAB2B和JcYAB3可能在种子的发育过程中起到重要的调控作用;同时,细胞分裂素、干旱或高盐胁迫处理抑制了大多数JcYABs成员的基因表达。此外,转录组测序结合q RT-PCR分析表明,低温处理有效诱导JcYAB2A和JcYAB2B的基因表达模式发生变化,并伴随着新的、截短的可变剪接转录本的动态积累。因此,推测JcYABs可能通过剪接体的功能竞争或功能互补参与低温响应的调节,这些结果有助于更好地了解YABBY家族成员的功能分化并阐明可变剪接如何调控...     

基因CfATG6和CfATG14参与调控果生刺盘孢细胞自噬和致病力

【目的】炭疽病是油茶的主要病害,由刺盘孢属的多种真菌引起,其中果生刺盘孢分布范围最广、分离率最高,是油茶炭疽病的主要致病菌。研究自噬相关蛋白CfAtg6和CfAtg14的生物学功能,为进一步揭示果生刺盘孢通过细胞自噬调控致病的分子机制,并为油茶炭疽病的防治提供理论基础。【方法】根据同源重组原理,通过聚乙二醇(polyethylene glycol, PEG)介导的方法,在果生刺盘孢中敲除基因CfATG6和CfATG14,并进一步获得回补菌株ΔCfatg6-C和ΔCfatg14-C。【结果】酵母双杂交试验结果显示,果生刺盘孢蛋白CfAtg6和CfAtg14可能存在互作关系。生物学表型测定结果表明,相较于野生型和回补菌株,突变体ΔCfatg6和ΔCfatg14均表现出营养生长速率显著减慢,附着胞形成率分别只有野生型的5%和18%;突变体ΔCfatg6和ΔCfatg14致病力均极显著减弱,造成的油茶叶片病斑面积少于野生型和回补菌株的1/3;CfATG6和CfATG14基因缺失突变体均丧失转运和降解CfAtg8蛋白的能力,并对细胞壁胁迫更敏感。突变体ΔCfatg6的分生孢子产量显著降低,仅为野生型的20%左右;氧化胁迫试验结果表明,相较于野生型和回补菌株,过氧化氢对突变体的生长抑制率升高10%左右。内质网压力胁迫试验表明,ΔCfatg14对二硫苏糖醇抑制率升高5%以上。【结论】自噬相关基因CfATG6和CfATG14参与调控了果生刺盘孢生长发育、细胞自噬和致病力。     

Gene structure, multiple alternative splicing, and expression in gonads of zebrafish Dmrt1

Many basic cellular processes are shared across vast phylogenetic distances, whereas sex-determining mechanisms are highly variable between phyla although the existence of two sexes is nearly universal in the animal kingdom. The only molecular similarity in sex determination found so far between phyla is among the fly doublesex, worm mab-3, and vertebrate Dmrt1/DMY, which contain a zinc-finger-like DNA-binding motif, DM domain. Here we report that three isoforms of the zebrafish Dmrt1 were generated in gonads by multiple alternative splicing, which encoded predicted proteins with 267, 246, and 132 amino acids, respectively. By cDNA cloning and genomic structure analysis, we found that there were seven exons of Dmrt1, which were alternatively spliced to generate the Dmrt1 isoforms. Northern blotting analysis revealed that expression of zebrafish Dmrt1 was higher in testis than ovary. Real time fluorescent quantitative RT-PCR indicated that expression of isoform a of Dmrt1 was dominantly higher than those of Dmrt1 b and c. Furthermore, in situ hybridization to gonads sections showed that Dmrt1 was expressed in developing germ cells of both testis and ovary, suggesting that the Dmrt1 gene is not only associated with testis development, but also, may be important in ovary differentiation of zebrafish.     

萝卜-芥蓝异源四倍体植物SR30基因转录本的选择性剪接分析

为了解异源多倍体形成后,其剪接因子基因SR30在各组织器官间的表达量以及选择性剪接模式与亲本的差异,选取萝卜-芥蓝异源四倍体(Raphanobrassica)及其亲本萝卜(Raphanus sativus)、芥蓝(Brassica oleracea var.alboglabra)为材料,运用RACE-PCR方法克隆到全长的编码序列(CDS)和3非编码区(3 UTR),运用q RT-PCR和半定量RT-PCR检测其在各组织器官中的表达量和各转录本表达量间的差异。结果表明,四倍体中萝卜同源的Rs SR30基因有5种转录本,芥蓝同源的Bo SR30基因有4种转录本。同时,SR30在3物种中的表达具有组织器官的差异,且在四倍体中的总体表达量显著低于亲本。根据克隆到的转录本,预测Rs SR30编码3种蛋白,Bo SR30编码2种,不同蛋白异构体的区别体现在C末端的丝氨酸-精氨酸富集(RS)结构域。因此,萝卜-芥蓝异源多倍体形成后,SR30基因在表达量和转录本选择性剪接方面都发生了改变。     

Gene expression in Plasmodium: from gametocytes to sporozoites

Completion of the complex developmental program of Plasmodium in the mosquito is essential for parasite transmission, yet this part of its life cycle is still poorly understood. In recent years, considerable progress has been made in the identification and characterization of genes expressed during parasite development in the mosquito. This line of investigation was greatly facilitated by the availability of the genome sequence of several Plasmodium, and by the application of approaches such as proteomics, microarrays, gene disruption by homologous recombination (gene knockout) and by use of subtraction libraries. Here, we review what is presently known about genes expressed in gametocytes and during the Plasmodium life cycle in the mosquito.     

鹅掌楸LcUGE基因的克隆和组织表达分析

为了解鹅掌楸(Liriodendron chinense)的UGE基因功能,采用RACE和EPIC-PCR技术克隆到2个UGE基因,命名为LcUGE1和LcUGE2。结果表明,LcUGE1基因的c DNA全长为1 531 bp,包含1 050 bp的开放阅读框,编码349个氨基酸, gDNA长度为11 920 bp;LcUGE2基因的c DNA长度为1 378 bp,包含1 056 bp的开放阅读框,编码351个氨基酸,g DNA长度为6544 bp。LcUGE1和LcUGE2基因均含有9个外显子和8个内含子,且外显子长度和内含子剪切位点序列几乎一致,但内含子片段长度存在显著差异。编码的LcUGE1和LcUGE2蛋白高度保守,保守性达到82%。LcUGE1基因在雄蕊中表达量最高,而LcUGE2基因则在花萼中表达量最高。这表明LcUGEs基因可能参与鹅掌楸的生殖发育过程。     

Rbfox-regulated alternative splicing is critical for zebrafish cardiac and skeletal muscle functions

Rbfox RNA binding proteins are implicated as regulators of phylogenetically-conserved alternative splicing events important for muscle function. To investigate the function of rbfox genes, we used morpholino-mediated knockdown of muscle-expressed rbfox1l and rbfox2 in zebrafish embryos. Single and double morphant embryos exhibited changes in splicing of overlapping sets of bioinformatically-predicted rbfox target exons, many of which exhibit a muscle-enriched splicing pattern that is conserved in vertebrates. Thus, conservation of intronic Rbfox binding motifs is a good predictor of Rbfox-regulated alternative splicing. Morphology and development of single morphant embryos were strikingly normal; however, muscle development in double morphants was severely disrupted. Defects in cardiac muscle were marked by reduced heart rate and in skeletal muscle by complete paralysis. The predominance of wavy myofibers and abnormal thick and thin filaments in skeletal muscle revealed that myofibril assembly is defective and disorganized in double morphants. Ultra-structural analysis revealed that although sarcomeres with electron dense M- and Z-bands are present in muscle fibers of rbfox1l/rbox2 morphants, they are substantially reduced in number and alignment. Importantly, splicing changes and morphological defects were rescued by expression of morpholino-resistant rbfox cDNA. Additionally, a target-blocking MO complementary to a single UGCAUG motif adjacent to an rbfox target exon of fxr1 inhibited inclusion in a similar manner to rbfox knockdown, providing evidence that Rbfox regulates the splicing of target exons via direct binding to intronic regulatory motifs. We conclude that Rbfox proteins regulate an alternative splicing program essential for vertebrate heart and skeletal muscle functions.     

毛竹PeSCL3基因表达特征及其启动子活性研究

为探讨毛竹(Phyllostachys edulis)SCL3基因的表达特征及其启动子活性,采用同源克隆的方法从毛竹中分离到SCL3同源基因Pe SCL3的编码区(ORF)和上游启动子序列(Pe SCL3p)。序列分析表明,Pe SCL3的ORF为1335 bp,推测编码含444氨基酸的蛋白,该蛋白与水稻(Oryza sativa)的SCL3同源性高达93.9%。Pe SCL3p长度为1358 bp,含有脱落酸(ABA)应答元件ABRE、赤霉素(GA3)应答元件GARE-motif和P-box、干旱诱导MYB结合位点等多种作用元件。实时定量PCR分析结果表明,Pe SCL3在毛竹叶中的表达丰度最高,其次是茎和根,而鞘中的最低;Pe SCL3的表达受GA3的抑制,受ABA、Na Cl和干旱的诱导。转Pe SCL3p∷GUS拟南芥(Arabidposis thaliana)的GUS染色结果表明,根尖、顶端生长点和子叶叶柄均被染成蓝色,尤其根尖的染色最深。这表明Pe SCL3对毛竹的生长发育,尤其是根系,可能起着重要的调控作用。     

Cat-2 gene expression

Summary Poly(A)⁴ RNA was isolated from maize scutella of different stages of post-germinative development and translated in vitro in a rabbit reticulocyte translation system. Immunoprecipitation of the translation products with CAT-2-specific antibody was used to quantitate the relative levels of translatable CAT-2 mRNA at each stage. The results show a close correlation between the developmental profile of Cat2 gene expression and the profile of CAT-2 mRNA levels. Evidence that the levels of CAT-2 mRNA are regulated by a temporal regulatory gene (Car1) is presented and the possible mechanism(s) of this regulation discussed.This work was supported by Research Grants No. GM22733 and No. GM33817 from the U.S. National Institutes of Health, Public Health Service to J.G.S. This is paper No. 9933 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695, USA     

巨桉BRI1基因的克隆和表达分析

为了解BRI1基因在巨桉中的功能,采用PCR技术克隆了EgrBRI1基因,分析了EgrBRI1的生物信息学和亚细胞定位,并对EgrBRI1基因响应激素和胁迫的差异表达进行了分析。结果表明,EgrBRI1基因全长3 893 bp,编码1 197个氨基酸。EgrBRI1蛋白稳定,空间结构复杂,存在3个motifs,主要定位于细胞膜。茉莉酸甲酯和油菜素内酯(BR)处理后,EgrBRI1基因在叶片中的表达上升,而水杨酸处理则没有明显的变化。盐胁迫和冷胁迫下,EgrBRI1基因表达表现为先下降后上升的趋势。因此,EgrBRI1基因能快速对外施激素做出响应,并在巨桉抗逆方面发挥重要作用,这可能是通过对BR信号的响应来实现的。     

AtBS14a and AtBS14b, two Bet1/Sft1-like SNAREs from Arabidopsis thaliana that complement mutations in the yeast SFT1 gene

SNAREs are membrane-associated proteins that play a central role in vesicle targeting and intra-cellular membrane fusion reactions in eukaryotic cells. Here we describe the identification of AtBS14a and AtBS14b, putative SNAREs from Arabidopsis thaliana that share 60% amino acid sequence identity. Both AtBS14a and BS14b are dosage suppressors of the temperature-sensitive growth defect in sft1-1 cells and over-expression of either AtBS14a or AtBS14b can support the growth of sft1Δ cells but not bet1Δ cells. These data together with structure–function and biochemical studies presented herein suggest that AtBS14a and AtBS14b share properties that are consistent with them being members of the Bet1/Sft1 SNARE protein family.     

巨桉EgrGBF1基因的克隆和表达模式分析

G-box结合蛋白(GBF)是一类能够识别并结合G-box的转录因子,广泛参与植物基因响应外界刺激的表达调控。通过巨桉(Eucalyptus grandis)初生生长到次生生长的转录组测序筛选出差异表达基因EgrGBF1,为探讨其在桉树生长发育中的功能,从巨桉中克隆了该基因,并进行了结构和进化分析。结果表明,EgrGBF1编码区长度为984 bp,编码327个氨基酸, 存在2个转录本,分别命名为EgrGBF1α和EgrGBF1β。实时荧光定量PCR结果表明,EgrGBF1α和EgrGBF1β在不同组织中,不同激素、胁迫处理下的表达模式不同,EgrGBF1α主要在茎尖表达,沿节间向下表达量逐渐降低,而EgrGBF1β在韧皮部高表达,在节间的表达量无显著差异。在水杨酸和缺硼处理下,EgrGBF1α和EgrGBF1β的表达趋势相反。EgrGBF1α在缺磷处理168 h的表达量最高,而EgrGBF1β在处理6 h的表达量最高。因此,EgrGBF1在桉树生长发育以及响应胁迫中发挥着重要作用,且转录本EgrGBF1α和EgrGBF1β可能具有不同的功能。     

橄榄CaICE1基因的克隆和表达分析

为了解橄榄(Canarium album)抗寒相关转录因子ICE1的调控功能,采用RT-PCR技术克隆了‘福榄1号’的ICE1,命名为CaICE1,并进行生物信息学、qRT-PCR表达模式和相关miRNA预测分析。结果表明,CaICE1 cDNA序列的开放阅读框长度为1 650 bp,可编码549个氨基酸(GenBank登录号MG459422)。Ca ICE1为不稳定亲水性蛋白质,含有跨膜结构、磷酸化位点以及HLH保守结构域,定位于细胞核,与枳的ICE1亲缘关系较近。CaICE1密码子偏好性较弱,AGA、AGG、TGG和CCA可能为其最优密码子群。CaICE1主要在橄榄花、种子和叶中大量表达,-3℃低温胁迫下CaICE1表达水平比常温显著上升。psRNAtarget预测结果表明,CaICE1可能是miR825、miR477、miR5658、miR1436和miR394等多个逆境响应miRNA的靶基因。因此,CaICE1可能在橄榄低温胁迫过程中发挥重要调控作用,且可能受miRNA的调控。