靶向基因编辑技术在秀丽隐杆线虫中的应用

遗传突变体和转基因是生物学研究的基础,是揭示生物体内各个基因相互作用的生物学研究对象.秀丽隐杆线虫(Caenorhabditis elegans)是遗传学研究的模式生物,如何有效地诱导特定基因发生突变和构建转基因线虫品系是秀丽线虫遗传学研究的两个重要方面.近些年来,靶向基因编辑技术迅速发展,使得科研人员可以在秀丽线虫中快速而高效地编辑特定基因.本文就线虫中靶向基因编辑的方法,特别是CRISPR/Cas9技术,以及目的线虫品系的筛选进行了综述.     

铜对秀丽隐杆线虫毒性效应的研究

为了阐明铜(Cu)对秀丽隐杆线虫Caenorhabditis elegans长期作用的毒性效应,对实验室多代筛选的耐铜型秀丽隐杆线虫进行了寿命、衰老、发育、生殖和运动等生物学指标的研究.结果显示耐铜型秀丽隐杆线虫与野生型秀丽隐杆线虫相比其寿命缩短、衰老提前、个体发育受到抑制,且出现繁殖率降低、生殖能力减弱、运动行为存在障碍等一系列生理变化.本文为理解与阐明Cu的毒性效应提供了实验资料,有助于深入开展Cu毒性机理的研究.     

BAZ-2和SET-6通过BLMP-1调控秀丽隐杆线虫衰老

该文使用秀丽隐杆线虫作为模式动物,探讨了两个表观遗传因子BAZ-2和SET-6通过BLMP-1调控编码线粒体功能蛋白核基因的表达,进而调控线虫衰老。利用JASPAR数据库,分析了baz-2和set-6突变体线虫中表达上调基因的启动子区域DNA序列,发现转录因子BLMP-1的特征结合位点在这些序列中富集。随后分别在baz-2和set-6突变体线虫中敲除blmp-1基因,检测blmp-1;baz-2和blmp-1;set-6双敲除突变体线虫的寿命、咽喉肌肉跳动能力、基础型和增强型食物诱导的缓慢运动反应、抗氧化应激能力和线粒体功能相关基因的表达水平,发现敲除blmp-1消除了baz-2和set-6突变体线虫寿命较长,咽喉肌肉跳动、基础型和增强型食物诱导的缓慢运动反应和抗氧化能力较好的行为表型,以及线粒体功能相关基因表达上调的现象。该研究阐明了BAZ-2和SET-6通过BLMP-1调控线虫衰老的机制。     

拟南芥雄性不育相关基因LDAD1&2的遗传定位(英文)

利用EMS诱变筛选手段分离到一株拟南芥类似花药不开裂雄性不育突变体(like-defective in anther de-hiscence,ldad),其果荚干瘪,花药不能开裂且花粉败育。遗传分析表明,突变体的表型受2个隐性基因控制;细胞学观察发现,在花药发育过程中伴随着小孢子的降解;通过图位克隆初步对ldad的2个突变位点分别定位,一个定位在1号染色体上SSLP标记F22L4与端粒之间171 kb的区间,另一个定位在5号染色体上SSLP标记T10O8与端粒间150 kb的区间内;生物信息学分析显示此区间内未见育性相关的已知基因。该研究的结果对进一步克隆LDAD1&2基因及探讨其在花药发育中的功能具有重要意义。     

必需氨基酸延缓秀丽隐杆线虫衰老的研究

利用模式生物秀丽隐杆线虫,考察8种人体必需氨基酸对衰老的影响。首先建立秀丽隐杆线虫寿命模型,以雷帕霉素为阳性对照药,分别考察8种必需氨基酸对线虫生存时间的影响。再用筛选出的氨基酸处理线虫21d,通过秀丽隐杆线虫-绿脓杆菌感染模型,考察氨基酸对线虫的抗感染能力的影响,利用实时荧光定量Real-Time RT-PCR方法检测氨基酸处理线虫后DAF-16/FOXO下游基因和免疫相关基因的表达水平。结果表明8种必需氨基酸中苏氨酸和异亮氨酸既能延长野生型线虫的寿命又能延长daf-16突变型线虫的寿命,同时还能增强秀丽隐杆线虫抗绿脓杆菌感染的能力,并提高免疫相关基因lys-7、clec-67的表达水平,而DAF-16/FOXO下游基因表达没有明显变化。因此苏氨酸和异亮氨酸能延长线虫寿命、提高抗感染能力,且对线虫寿命的延长作用不完全依赖于DAF-16/FOXO转录因子。     

细菌与秀丽隐杆线虫之间互作关系的研究进展

秀丽隐杆线虫(Caenorhabditis elegans)以其个体小、易培养、生活周期短等优势成为生物发育、衰老、神经及免疫相关机制研究的模式生物.它在实验室培养时主要靠饲喂大肠杆菌OP50,有报道,细菌及其代谢物对线虫的代谢、行为和寿命有至关重要的影响.因此,作为一个遗传模型,秀丽隐杆线虫可以帮助研究微生物与宿主相...     

利用改进的MutMap方法克隆水稻雄性不育基因

通过对籼稻黄华占EMS(甲磺酸乙酯)诱变, 筛选得到一隐性核不育的水稻雄性不育突变体osms55, 遗传分析表明该突变体为单基因控制的隐性核不育, 采用高通量的Illumina Infinium iSelect SNP(50 K)芯片检测技术鉴定该突变体的遗传背景, 确认该突变体的遗传背景与黄华占一致。文章利用改进的MutMap方法成功克隆该雄性不育基因, 突变位点与突变表型的共分离分析表明LOC_Os02g40450(MER3)是控制osms55突变体雄性不育的基因, 该基因的剪切识别位点发生变异后导致剪切异常, 造成第5外显子缺失15个碱基, 从而产生雄性不育。改进的MutMap方法无需精确组装的野生型基因组序列作对照, 而是通过将定位群体中有突变表型植株的DNA pool和野生型植株DNA的重测序结果分别与日本晴参考基因组进行比对, 然后再比较突变体和野生型的差异SNP来确定候选基因, 该方法大大降低了野生型基因组测序和组装成本, 进一步扩大了MutMap方法的应用范围。     

维生素C通过胰岛素信号通路增强秀丽隐杆线虫抗氧化作用

目的：利用秀丽隐杆线虫为模式生物,研究维生素C在秀丽隐杆线虫体内的抗氧化效应及其机制。方法：分别以含有0.05、0.25、0.5 mg/mL维生素C的NGM培养基饲养秀丽隐杆线虫,测定不同浓度维生素C饲养线虫体内超氧化物歧化酶（SOD）和过氧化氢酶（CAT）的含量,同时检测0.25 mg/mL的维生素C饲养线虫age-1、daf-2、daf-16、sir-2.1、clt-1基因mRNA变化。在高氧环境中,干扰0.25 mg/mL维生素C饲养线虫daf-2、daf-16基因表达检测线虫的存活情况,观察0.25 mg/mL维生素C饲养线虫DAF-16入核情况。结果：0.25 mg/mL的维生素C提高秀丽隐杆线虫体内SOD和CAT活力,在高氧环境中,0.25 mg/mL的维生素C降低age-1、daf-2基因表达,提高daf-16基因表达,同时增加DAF-16蛋白入核。结论：维生素C通过DAF-16胰岛素信号通路增强秀丽隐杆线虫抗氧化作用。     

基于微流控芯片的秀丽隐杆线虫的研究进展

微流控芯片技术作为近年来最前沿的分析技术之一,已经在化学、生物学、医药学等研究领域取得了突破性的进展.微流控芯片具有高通量、微型化和多功能集成化等独特优势,已经成为生物医学研究的新平台之一,被越来越多地应用于秀丽隐杆线虫的研究.综述了基于微流控芯片上的秀丽隐杆线虫在生物医学领域中的研究进展,侧重介绍了微流控芯片在线虫的自动化固定、行为学、衰老与发育学、神经学、药物筛选及基因筛选等六大方面所取得的最新进展,并展望了微流控芯片的应用前景.     

MS1521是拟南芥花药发育的必需基因

通过对3个拟南芥（Arabidopsis thaliana）雄性不育突变体（ms1521,st350,st454）的分析,研究了MS1521基因在花药发育过程中的功能。ms1521是通过EMS诱变野生型拟南芥得到的一株突变体,遗传分析表明ms1521是隐性单核基因控制的。利用图位克隆的方法对不育基因MS1521进行了定位,结果将MS1521定位于拟南芥第一条染色体上26kb的区间内,该定位区间内有一个影响花器官形态建成的基因UFO。测序结果表明在ms1521突变体中UFO基因编码区的958bp处发生了单碱基突变,导致MS1521该位点的氨基酸由天冬酰胺变成了天冬氨酸。另外两个表型与ms1521相似的突变体st350和st454来自T-DNA插入突变体群体。测序结果表明突变体st350和st454分别在UFO基因编码区发生了提前终止突变。等位分析表明它们与MS1521基因是等位的。3个突变体营养生长期发育正常,但生殖生长发育出现异常：有的雄蕊只有花丝没有花药;或者有花药但花丝变短;或者雄蕊有正常的花丝和花药,花药中有可育的花粉,但药室不能开裂;最终导致突变体不育的表型。进一步细胞学观察发现药室不能开裂是由于药室内壁细胞纤维化和木质化增厚不明显造成的。以上这些结果表明MS1521基因在花药发育过程中起重要作用。     

Acetate non-utilizing mutants of Arabidopsis: evidence that organic acids influence carbohydrate perception in germinating seedlings

A phenotypic screen was employed to isolate Arabidopsis plants that are deficient in their ability to utilize or sense acetate. The screening strategy, based on resistance to the toxic acetate analogue monofluoroacetic acid, was adapted from one that has been used successfully to identify important metabolic and regulatory genes involved in acetate metabolism in fungi. Following conventions established from the fungal work, the mutants were called acn mutants for ac etate n on-utilization. Three highly resistant plant lines were the focus of genetic and physiological studies. Mutant acn1 appears to be a true acetate non-utilizing mutant, as it displays increased sensitivity to exogenous acetate. The progeny of the original acn2 mutant did not germinate, even in the presence of sucrose as an exogenous carbon source. The germination of seeds from the F3 generation depended on the sucrose concentration in the medium. Only a small proportion of seeds germinated in the absence of exogenous sucrose and in the presence of 100 mM sucrose, but up to 70% of seeds germinated on 20 mM sucrose. Mutant acn3 exhibited sensitivity to exogenous sucrose, showing significant chlorosis on medium containing 20 mM sucrose, but no chlorosis when grown in the absence of exogenous sucrose. This phenotype was alleviated if acetate was provided. The acn mutants demonstrate that disrupting organic acid utilization can have profound affects on carbohydrate metabolism.Communicated by G. Jürgens     

Identification of mutations in Caenorhabditis elegans that cause resistance to high levels of dietary zinc and analysis using a genomewide map of single nucleotide polymorphisms scored by pyrosequencing

Zinc plays many critical roles in biological systems: zinc bound to proteins has structural and catalytic functions, and zinc is proposed to act as a signaling molecule. Because zinc deficiency and excess result in toxicity, animals have evolved sophisticated mechanisms for zinc metabolism and homeostasis. However, these mechanisms remain poorly defined. To identify genes involved in zinc metabolism, we conducted a forward genetic screen for chemically induced mutations that cause Caenorhabditis elegans to be resistant to high levels of dietary zinc. Nineteen mutations that confer significant resistance to supplemental dietary zinc were identified. To determine the map positions of these mutations, we developed a genomewide map of single nucleotide polymorphisms (SNPs) that can be scored by the high-throughput method of DNA pyrosequencing. This map was used to determine the approximate chromosomal position of each mutation, and the accuracy of this approach was verified by conducting three-factor mapping experiments with mutations that cause visible phenotypes. This is a generally applicable mapping approach that can be used to position a wide variety of C. elegans mutations. The mapping experiments demonstrate that the 19 mutations identify at least three genes that, when mutated, confer resistance to toxicity caused by supplemental dietary zinc. These genes are likely to be involved in zinc metabolism, and the analysis of these genes will provide insights into mechanisms of excess zinc toxicity.     

Genetics of metalaxyl resistance in Phytophthora infestans.

Several sexual crosses involving isolates of Phytophthora infestans of diverse sensitivities to metalaxyl were studied. Metalaxyl sensitivity was determined by comparing the growth of an isolate on metalaxyl-amended agar medium (5 microg/ml) with growth on medium containing no metalaxyl. When both parents had the same phenotype for metalaxyl sensitivity (both resistant or both sensitive), all F1 progeny had the parental phenotype. In two crosses (75 and 76) each involving one sensitive and one resistant parent, however, the progeny segregated 1:1, suggesting that the common resistant parent (Bg8) was heterozygous for metalaxyl sensitivity. When an F2 progeny was constructed from resistant F1 isolates in cross 76, the progeny segregated 1:3 (sensitive:resistant), indicating that metalaxyl resistance in Bg8 is conferred by a single dominant gene. Variation in the progeny sensitivity appears to involve minor genes. A correlation study between metalaxyl resistance and fitness components did not reveal any association.     

Zebrafish Mutants calamity and catastrophe Define Critical Pathways of Gene–Nutrient Interactions in Developmental Copper Metabolism

Nutrient availability is an important environmental variable during development that has significant effects on the metabolism, health, and viability of an organism. To understand these interactions for the nutrient copper, we used a chemical genetic screen for zebrafish mutants sensitive to developmental copper deficiency. In this screen, we isolated two mutants that define subtleties of copper metabolism. The first contains a viable hypomorphic allele of atp7a and results in a loss of pigmentation when exposed to mild nutritional copper deficiency. This mutant displays incompletely penetrant skeletal defects affected by developmental copper availability. The second carries an inactivating mutation in the vacuolar ATPase that causes punctate melanocytes and embryonic lethality. This mutant, catastrophe, is sensitive to copper deprivation revealing overlap between ion metabolic pathways. Together, the two mutants illustrate the utility of chemical genetic screens in zebrafish to elucidate the interaction of nutrient availability and genetic polymorphisms in cellular metabolism.     

Molecular cloning, chromosomal mapping, and sequence analysis of copper resistance genes from Xanthomonas campestris pv. juglandis: homology with small blue copper proteins and multicopper oxidase.

Copper-resistant strains of Xanthomonas campestris pv. juglandis occur in walnut orchards throughout northern California. The copper resistance genes from a copper-resistant strain C5 of X. campestris pv. juglandis were cloned and located on a 4.9-kb ClaI fragment, which hybridized only to DNA of copper-resistant strains of X. campestris pv. juglandis, and was part of an approximately 20-kb region which was conserved among such strains of X. campestris pv. juglandis. Hybridization analysis indicated that the copper resistance genes were located on the chromosome. Plasmids conferring copper resistance were not detected in copper-resistant strains, nor did mating with copper-sensitive strains result in copper-resistant transconjugants. Copper resistance genes from X. campestris pv. juglandis shared nucleotide sequence similarity with copper resistance genes from Pseudomonas syringae pv. tomato, P. syringae, and X. campestris pv. vesicatoria. DNA sequence analysis of the 4.9-kb fragment from strain C5 revealed that the sequence had an overall G+C content of 58.7%, and four open reading frames (ORF1 to ORF4), oriented in the same direction. All four ORFs were required for full expression of copper resistance, on the basis of Tn3-spice insertional inactivation and deletion analysis. The predicted amino acid sequences of ORF1 to ORF4 showed 65, 45, 47, and 40% identity with CopA, CopB, CopC, and CopD, respectively, from P. syringae pv. tomato. The most conserved regions are ORF1 and CopA and the C-terminal region (166 amino acids from the C terminus) of ORF2 and CopB. The hydrophobicity profiles of each pair of predicted polypeptides are similar except for the N terminus of ORF2 and CopB. Four histidine-rich polypeptide regions in ORF1 and CopA strongly resembled the copper-binding motifs of small blue copper proteins and multicopper oxidases, such as fungal laccases, plant ascorbate oxidase, and human ceruloplasmin. Putative copper ligands of the ORF1 polypeptide product are proposed, indicating that the polypeptide of ORF1 might bind four copper ions: one type 1, one type 2, and two type 3.     

Defects in glycosylphosphatidylinositol (GPI) anchor synthesis activate Hog1 kinase and confer copper-resistance in Saccharomyces cerevisisae

Las21/Gpi7 contains a heavy-metal-associated motif at its N-terminus. When this motif was disrupted by amino acid substitution, the cells acquired weak copper-resistance. We found that the previously isolated las21 mutants were strongly resistant to copper. Metallothionein is necessary for the expression of the copper-resistance of the las21 mutants. However, hyper-production of metallothionein is unlikely to be the cause of copper-resistance of the las21 mutants. Copper-sensitive mutants (collectively called Cus mutants) were isolated from the las21delta and characterized. One of the Cus genes was found to be PBS2, which encodes Hog1 MAP kinase kinase, indicating that the Hog1 MAP kinase pathway is needed for the expression of copper-resistance of the las21 mutants. As expected, the las21delta hog1delta strain was no longer copper-resistant. We found that Hog1 was constitutively activated in las21delta cells and in ssk1delta las21delta cells but not in sho1delta las21delta cells. Inactivation of either FSR2/MCD4 or MPC1/GPI13, both of which are involved in GPI anchor synthesis, like LAS21, caused a similar level of constitutive activation of Hog1 kinase and copper-resistance as found in the las21delta strain. The constitutive activation was canceled by introducing the sskl mutation, but not the sho1 mutation, in each GPI anchor mutant tested, suggesting that the defect in GPI anchor synthesis specifically affects the Slnl branch of the MAP kinase pathway. Since the wild-type cells grown in YPD containing 0.5 M NaCl do not show copper-resistance, mere activation of Hog1 is not sufficient for expression of copper-resistance. We propose that a defect in GPI anchor synthesis has multiple consequences, including activation of the Hog1 MAP kinase cascade and conferring copper-resistance.     

Use of homeotic mutation tasselseed2 for investigation of the action of maize meiotic genes during micro- and megasporogenesis

The manifestation of the ms43 maize meiotic mutation in the megasporogenesis of ts2 ms43 double mutants has been studied. Combined genetical and cytological analysis of the progeny of diheterozygote selfing showed that the ms43 mei gene was not microsporogenesis-specific. The manifestation of ms43 in megasporogenesis of the double mutants proved to be affected by the ts2 mutation. It prevented formation of the phenotype characteristic of ms43 and distorted early developmental stages of the entire ovule. It is the first study of megasporogenesis in tasselseed2 mutant tassels. Cytological data on the afd1 mutation in single and double mutants are presented. Possible mechanisms of the interaction between the ms43 and ts2 mutations are discussed.     

DDT resistance in Drosophila correlates with Cyp6g1 over-expression and confers cross-resistance to the neonicotinoid imidacloprid

Mutagenesis can be used as a means of predicting likely mechanisms of resistance to novel classes of insecticides. We used chemical mutagenesis in Drosophila to screen for mutants that had become resistant to imidacloprid, a neonicotinoid insecticide. Here we report the isolation of two new dominant imidacloprid-resistant mutants. By recombinational mapping we show that these map to the same location as Rst(2)DDT. Furthermore, we show that pre-existing Rst(2)DDT alleles in turn confer cross-resistance to imidacloprid. In order to localize the Rst(2)DDT gene more precisely, we mapped resistance to both DDT and imidacloprid with respect to P-element markers whose genomic location is known. By screening for recombinants between these P-elements and resistance we localized the gene between 48D5-6 and 48F3-6 on the polytene chromosome map. The genomic sequence in this interval shows a cluster of cytochrome P450 genes, one of which, Cyp6g1, is over-expressed in all resistant strains examined. We are now testing the hypothesis that resistance to both compounds is associated with over-expression of this P450 gene.     

Mitochondrial Dysfunction Confers Resistance to Multiple Drugs in Caenorhabditis elegans

In a previous genetic screen for Caenorhabditis elegans mutants that survive in the presence of an antimitotic drug, hemiasterlin, we identified eight strong mutants. Two of these were found to be resistant to multiple toxins, and in one of these we identified a missense mutation in phb-2, which encodes the mitochondrial protein prohibitin 2. Here we identify two additional mutations that confer drug resistance, spg-7 and har-1, also in genes encoding mitochondrial proteins. Other mitochondrial mutants, isp-1, eat-3, and clk-1, were also found to be drug-resistant. Respiratory complex inhibitors, FCCP and oligomycin, and a producer of reactive oxygen species (ROS), paraquat, all rescued wild-type worms from hemiasterlin toxicity. Worms lacking mitochondrial superoxide dismutase (MnSOD) were modestly drug-resistant, and elimination of MnSOD in the phb-2, har-1, and spg-7 mutants enhanced resistance. The antioxidant N-acetyl-l-cysteine prevented mitochondrial inhibitors from rescuing wild-type worms from hemiasterlin and sensitized mutants to the toxin, suggesting that a mechanism sensitive to ROS is necessary to trigger drug resistance in C. elegans. Using genetics, we show that this drug resistance requires pkc-1, the C. elegans ortholog of human PKCε.     

A mutation in Arabidopsis that leads to constitutive expression of systemic acquired resistance.

Systemic acquired resistance (SAR) is a nonspecific defense response in plants that is associated with an increase in the endogenous level of salicylic acid (SA) and elevated expression of pathogenesis-related (PR) genes. To identify mutants involved in the regulation of PR genes and the onset of SAR, we transformed Arabidopsis with a reporter gene containing the promoter of a beta-1,3-glucanase-encoding PR gene (BGL2) and the coding region of beta-glucuronidase (GUS). The resulting transgenic line (BGL2-GUS) was mutagenized, and the M2 progeny were scored for constitutive GUS activity. We report the characterization of one mutant, cpr1 (constitutive expressor of PR genes), that was identified in this screen and shown by RNA gel blot analysis also to have elevated expression of the endogenous PR genes BGL2, PR-1, and PR-5. Genetic analyses indicated that the phenotype conferred by cpr1 is caused by a single, recessive nuclear mutation and is suppressed in plants producing a bacterial salicylate hydroxylase, which inactivates SA. Furthermore, biochemical analysis showed that the endogenous level of SA is elevated in the mutant. Finally, the cpr1 plants were found to be resistant to the fungal pathogen Peronospora parasitica NOCO2 and the bacterial pathogen Pseudomonas syringae pv maculicola ES4326, which are virulent in wild-type BGL2-GUS plants. Because the cpr1 mutation is recessive and associated with an elevated endogenous level of SA, we propose that the CPR1 gene product acts upstream of SA as a negative regulator of SAR.