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1.
Insect Sci ; 27(1): 14-21, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31246335

RESUMO

Manipulating an exogenous or endogenous gene of interest at a defined level is critical for a wide variety of experiments. The Gal4/UAS system has been widely used to direct gene expression for studying complex genetic and biological problems in Drosophila melanogaster and other model organisms. Driven by a given tissue-specific Gal4, expressing UAS-transgene or UAS-RNAi (RNA interference) could be used to up- or down-regulate target gene expression, respectively. However, the efficiency of the Gal4/UAS system is roughly predefined by properties of transposon vector constructs and the insertion site in the transgenic stock. Here, we describe a simple way to modulate optomotor blind (omb) expression levels in its endogenous expression region of the wing disc. We co-expressed UAS-omb and UAS-omb-RNAi together under the control of dpp-Gal4 driver which is expressed in the omb expression region of the wing pouch. The repression effect is more sensitive to temperature than that of overexpression. At low temperature, overexpression plays a dominant role but the efficiency is attenuated by UAS-omb-RNAi. In contrast, at high temperature RNAi predominates in gene expression regulation. By this strategy, we could manipulate omb expression levels at a moderate level. It allows us to manipulate omb expression levels in the same tissue between overexpression and repression at different stages by temperature control.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/genética , Proteínas com Domínio T/genética , Asas de Animais/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Proteínas com Domínio T/metabolismo
2.
Insect Sci ; 25(2): 211-221, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27696733

RESUMO

In Locusta migratoria, we found that two chitin biosynthesis genes, UDP N-acetylglucosamine pyrophosphorylase gene LmUAP1 and chitin synthase gene LmCHS1, are expressed mainly in the integument and are responsible for cuticle formation. However, whether these genes are regulated by 20-hydroxyecdysone (20E) is still largely unclear. Here, we showed the developmental expression pattern of LmUAP1, LmCHS1 and the corresponding 20E titer during the last instar nymph stage of locust. RNA interference (RNAi) directed toward a common region of the two isoforms of LmEcR (LmEcRcom) reduced the expression level of LmUAP1, while there was no difference in the expression of LmCHS1. Meantime, injection of 20E in vivo induced the expression of LmUAP1 but not LmCHS1. Further, we found injection-based RNAi of LmEcRcom resulted in 100% mortality. The locusts failed to molt with no apolysis, and maintained in the nymph stage until death. In conclusion, our preliminary results indicated that LmUAP1 in the chitin biosynthesis pathway is a 20E late-response gene and LmEcR plays an essential role in locust growth and development, which could be a good potential target for RNAi-based pest control.


Assuntos
Quitina Sintase/metabolismo , Ecdisterona/metabolismo , Regulação da Expressão Gênica , Locusta migratoria/metabolismo , Nucleotidiltransferases/metabolismo , Animais , Quitina/biossíntese , Quitina Sintase/genética , Feminino , Hemolinfa/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Locusta migratoria/genética , Locusta migratoria/crescimento & desenvolvimento , Masculino , Nucleotidiltransferases/genética , Ninfa/enzimologia
3.
Insect Sci ; 23(3): 406-16, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26792119

RESUMO

Insect chitinases are involved in degradation of chitin from the exoskeleton or peritrophic metrix of midgut. In Locusta migratoria, two duplicated Cht5s (LmCht5-1 and LmCht5-2) have been shown to have distinct molecular characteristics and biological roles. To explore the protein properties of the two LmCht5s, we heterologously expressed both enzymes using baculovirus expression system in SF9 cells, and characterized kinetic and carbohydrate-binding properties of purified enzymes. LmCht5-1 and LmCht5-2 exhibited similar pH and temperature optimums. LmCht5-1 has lower Km value for the oligomeric substrate (4MU-(GlcNAc)3 ), and higher Km value for the longer substrate (CM-Chitin-RBV) compared with LmCht5-2. A comparison of amino acids and homology modeling of catalytic domain presented similar TIM barrel structures and differentiated amino acids between two proteins. LmCht5-1 has a chitin-binding domain (CBD) tightly bound to colloidal chitin, but LmCht5-2 does not have a CBD for binding to colloidal chitin. Our results suggested both LmCht5-1 and LmCht5-2, which have the critical glutamate residue in region II of catalytic domain, exhibited chitinolytic activity cleaving both polymeric and oligomeric substrates. LmCht5-1 had relatively higher activity against the oligomeric substrate, 4MU-(GlcNAc)3 , whereas LmCht5-2 exhibited higher activity toward the longer substrate, CM-Chitin-RBV. These findings are helpful for further research to clarify their different roles in insect growth and development.


Assuntos
Quitinases/metabolismo , Proteínas de Insetos/metabolismo , Locusta migratoria/enzimologia , Animais , Quitina/química , Quitinases/química , Quitinases/genética , Proteínas de Insetos/química , Proteínas de Insetos/genética , Cinética , Ligação Proteica , Células Sf9
4.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 44(3): 258-63, 2015 May.
Artigo em Chinês | MEDLINE | ID: mdl-26350005

RESUMO

OBJECTIVE: To investigate the risks of recurrent preeclampsia and observe the incidence and long-term prognosis of recurrent preeclampsia. METHODS: One hundred and fifteen women with preeclampsia history admitted in Women's Hospital, Zhejiang University School of Medicine from January 2009 to December 2013 were enrolled in the study. The clinical data were retrospectively analyzed. RESULTS: Among 115 women with preeclampsia, 82 cases (71.3%)had recurrent preeclampsia. The onset age, the pregnant interval time, regular prenatal check-up, weight gain during pregnancy, body mass index (BMI), hyperlipidemia, fetal growth restriction (FGR), maternal family history were closely associated with recurrent preeclampsia (P<0.05). Logistic regression analysis showed that the pregnant interval time, BMI, FGR were independent risk factors for preeclampsia recurrence. Compared with the preeclampsia in first pregnancy, the mather had earlier onset and termination of pregnancy, higher blood pressure, higher rate of urine protein ≥ 2+ and higher rate of complications in recurrent preeclampsia. The offspring had higher rate of preterm birth, especially the time of birth for 34 weeks or earlier and lower birth weight (P<0.05). The incidence of chronic hypertension in recurrent preeclampsia group was higher than that in no recurrence group (47.5% vs 23.3%, P<0.05); the overweight rate of first pregnant offspring in recurrence group was higher than that in no recurrence group (31.25% vs 6.70%, P<0.05). CONCLUSION: The onset age, time interval, regular prenatal check-up, weight gain during pregnancy, BMI, hyperlipidemia, FGR, maternal family history are closely associated with recurrent preeclampsia, and the pregnant interval time, BMI, FGR are independent risk factors for preeclampsia recurrence. Recurrent preeclampsia has more serious clinical outcome and complications, and prevention need to be emphasized.


Assuntos
Pré-Eclâmpsia/epidemiologia , Complicações na Gravidez/epidemiologia , Índice de Massa Corporal , Feminino , Retardo do Crescimento Fetal/epidemiologia , Humanos , Hiperlipidemias/epidemiologia , Recém-Nascido , Gravidez , Recidiva , Estudos Retrospectivos , Fatores de Risco
5.
Insect Sci ; 20(1): 53-60, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23955825

RESUMO

MicroRNAs (miRNAs) are small noncoding RNAs, which participate in many biological processes. The small RNA transcriptome in the migratory locust has been characterized and 50 conserved miRNA families and 185 potential locust-specific miRNA family candidates have been identified using high-throughput sequencing. However, it is unclear whether miRNAs influence a wide variety of locusts' biological processes, such as growth or development. In insects, Dicer1 ribonuclease transforms miRNA precursors into mature miRNAs. Thus, using systemic RNA interference (RNAi) to silence the expression of Dicer1 in the migratory locust, Locusta migratoria, we reduced miRNA contents in the locust and disrupted two types of molt (nymph-nymph, and nymph-adult). The RNAi of LmDicer1 also resulted in a high mortality in L. migratora. Our study revealed that LmDicer1 was essential for miRNA regulation and development of L. migratoria. These results further support our notion that LmDicer1 could serve as an excellent target for developing novel strategies for controlling this important insect pest.


Assuntos
Gafanhotos/enzimologia , Gafanhotos/genética , Proteínas de Insetos/genética , MicroRNAs/genética , Interferência de RNA , Ribonuclease III/genética , Animais , Gafanhotos/classificação , Proteínas de Insetos/metabolismo , MicroRNAs/metabolismo , Dados de Sequência Molecular , Filogenia , Ribonuclease III/metabolismo
6.
Insect Sci ; 20(1): 109-19, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23955831

RESUMO

ß-N-acetylglucosaminidases are crucial enzymes involved in chitin degradation in insects. We identified a ß-N-acetylglucosaminidase gene (LmNAG1) from Locusta migratoria. The full-length complementary DNA (cDNA) of LmNAG1 consists of 2 667 nucleotides, including an open reading frame (ORF) of 1 845 nucleotides encoding 614 amino acid residues, and 233- and 589-nucleotide non-coding regions at the 5'- and 3'-ends, respectively. Phylogenetic analysis grouped the cDNA-deduced LmNAG1 protein with the enzymatically characterized ß-N-acetylglucosaminidases in group I. Analyses of stage- and tissue-dependent expression patterns of LmNAG1 were carried out by real-time quantitative polymerase chain reaction. Our results showed that LmNAG1 transcript level in the integument was significantly high in the last 2 days of the fourth and fifth instar nymphs. LmNAG1 was highly expressed in foregut and hindgut. RNA interference of LmNAG1 resulted in an effective silence of the gene and a significantly reduced total LmNAG enzyme activity at 48 and 72 h after the injection of LmNAG1 double-stranded RNA (dsRNA). As compared with the control nymphs injected with GFP dsRNA, 50% of the dsLmNAG1-injected nymphs were not able to molt successfully and eventually died. Our results suggest that LmNAG1 plays an essential role in molting process of L. migratoria.


Assuntos
Acetilglucosaminidase/genética , Proteínas de Insetos/genética , Locusta migratoria/enzimologia , Muda , Interferência de RNA , Acetilglucosaminidase/metabolismo , Animais , Sequência de Bases , Proteínas de Insetos/metabolismo , Locusta migratoria/classificação , Locusta migratoria/genética , Locusta migratoria/crescimento & desenvolvimento , Dados de Sequência Molecular , Filogenia
7.
Yi Chuan Xue Bao ; 31(2): 159-65, 2004 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-15473306

RESUMO

Random Amplified Polymorphic DNA (RAPD) markers were applied to analyze genetic relationships of five populations of Oxya chinensis collected from Shanxi Province and laner Mongolia, Oxya japonica from Guangxi was used as an outgroup. Genomic DNA of sixty-four individuals was extracted from dissected leg muscle using phenol-chloroform procedure, and then amplified by 10 random primers (10 bp), the amplified products were separated by agarose gel electrophoresis. The results were as follows: (1) a total of 115 clear and reproducible bands were generated, molecular size was 200 - 2500 bp. The obtaining segments of individual primer were among 5 - 15, on average, about 12 bands per primer. (2) The dendrogram based on 115 RAPD markers was constructed and clustered using between-groups linkage method. The cluster analysis indicated strong similarities within populations, firstly, the individuals in each population closely clustered together;and then five populations of Oxya chinensis could be distinguished with RAPD markers and were grouped into two distinct clusters. The dendrogram showed that Shanxi Linyi population and Tunliu population were the most similar,which were clustered with Taiyuan population Shanxi into one cluster, while, Daixian population in Shanxi was closely related to laner Mongolia population, both of which belonged to the other cluster. Nevertheless, All the five populations of Oxya chinensis had far genetic distance with Oxya japonica. In the dendrogram, a tendency of clustering following a North-South gradient could be observed, the results implied that genetic distance of five populations of Oxya chinensis correlated with geographical distance to some degree.


Assuntos
Ortópteros/genética , Técnica de Amplificação ao Acaso de DNA Polimórfico , Animais , Genética Populacional , Ortópteros/classificação
8.
Yi Chuan Xue Bao ; 30(6): 533-9, 2003 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-12939798

RESUMO

The molecular phylogenetic relationships of five species of Oxya Audient-serville including O. chinensis, O. brachyptera, O. agavisa, O. japonica and O. intricata were studied with RAPD. Genomic DNA of forty-one individuals were amplified with eight oligonucleotide (10 mer) primers which were previously selected, the specifical bands occured in all them, a total of 96 clear and reproducible bands (rang from 200-2500 bp) were generated, 95 of which were polymorphic band, the only one band (850 bp) which was amplified with S362 was common to five species of Oxya. The obtaining segments of individual primer were between 8-16, the average was 12. A molecular phylogenetic tree based on was constructed Euclidean distance among five species of rice grasshopper by between-groups linkage method, the result indicated O. chinensis was closely related to O. brachyptera, the genetic relationship of O. japonica and O. agavisa was also close, whereas O. intricata had far phylogenetic relationship with them. The results of dendrogram were consistent with the previous conclusion of morphologic classification and cytotaxonomy, and suggested RAPD was suitable for analysis of phylogenetic relationships among species of Oxya.


Assuntos
Gafanhotos/genética , Filogenia , Técnica de Amplificação ao Acaso de DNA Polimórfico/métodos , Animais , DNA/genética , DNA/isolamento & purificação , Gafanhotos/classificação , Especificidade da Espécie
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