Identification of ARF genes in Cucurbita pepo L and analysis of expression patterns, and functional analysis of CpARF22 under drought, salt stress.

BACKGROUND: Auxin transcription factor (ARF) is an important transcription factor that transmits auxin signals and is involved in plant growth and development as well as stress response. However, genome-wide identification and responses to abiotic and pathogen stresses of the ARF gene family in Cucurbita pepo L, especially pathogen stresses, have not been reported. RESULTS: Finally, 33 ARF genes (CpARF01 to CpARF33) were identified in C.pepo from the Cucurbitaceae genome database using bioinformatics methods. The putative protein contains 438 to 1071 amino acids, the isoelectric point is 4.99 to 8.54, and the molecular weight is 47759.36 to 117813.27 Da, the instability index ranged from 40.74 to 68.94, and the liposoluble index ranged from 62.56 to 76.18. The 33 genes were mainly localized in the nucleus and cytoplasm, and distributed on 16 chromosomes unevenly. Phylogenetic analysis showed that 33 CpARF proteins were divided into 6 groups. According to the amino acid sequence of CpARF proteins, 10 motifs were identified, and 1,3,6,8,10 motifs were highly conserved in most of the CpARF proteins. At the same time, it was found that genes in the same subfamily have similar gene structures. Cis-elements and protein interaction networks predicted that CpARF may be involved in abiotic factors related to the stress response. QRT-PCR analysis showed that most of the CpARF genes were upregulated under NaCl, PEG, and pathogen treatment compared to the control. Subcellular localization showed that CpARF22 was localized in the nucleus. The transgenic Arabidopsis thaliana lines with the CpARF22 gene enhanced their tolerance to salt and drought stress. CONCLUSION: In this study, we systematically analyzed the CpARF gene family and its expression patterns under drought, salt, and pathogen stress, which improved our understanding of the ARF protein of zucchini, and laid a solid foundation for functional analysis of the CpARF gene.

Identification of an allatostatin C signaling system in mollusc Aplysia.

Neuropeptides, as pervasive intercellular signaling molecules in the CNS, modulate a variety of behavioral systems in both protostomes and deuterostomes. Allatostatins are neuropeptides in arthropods that inhibit the biosynthesis of juvenile hormones. Based on amino acid sequences, they are divided into three different types in arthropods: allatostatin A, allatostatin B, allatostatin C. Allatostatin C (AstC) was first isolated from Manduca sexta, and it has an important conserved feature of a disulfide bridge formed by two cysteine residues. Moreover, AstC appears to be the ortholog of mammalian somatostatin, and it has functions in common with somatostatin, such as modulating feeding behaviors. The AstC signaling system has been widely studied in arthropods, but minimally studied in molluscs. In this study, we seek to identify the AstC signaling system in the marine mollusc Aplysia californica. We cloned the AstC precursor from the cDNA of Aplysia. We predicted a 15-amino acid peptide with a disulfide bridge, i.e., AstC, using NeuroPred. We then cloned two putative allatostatin C-like receptors and through NCBI Conserved Domain Search we found that they belonged to the G protein-coupled receptor (GPCR) family. In addition, using an inositol monophosphate 1 (IP1) accumulation assay, we showed that Aplysia AstC could activate one of the putative receptors, i.e., the AstC-R, at the lowest EC50, and AstC without the disulfide bridge (AstC') activated AstC-R with the highest EC50. Moreover, four molluscan AstCs with variations of sequences from Aplysia AstC but with the disulfide bridge activated AstC-R at intermediate EC50. In summary, our successful identification of the Aplysia AstC precursor and its receptor (AstC-R) represents the first example in molluscs, and provides an important basis for further studies of the AstC signaling system in Aplysia and other molluscs.

Synaptic mechanisms for motor variability in a feedforward network.

Behavioral variability often arises from variable activity in the behavior-generating neural network. The synaptic mechanisms underlying this variability are poorly understood. We show that synaptic noise, in conjunction with weak feedforward excitation, generates variable motor output in the Aplysia feeding system. A command-like neuron (CBI-10) triggers rhythmic motor programs more variable than programs triggered by CBI-2. CBI-10 weakly excites a pivotal pattern-generating interneuron (B34) strongly activated by CBI-2. The activation properties of B34 substantially account for the degree of program variability. CBI-10- and CBI-2-induced EPSPs in B34 vary in amplitude across trials, suggesting that there is synaptic noise. Computational studies show that synaptic noise is required for program variability. Further, at network state transition points when synaptic conductance is low, maximum program variability is promoted by moderate noise levels. Thus, synaptic strength and noise act together in a nonlinear manner to determine the degree of variability within a feedforward network.

Newly Identified Aplysia SPTR-Gene Family-Derived Peptides: Localization and Function.

When individual neurons in a circuit contain multiple neuropeptides, these peptides can target different sets of follower neurons. This endows the circuit with a certain degree of flexibility. Here we identified a novel family of peptides, the Aplysia SPTR-Gene Family-Derived peptides (apSPTR-GF-DPs). We demonstrated apSPTR-GF-DPs, particularly apSPTR-GF-DP2, are expressed in the Aplysia CNS using immunohistochemistry and MALDI-TOF MS. Furthermore, apSPTR-GF-DP2 is present in single projection neurons, e.g., in the cerebral-buccal interneuron-12 (CBI-12). Previous studies have demonstrated that CBI-12 contains two other peptides, FCAP/CP2. In addition, CBI-12 and CP2 promote shortening of the protraction phase of motor programs. Here, we demonstrate that FCAP shortens protraction. Moreover, we show that apSPTR-GF-DP2 also shortens protraction. Surprisingly, apSPTR-GF-DP2 does not increase the excitability of retraction interneuron B64. B64 terminates protraction and is modulated by FCAP/CP2 and CBI-12. Instead, we show that apSPTR-GF-DP2 and CBI-12 increase B20 excitability and B20 activity can shorten protraction. Taken together, these data indicate that different CBI-12 peptides target different sets of pattern-generating interneurons to exert similar modulatory actions. These findings provide the first definitive evidence for SPTR-GF's role in modulation of feeding, and a form of molecular degeneracy by multiple peptide cotransmitters in single identified neurons.

[Screening, identification and biocontrol effect of antagonistic actinomycetes against the pathogen of Cytospora sp. for apple tree]. / è‹¹æžœæ ‘è çƒ‚ç— èŒæ‹®æŠ—æ”¾çº¿èŒçš„ç­›é€‰ã€é‰´å®šåŠé˜²æ•ˆ.

The pathogen of Cytospora sp. was considered as the target fungus to evaluate the bio-control potential of antagonistic actinomycetes against Cytospora sp. in the present study, which was isolated from the rhizosphere soil of apple tree. Moreover, the antagonistic effect of actinomycetes against Cytospora sp. growth was determined by the method of confrontation and growth rate, and the screened antagonistic strain was identified by the method of morphology and molecular biology. Also, the inhibitory activity of strain ZZ-9 against the conidium germination and mycelia growth of Cytospora sp. was determined in vivo, and its bio-control effect on Cytospora sp. growth was determined in detached apple twigs. The results showed that fifteen strains of actinomycetes had the inhibitory effect on Cytospora sp. growth, among all the isolated strains, which accounted for 18.8% and especially the inhibitory rate of eight strains was more than 50%. In addition, among all the screened strains, the strain ZZ-9 presented the highest inhibitory rate of 96.4%, which was significantly higher than those of the other isolated strains. The strain ZZ-9 was initially identified as Streptomyces rochei according to cultural characteristics, physiological and biochemical properties and 16S rDNA analysis. The nucleotide sequences in GenBank accession number was registered as KT986228. Different dilution of ZZ-9 fermentation had significant inhibitory effect on Cytospora sp. conidium germination and mycelia growth. The inhibitory rates of the 50 times fermentation on both conidium germination and mycelia growth were more than 80%. The inhibited mycelia's color was deepened, the mycelia branches were increased, and the ends of hyphae were swelled and deformed, even the protoplasm was concentrated and released. The bio-control effect of the ZZ-9 stock solution on Cytospora sp. growth was more than 75% in detached apple twigs. Thus, our results indicated that the strain ZZ-9 could be used for controlling apple tree Valsa canker in vivo and vitro.

[Efficiency of Trichoderma longibrachiatum T6 in the control of Meloidogyne incognita and its rhizosphere colonization in cucumber].

Efficiency of different concentrations of Trichoderma longibrachiatum T6 against Meloidogyne incognita and its rhizosphere colonization in cucumber were determined in greenhouse experiments. The results of rhizosphere colonization experiments showed that the number of colonies in cucumber soil and root increased significantly ten weeks after inoculation with the second stage juveniles of M. incognita and different concentrations of T. longibrachiatum T6, and there was significant difference in different concentrations of T. longibrachiatum T6, e.g., the maximum numbers of colonies in soil and root were 7.8 x 107 and 6.3 x 105 CFU · mL⁻¹ respectively after treated with the spore suspension of 1.5 x 107 CFU · mL⁻¹. Greenhouse experiments results showed that different concentrations of T. longibrachiatum T6 had significant control effect on different life stages of M. incognita, and the control effect increased with the concentration of T. longibrachiatum T6. T. longibrachiatum T6 significantly increased plant height, root length, above-ground and root fresh mass o cucumber inoculated by M. incognita. T. longibrachiatum T6 could colonize in cucumber rhizosphere, had control effect on M. incognita, and significantly improved the growth of cucumber.

[Lethal effects of Trichoderma longibrachiatum on Heterodera avenae].

The lethal effect of the conidia suspension of Trichoderma longibrachiatum against second stage juveniles of Heterodera avenae was observed under microscopic conditions and studied in vitro to preliminarily determine the potential and mechanism of the conidia suspension of T. longibrachiatum against H. avenae. Microscopic observation results showed that the conidia suspension of T. longibrachiatum adhered to or parasitized on the surface of second stage juveniles, even some parasitized nematodes were deformed at the initial stage. Later, the second stage juveniles were wrapped and the integuments were penetrated by a large number of hyphae germinated from the conidia suspension of T. longibrachiatum. Even the body of dead second stage juveniles was completely lysed. In vitro studies showed that different concentrations (1.5 x 10(5)-1.5 x 10(7) cfu x mL(-1)) of T. longibrachiatum (conidia suspension) had strong lethal and parasitic effects on the second stage juveniles of H. avenae, and significant differences existed among the treatments with different concentrations of T. longibrachiatum. In addition, the lethal and parasitic effects increased with increasing the T. longibrachiatum concentration. The mortality and corrected mortality of the second stage juveniles treated with the concentrations of 1.5 x 10(7) cfu x mL(-1) were 91.3% and 90.4% after 72 hours, and the second stage juveniles were parasitized by 88.7% after 14 days. Therefore, the conidia suspension of T. longibrachiatum had a great lethal effect on H. avenae, and the strain could be considered as a potential bio-control agent against H. avenae.

[Parasitic and lethal effects of Trichoderma longibrachiatum on Heterodera avenae: microscopic observation and bioassay].

A laboratory experiment was conducted to study the parasitic and lethal effects of Trichoderma longibrachiatum conidia suspension on Heterodera avenae cysts. Different concentrations (1.5 x 10(5)-1.5 x 10(7) cfu x mL(-1)) of T. longibrachiatum conidia suspension had strong parasitic and lethal effects on H. avenae cysts, and the effects differed significantly among the different concentrations. When treated with the T. longibrachiatum conidia suspension at a concentration of 1.5 x 10(7) cfu x mL(-1), 96.7% of the H. avenae cysts were parasitized by the conidia at the 18th day, and the hatching rate of the cysts was inhibited by 91.2% at the 22nd day. The microscopic observation showed that at the initial parasitic stage, T. longibrachiatum conidia suspension adhered or parasitized on the cyst surface, germinated a large number of hyphae, and grew on the cyst surface, making the development of cyst embryo stopped and the contents in cysts flocculated, and even, some cysts started to deform, and small dark brown vacuoles formed on the cyst surface. At the later parasitic stage, the cysts were penetrated by dense mycelium, cysts were broken, their contents exosmosed, and the mycelium on the integument of some cysts produced conidiophores, on which, conidium were adhered or parasitized. It was considered that T. longibrachiatum could be used as a potential high-efficient bioagent to control the occurrence and damage of H. avenae.

Study on hepatitis C virus genotypes in Yantai district, Shandong province / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To understand the hepatitis C virus (HCV) genotype distribution in Yantai district of Shandong province, and to explore whether the HCV genotypes was relevant to the injure of liver through the index of liver function.</p><p><b>METHODS</b>Using specific PCR primers to amplify the HCV RNA 5' UTR/NS5B,then PCR products were sequenced by genetic analyzer. The genotypes were identified by alignment to the GenBank reference sequences and construction the phylogenetic tree of 5' UTR.</p><p><b>RESULTS</b>Among 9 unpaid blood donors we detected two kinds of genotypes of 1b and 3a, respectively, 8 cases (88.9%) and 1 case (11.1%). Among 33 cases of hepatitis C patients we detected the 1b, 2a and 6a the three kinds of genotypes, respectively, 22 (66.7%), 10 (30.3%) and 1 (3.03%) cases. Subtype 1b is the advantage of popular genotype in HCV carriers from Yantai district, and the distribution of 1b was no significant difference in the different population (chi2 = 0.796, P = 0.373); The difference of indicator of liver damage in the different genotypes of subjects were significant (P < 0.05), the mean of ALT, AST of 2a-subtype carriers was significantly higher than the 1b-subtype population.</p><p><b>CONCLUSIONS</b>The genetic diversity of HCV in Shandong Yantai district was presented. The main genotypes were 1b-subtype, and 3a and 6a-subtype was detected firstly. The genotype of HCV were relevant to the liver damage indicators, 2a-subtype hepatitis C virus infection in the liver cells may play an important role in the disease process.</p>

Detection and analysis of gene polymorphism in hepatitis B virus C region / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To explore the variations of gene C in hepatitis B viruses between hepatitis B patients and healthy carriers, and provide experimental evidences for analysis of virus gene mutations acting on the virus material science and response of the body to the virus.</p><p><b>METHODS</b>The virus DNA load in hepatitis B patients and healthy blood donors was investigated by real-time polymerase chain reaction (PCR). Gene sequence analysis was taken to detect gene polymorphism, and all the success samples were compaired with standard strain by DNAstar.</p><p><b>RESULTS</b>(1)G Compared with standard strain, C region in all samples had mutations, there were 31 mutations in at least 2 samples (3 mutations in gene PreC and 28 mutations in gene C), including 9 missense mutations, 1 chain termination mutation and 21 synonymous mutation. Mutations nt 1827 c-->a and nt 2221 c-->t existed in all the samples, and most samples had 6 synonymous mutations. Four hepatitis B patients had mutation nt1896 g-->a, and another 4 patients had 2 mutations, namely, S87G and I97F (or 197L) in HBcAg CTL recognition episome. (2) The success ratio of amplification and sequencing of HBV DNA was closely associated with its copy numbers. In the present study, copy numbers of HBV DNA which were successfully amplified and sequenced were almost more than 40 193/ml.</p><p><b>CONCLUSIONS</b>HBV genome were easily affected by nucleotide mutations, 2 residues had mutations in gene of C region, which is firstly reported, suggesting these mutations may be geographical restricted. Mutations in gene of C region may either change the structure and function of HBeAg and HBcAg, which may further induce the escape of immune clearance for HBV or influence the detection of HBsAg or HBeAg, which may creat new problems for the prevention, diagnosis and treatment of hepatitis B.</p>

Change of T lymphocyte and its activated subsets in SARS patients / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study abnormal changes of T lymphocyte and its activated subsets in severe acute respiratory syndrome (SARS) patients.</p><p><b>METHODS</b>Flow cytometer with multi-color flouroscence and hematology analyzer were used to detect the expression of T lymphocyte and its activated a subsets in 240 SARS patients including 50 cases of critical type and 190 cases of common type.</p><p><b>RESULTS</b>Statistical analysis by means of SAS software showed that there was significant decrease in absolute counts (AC) of T lymphocyte and its subsets in SARS patients when compared with normal people, while percentages (PC) of CD3+CD25+ and CD3+ HLA-DR+ subsets were increased markedly. Compared with common type, there was significant decrease in absolute counts of critical type of T lymphocyte, CD4+, CD25+CD3+, CD28+CD4+, and CD95+CD4+subsets. The ACs of T lymphocytes including CD4 and CD8 subsets in different phases were as below: III > II > I. The ACs of subsets involved in activation such as CD3+ HLA-DR+/lym, CD3+CD25+/lym, CD28+CD4+/CD4, CD28+CD8+/CD8, and CD38+CD4+/CD4 all were highest in group III. In addition, the AC and PC of CD95+CD4+/CD4 and CD95+CD8/CD8 subset in group III were highest while group I was lowest.</p><p><b>CONCLUSIONS</b>With depressing cellular immunity, the activation of T lymphocytes were suppressed obviously in SARS patients, especially for critical patients.</p>