Essential roles for the Dhr78 orphan nuclear receptor during molting of the Drosophila tracheal system

The Drosophila Dhr78 orphan nuclear receptor has been proposed to play a role in molting of the tracheal cuticle and regulate gene expression during the third larval instar, possibly in response to a novel systemic hormonal signal. Here, we show that there are no essential maternal functions for Dhr78 during development, and that mutants missing both maternal and zygotic Dhr78 function die primarily during second and third instar larval development. We show that defects in the tracheal system can be observed as early as the first instar, manifested as regions of fluid in the dorsal tracheal trunks. In addition, Dhr78 mutant tracheae show a highly penetrant defect in gas filling at the first-to-second instar larval molt. Dhr78 expression in only the tracheal system is sufficient to rescue the lethality of Dhr78 mutants, and selective inactivation of Dhr78 function in the tracheae by targeted RNAi is sufficient to result in tracheal defects. Finally, we see no evidence for widespread activation of the Dhr78 ligand binding domain in third instar larvae using the GAL4-LBD system, arguing against a systemic hormone for the receptor at this stage in development. Taken together, our results indicate that Dhr78 exerts its essential functions during molting of the tracheal cuticle in Drosophila.     

A Conditional Knockout Toolkit for Caenorhabditis elegans Based on the Cre/loxP Recombination

Conditional knockout (cKO) based on site-specific recombination (SSR) technology is a powerful approach for estimating gene functions in a spatially and temporally specific manner in many model animals. In Caenorhabditis elegans (C. elegans), spatial- and temporal-specific gene functions have been largely determined by mosaic analyses, rescue experiments and feeding RNAi methods. To develop a systematic and stable cKO system in C. elegans, we generated Cre recombinase expression vectors that are driven by various tissue-specific or heat-shock promoters. Validation using Cre-mediated fluorescence protein inactivation or activation systems demonstrated successful Cre-dependent loxP excision. We established a collection of multi-copy Cre transgenic strains for each evaluated vector. To evaluate our Cre/loxP-based cKO system, we generated sid-1 deletion mutants harboring floxed sid-1 single-copy integration (SCI) using ultraviolet trimethylpsoralen (UV/TMP) methods. sid-1 mutants that were rescued by the floxed sid-1 SCI were then crossed with the Pdpy-7::Cre strain for cKO in the hypodermis. The sid-1 cKO animals were resistant to bli-3 RNAi, which causes the Bli-phenotyple in the hypodermis, but they were sensitive to unc-22 RNAi, which leads to twitching of the body wall muscle. Our system, which is based on the combination of a transgenic Cre collection, pre-existing deletion mutants, and UV/TMP SCI methods, provided a systematic approach for cKO in C. elegans.     

Drosophila R2D2 mediates follicle formation in somatic tissues through interactions with Dicer-1

The miRNA pathway has been shown to regulate developmentally important genes. Dicer-1 is required to cleave endogenously encoded microRNA (miRNA) precursors into mature miRNAs that regulate endogenous gene expression. RNA interference (RNAi) is a gene silencing mechanism triggered by double-stranded RNA (dsRNA) that protects organisms from parasitic nucleic acids. In Drosophila, Dicer-2 cleaves dsRNA into 21 base-pair small interfering RNA (siRNA) that are loaded into RISC (RNA induced silencing complex) that in turn cleaves mRNAs homologous to the siRNAs. Dicer-2 co-purifies with R2D2, a low-molecular weight protein that loads siRNA onto Ago-2 in RISC. Loss of R2D2 results in defective RNAi. However, unlike mutants in other RNAi components like Dicer-2 or Ago-2, we report here that r2d2¹ mutants have striking developmental defects. r2d2¹ mutants have reduced female fertility, producing less than 1/10 the normal number of progeny. These escapers have normal morphology. We show R2D2 functions in the ovary, specifically in the somatic tissues giving rise to the stalk and other follicle cells critical for establishing the cellular architecture of the oocyte. Most interestingly, the female fertility defects are dramatically enhanced when one copy of the dcr-1 gene is missing and Dicer-1 protein co-immunoprecipitates with R2D2 antisera. These data show that r2d2¹ mutants have reduced viability and defective female fertility that stems from abnormal follicle cell function, and Dicer-1 impacts this process. We conclude that R2D2 functions beyond its role in RNA interference to include ovarian development in Drosophila.     

Expression of the tyrosinase-encoding gene in a colorless melanophore mutant of the medaka fish, Oryzias latipes

In the medaka fish Oryzias latipes many mutants for body colors have been isolated. Among them, a colorless melanophore mutant b, carrying b alleles homozygously, has pigmented black eyes but orange-colored skin with amelanotic melanophores, suggesting the presence of a tissue-specific mechanism of melanin formation. To cast light on the molecular basis of the mechanism, we have cloned cDNAs for tyrosinase (Tyr), a key enzyme in melanin biosynthesis, from the wild-type (wt) fish. DNA sequence analysis revealed that all clones encode a protein of 540 amino acids, having five potential glycosylation sites and two copper-binding sites that are characteristic features of Tyr. Genomic DNA blot analysis disclosed that the Tyr gene is present as a single copy in the fish genome. Using a cDNA clone as a probe, RNA blot analysis was carried out. In the wt, the 2.2-kb Tyr mRNA was expressed in eyes and skin but not in liver, corresponding to tissue-specific melanin formation. In the b mutant, contrary to expectation, the mRNA was detected not only in eyes but also in amelanotic skin. Therefore, pigmentation of the skin controlled by b is not directly related to expression of the Tyr gene.     

Abscisic acid-dependent PMT1 expression regulates salt tolerance by alleviating abscisic acid-mediated reactive oxygen species production in Arabidopsis

Phosphocholine(PCho) is an intermediate metabolite of nonplastid plant membranes that is essential for salt tolerance. However, how PCho metabolism modulates response to salt stress remains unknown. Here, we characterize the role of phosphoethanolamine N-methyltransferase 1(PMT1) in salt stress tolerance in Arabidopsis thaliana using a T-DNA insertional mutant, geneediting alleles, and complemented lines. The pmt1 mutants showed a severe inhibition of root elongation when exposed to salt stress,...     

新生隐球菌RBP1基因克隆与功能分析

新生隐球菌是自然界广泛存在的具荚膜的酵母型病原真菌,能侵染人类中枢神经系统引起真菌性脑膜炎,每年导致全球大约18万人死亡。本研究在前期隐球菌交配表达谱的基础上,选择一上调表达的RNA结合蛋白基因（CNAG_04772）,进行克隆和功能分析。结果表明该基因全长2 247bp,cDNA全长1 518bp,编码505个氨基酸组成的蛋白,含有2个RNA识别基序RRM1和RRM2,命名为RBP1。基因表达模式分析表明RBP1在隐球菌酵母细胞、担子以及担孢子阶段都有表达,交配菌丝阶段不表达;亚细胞定位分析表明Rbp1蛋白定位于隐球菌的细胞核和细胞质中。与野生型菌株H99相比,敲除突变体菌株能够交配并产生双核菌丝,但丧失产生担孢子的能力,而互补菌株与野生型菌株H99间无显著差异。致病力测定结果显示,敲除突变体菌株致病性显著降低。     

刺槐核糖体蛋白同源基因RpRPL22在共生结瘤过程中功能研究

目的: 核糖体蛋白(RPs)属于多功能蛋白,能够参与调控细胞生长和响应胁迫条件。RpRPL22是一个从豆科植物刺槐中分离得到的结瘤相关基因,通过序列比对发现其与核糖体大亚基蛋白RPL22高度同源。对其如何通过调控根瘤菌侵染而在共生结瘤过程中发挥重要作用进行了较为深入的探索。方法: 利用实时荧光定量PCR技术(qRT-PCR)分析RpRPL22在接菌后不同时间及不同植物组织的表达变化。利用cDNA末端快速扩增技术(RACE)获得目的基因cDNA全长。通过GFP报告基因进行RpRPL22亚细胞定位分析。通过Gateway BP重组技术构建RNA干扰(RNAi)重组载体,借助电转化法将重组载体转至农杆菌K599,利用农杆菌介导植物根部,接菌后观察和测量植株表型。首先从宏观水平统计观察目的基因是否对结瘤过程有影响,其次从分子水平揭示目的基因在共生结瘤过程的重要功能。结果: 不同接菌时期、不同植物组织目的基因qRT-PCR相对表达量结果显示,几乎在所有取样的接菌时间,目的基因RpRPL22在接菌根中的相对表达量都低于未接菌对照根,只有接菌后第25天除外。在成熟的根瘤中,接菌后第25天该基因的表达量也最高。洋葱表皮和毛状根亚细胞定位结果均显示在椰菜花叶病毒(CaMV)的35S启动子控制下,RpRPL22融合绿色荧光蛋白GFP的荧光信号在细胞核和细胞质有明显的表达。RNAi转化植株的表型统计观察结果,比如植株鲜重、植株的有效结瘤数目较对照组均有明显的降低;同时RNAi转化植株在根瘤菌侵染过程形成的侵染线数目和根瘤原基数目较对照均显著降低。根瘤切片实验用于观察根瘤显微超微结构,结果显示RNAi植株根瘤中固氮区的受菌侵染细胞数目与对照相比明显减少。电镜观察根瘤单个受菌侵染细胞中类菌体形态显示,RNAi根瘤中类菌体侵染细胞胞体多呈不规则形状,皱缩变形严重,环类菌体周间隙空间增大,多共生体融合,表现出细胞凋亡的迹象。对照根瘤中的受菌侵染细胞胞体多呈圆形椭圆形,胞质饱满丰富且分布均匀,细胞发育正常,表明RNAi植株根瘤发育过程明显受阻。结论: 核糖体蛋白(RP)能够参与调控豆科植物共生结瘤过程,相关同源基因RpRPL22可能在起始根瘤菌侵染植物和阻止类菌体降解过程中起重要作用。     

高通量测序检测金针菇RNAi转化子插入位点及拷贝数

本研究对金针菇Flammulina velutipes的一个RNAi转化子菌株1382R3进行了高通量测序,以本实验室先前获得的野生型W23基因组数据为参考,分析了该转化子的基因插入位点以及拷贝数。转化子菌株1382R3是通过农杆菌介导将fv-hmg1-RNAi载体转化至金针菇菌株并通过PCR检测筛选标记而得到。通过BLAST将转化子测序的reads对外源载体和基因组定位,找到具有基因组序列（GS）和外源载体序列（ES）两种序列的临界reads,并据此使用PERL语言程序成功在转化子1382R3菌株中找到两个插入位点。对两个插入位置的序列分析表明：在插入位点1,T-DNA片段部分插入;在插入位点2,T-DNA全部插入到基因组。两个插入位点都对基因组内源基因的表达造成了一定的干扰。此方法拓宽了高通量测序技术的应用范围,将其运用到遗传转化插入位置和拷贝数的研究中,有利于食用菌的功能基因组及基因工程研究。     

The pentatricopeptide repeat protein EMB1270 interacts with CFM2 to splice specific group II introns in Arabidopsis chloroplasts

Chloroplast biogenesis requires the coordinated expression of chloroplast and nuclear genes. Here, we show that EMB1270, a plastid-localized pentatricopeptide repeat (PPR) protein, is required for chloroplast biogenesis in Arabidopsis thaliana. Knockout of EMB1270 led to embryo arrest, whereas a mild knockdown mutant of EMB1270 displayed a virescent phenotype. Almost no photosynthetic proteins accumulated in the albino emb1270 knockout mutant. By contrast, in the emb1270 knockdown mutant, the levels of ClpP1 and photosystem I (PSI) subunits were significantly reduced, whereas the levels of photosystem II (PSII) subunits were normal. Furthermore, the splicing efficiencies of the clpP1.2, ycf3.1, ndhA, and ndhB plastid introns were dramatically reduced in both emb1270 mutants. RNA immunoprecipitation revealed that EMB1270 associated with these introns in vivo. In an RNA electrophoretic mobility shift assay (REMSA), a truncated EMB1270 protein containing the 11 N-terminal PPR motifs bound to the predicted sequences of the clpP1.2, ycf3.1, and ndhA introns. In addition, EMB1270 specifically interacted with CRM Family Member 2 (CFM2). Given that CFM2 is known to be required for splicing the same plastid RNAs, our results suggest that EMB1270 associates with CFM2 to facilitate the splicing of specific group II introns in Arabidopsis.     

Circadian period lengths of lipid synthesis mutants (cel,chol-1) of Neurospora show defective temperature,but intact pH-compensation

The influence of extracellular pH on the circadian sporulation rhythm of Neurospora crassa has been investigated for the mutants chol-1 and cel. Both mutants have a defect in the lipid synthesis pathway and require either choline or palmitate, respectively, as supplements for normal growth. The chol-1 and cel mutants also show an impaired temperature-compensation when growing on minimal medium. We investigated the possible correlation between loss of temperature- and pH-compensation in cel and chol-1 similar to the correlation found earlier for the frq⁷ mutant. Our results show that the cel and the chol-1 mutants, although defective in temperature-compensation have an intact pH-compensation of their circadian rhythms. At present, the products of the frq-locus are the only components of the clock that affect the sporulation rhythm of Neurospora both through pH- and temperature-compensation.     

In vivo and in vitro evidence that the four essential intermediate filament (IF) proteins A1, A2, A3 and B1 of the nematode Caenorhabditis elegans form an obligate heteropolymeric IF system

The in vitro polymerization and tissue-specific expression patterns of the four essential intermediate filament (IF) proteins (A1, A2, A3, B1) and the non-essential IF protein A4 were analyzed. Recombinant B1, used as a probe in blot overlay assays of the 11 Caenorhabditis elegans IF proteins, reacted strongly with proteins A1 to A4, indicating a heterotypic interaction. Obligate heteropolymeric filament assembly in vitro was confirmed by electron microscopy. Protein B1 formed long IF when mixed with an equimolar amount of A1, A2 or A3. Developmentally regulated coexpression of B1 and one or more members of the A family was found with GFP-promoter reporters. This coexpression pattern argues for a heteropolymer system in vivo. One or both splice variants of the B1 gene are always coexpressed in a tissue-specific manner with at least one member of the A family in hypodermis, pharynx, pharyngeal-intestinal valve, excretory cells, uterus, vulva and rectum. Interestingly, while the intestine normally lacks a B1/A pair, the dauer larva shows intestinal B1 and A4. These results are in line with similar postembryonic phenotypes of the hypodermis induced by RNA interference (RNAi) of genes B1, A2 and A3. Similarly, defects of the pharynx and its A1-GFP containing tonofilaments observed in the postembryonic B1 RNAi phenotype are consistent with the coexpression of B1 and A1 in the marginal cells. Thus RNAi analyses provide independent evidence for the existence of the B1/A obligate heteropolymer system in vivo. Proteins A1 and B1 have a similar and rather slow turnover rate in photobleaching experiments of the pharynx tonofilaments.     

水稻黄绿叶调控基因YGL18的克隆与功能解析

叶色突变体往往伴随着叶绿素含量变化及叶绿体结构异常,是研究叶绿体发育与光合作用相关基因功能的重要材料。该研究通过甲基磺酸乙酯(EMS)诱变籼稻(Oryzasativasubsp.indica)品种华占(HZ)获得黄绿叶突变体,将其命名为ygl18 (yellow-green leaf 18)。与野生型相比,黄绿叶突变体ygl18自三叶期起叶片开始变黄且程度不断加深,同时伴随着光合速率与叶绿素含量下降,且结实率、千粒重及有效穗数均显著降低。透射电镜观察结果显示, ygl18的叶绿体结构紊乱,基质片层疏松,发育受到抑制,与叶片出现黄绿色表型一致。遗传分析表明, ygl18突变性状受1对隐性等位核基因控制,这对等位基因位于水稻第3号染色体长臂标记InDel2和InDel3之间115.2 kb范围内。进一步研究发现该突变体表型是编码铁氧还蛋白FdC2的基因LOC＿Os03g48040的5’UTR发生突变所致。通过CRISPR转基因实验验证了该基因对表型的控制作用。研究结果揭示了叶色调控网络的遗传基础,可为今后选育高光效水稻品种提供新线索。