Effect of Gonadotropic Hormones on Stress Resistance of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> Females Infected with Different <Emphasis Type="Italic">Wolbachia pipientis</Emphasis> Genotypes

The effect of gonadotropic hormones (juvenile (JH) and 20-hydroxyecdysone (20E)) on heat stress resistance was for the first time studied in wild type D. melanogaster line females infected with different genotypes of the Wolbachia pipientis alpha-proteobacterium. It was found that an experimental increase in JH level induces a decrease in the heat stress resistance, while an increase in 20E level induces its increase in sixday females both uninfected with the Wolbachia and infected with different bacterium strains (wMelCS, wMelPop, and wMel). However, the intensity of response differs: a decrease in the survival with an increase in JH level and its increase with an increase in 20E level are more pronounced in females infected with pathogenic wMelPop strain and less pronounced in females infected with the wMelCS genotype than in uninfected females and females infected with the wMel genotype. Data obtained suggest that the wMelCS genotype induces a decrease and wMelPop induces an increase in the level of stress hormone (dopamine), since previously we demonstrated that an increase in the JH level in mature females increases the dopamine level, an increase in the 20E level decreases it, and an increase/decrease in the dopamine level, in turn, leads to a decrease/increase of the Drosophila female resistance to heat stress.     

The insulin-like receptor gene expression in the tissues synthesizing gonadotropic hormones at sexual maturation of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> females

The insulin/insulin-like growth factor signaling pathway is involved in the regulation of the synthesis of insect gonadotropic hormones, juvenile (JH) and 20-hydroxyecdysone (20E). We carried out the immunohistochemical analysis of the insulin receptor (InR) expression in the corpus allatum (the JH-producing gland) and in the ovarian follicular cells (a site for the synthesis of 20E precursor, ecdysone) in the process of sexual maturation of D. melanogaster females and examined the influence of exogenous JH on the InR expression in these tissues. For the first time, it was demonstrated that InR was expressed in follicular cells and that its expression in corpus allatum and follicular cells of Drosophila females was stage-specific, i.e., the expression intensity in young females greatly exceeded that in mature individuals. We also found a negative feedback loop in the regulation of JH levels by the insulin signaling pathway in Drosophila adults: the experimental increase in the JH titers in young females dramatically reduced the InR expression intensity in corpus allatum and follicular cells.     

A novel <Emphasis Type="Italic">TaSST</Emphasis> gene from wheat contributes to enhanced resistance to salt stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis>

In this research, through the analyzing of the Triticum aestivum salt-tolerant mutant gene expression profile, under salt stress. A brand new gene with unknown functions induced by salt was cloned. The cloned gene was named Triticum aestivum salt stress protein (TaSST). GenBank accession number of TaSST is ACH97119. Quantitative polymerase chain reaction (qPCR) results exhibited that the expression TaSST was induced by salt, abscisic acid (ABA), and polyethylene glycol (PEG). TaSST could improve salt tolerance of Arabidopsis-overexpressed TaSST. After salt stress, physiological indexes of transgenic Arabidopsis were better compared with WT (wild-type) plants. TaSST was mainly located in the cytomembrane. qPCR analyzed the expression levels of nine tolerance-related genes of Arabidopsis in TaSST-overexpressing Arabidopsis. Results showed that the expression levels of SOS3, SOS2, KIN2, and COR15a significantly increased, whereas the expression of the five other genes showed no obvious change. OsI_01272, the homologous gene of TaSST in rice, was interfered using RNA interference (RNAi) technique. RNAi plants became more sensitive to salt than control plants. Thus, we speculate that TaSST can improve plant salt tolerance.     

Silencing of <Emphasis Type="Italic">Mythimna separata chitinase</Emphasis> genes via oral delivery of <Emphasis Type="Italic">in planta</Emphasis>-expressed RNAi effectors from a recombinant plant virus

Objectives

To evaluate transient expression of RNA interference (RNAi) effectors in Nicotiana benthamiana plants by using recombinant virus vectors and also oral delivery of the effectors for silencing of Mythimna separata endogenous gene expression.

Results

Mythimna separata is a serious pest of corn production in China. To evaluate RNAi approaches to target specific RNAs in M. separate, we cloned fragments of the M. separata chitinase sequences into a virus vector in order to produce RNAi effectors during virus infection and replication in plants. When the infected plants were fed to M. separata, expression levels of target MseChi1 and MseChi2 genes were down-regulated by 76 and 45 %, respectively, and sequence-specific siRNAs were detected in recipient insects. RNAi-based silencing of chitinase genes also led to body weight decreases by 43 %.

Conclusion

Our research demonstrates target mRNA knockdown and suggests a promising application for controlling of M. separata by in planta expression of RNAi effectors using a recombinant plant virus.

     

Contributions of <Emphasis Type="Italic">TaSUTs</Emphasis> to grain weight in wheat under drought

Key message

The homologous genes to OsSUT1-5 in wheat were identified and detailed analysed. TaSUT1 was the predominant sucrose transporter group and it illustrated the genotypic variations towards drought during grain filling.

Abstract

Sucrose transporters (SUT) play crucial roles in wheat stem water soluble carbohydrate (WSC) remobilization to grain. To determine the major functional SUT gene groups in shoot parts of wheat during grain development, drought tolerant varieties, Westonia and Kauz, were investigated in field drought experiments. Fourteen homologous genes to OsSUT1-5 were identified on five homeologous groups, namely TaSUT1_4A, TaSUT1_4B, TaSUT1_4D; TaSUT2_5A, TaSUT2_5B, TaSUT2_5D; TaSUT3_1A, TaSUT3_1D; TaSUT4_6A, TaSUT4_6B, TaSUT4_6D; TaSUT5_2A, TaSUT5_2B, and TaSUT5_2D, and their gene structures were analysed. Wheat plants above the ground were harvested from pre-anthesis to grain maturity and the stem, leaf sheath, rachis, lemma and developing grain were used for analysing TaSUT gene expression. Grain weight, thousand grain weight, kernel number per spike, biomass and stem WSC were characterized. The study showed that among the five TaSUT groups, TaSUT1 was the predominant sucrose transporting group in all organs sampled, and the expression was particularly high in the developing grain. In contrast to TaSUT1, the gene expression levels of TaSUT2, TaSUT3 and TaSUT4 were lower, except for TaSUT3 which showed preferential expression in the lemma before anthesis. The TaSUT5 gene group was very weakly expressed in all tissues. The upregulated gene expression of TaSUT1 Westonia type in stem and grain reveal a crucial role in stem WSC remobilization to grain under drought. The high TaSUT1 gene expression and the significant correlations with thousand grain weight (TGW) and kernel number per spike demonstrated the contribution in Kauz’s high grain yield in an irrigated environment and high TGW in Westonia under drought stress. Further molecular level identification is required for gene marker development.

     

<Emphasis Type="Italic">ZmFKBP20-1</Emphasis> improves the drought and salt tolerance of transformed Arabidopsis

FK506-binding proteins (FKBPs), which belong to the peptidyl-prolyl cis/trans isomerase superfamily, are involved in plant response to abiotic stresses. A number of FKBP family genes have been isolated in plants, but little has been reported of FKBP genes in maize. In this study, a drought-induced FKBP gene, ZmFKBP20-1, was isolated from maize and was characterized for its role in stress responses using gene expression, protein subcellular localization, transformation in Arabidopsis, expression patterns of the stress-responsive genes, and physiological parameter analysis. During drought and salt stresses, ZmFKBP20-1 transgenic Arabidopsis plants exhibited enhanced tolerance, which was concomitant with the altered expression of stress/ABA-responsive genes, such as COR15a, COR47, ERD10, RD22, KIN1, ABI1, and ABI2. The resistance characteristics of ZmFKBP20-1 overexpression were associated with a significant increase in survival rate. These results suggested that ZmFKBP20-1 plays a positive role in drought and salt stress responses in Arabidopsis and provided new insights into the mechanisms of FKBP in response to abiotic stresses in plants.     

Reliability of the search for 19 common mutations in the <Emphasis Type="Italic">CFTR</Emphasis> gene in Russian cystic fibrosis patients and the calculated frequency of the disease in Russian Federation

A study of Russian cystic fibrosis (CF) patient DNA was conducted to assess the incidence frequency of 19 mutations, namely CFTRdele2,3(21kb), F508del, I507del, 1677delTA, 2143delT, 2184insA, 394delTT, 3821delT, L138ins, 604insA, 3944delGT, G542X, W1282X, N1303K, R334W, and 3849 + 10kbC > T, S1196X, 621 + 1g > t, and E92K of the CFTR gene. We also sought to determine the estimated CF frequency in Russian Federation. In addition, we determined the total information content of the approach for 19 common mutations registration in the CFTR gene, 84.6%, and the allelic frequencies of the examined mutations: three mutations were observed with a frequency exceeding 5% (F508del, 53.98%, E92K, 6.47%, CFTRdele2,3(21kb), 5.35%); other mutations were observed with frequencies ranging from 0.13 to 3.0%. The CF population carrier frequency was 1 in 38 subjects, while the predicted CF frequency was 1 in 5776 newborns.     

Characterization of <Emphasis Type="Italic">GA20ox</Emphasis> genes in tall and dwarf types coconut (<Emphasis Type="Italic">Cocos nucifera</Emphasis> L.)

Coconuts (Cocos nucifera L.) are divided by the height into tall and dwarf types. In many plants the short phenotype was emerged by mutation of the GA20ox gene encoding the enzyme involved in gibberellin (GA) biosynthesis. Two CnGA20ox genes, CnGA20ox1 and CnGA20ox2, were cloned from tall and dwarf types coconut. The sequences, gene structures and expressions were compared. The structure of each gene comprised three exons and two introns. The CnGA20ox1 and CnGA20ox2 genes consisted of the coding region of 1110 and 1131 bp, encoding proteins of 369 and 376 amino acids, respectively. Their amino acid sequences are highly homologous to GA20ox1 and GA20ox2 genes of Elaeis guineensis, but only 57% homologous to each other. However, the characteristic amino acids two histidines and one aspartic acid which are the two iron (Fe²⁺) binding residues, and arginine and serine which are the substrate binding residues of the dioxygenase enzyme in the 20G-FeII_Oxy domain involved in GA biosynthesis, were found in the active site of both enzymes. The evolutionary relationship of their proteins revealed three clusters in vascular plants, with two subgroups in dicots and three subgroups in monocots. This result confirmed that CnGA20ox was present as multi-copy genes, and at least two groups CnGA20ox1 and CnGA20ox2 were found in coconut. The nucleotide sequences of CnGA20ox1 gene in both coconut types were identical but its expression was about three folds higher in the leaves of tall coconut than in those of dwarf type which was in good agreement with their height. In contrast, the nucleotide sequences of CnGA20ox2 gene in the two coconut types were different, but the expression of CnGA20ox2 gene could not be detected in either coconut type. The promoter region of CnGA20ox1 gene was cloned, and the core promoter sequences and various cis-elements were found. The CnGA20ox1 gene should be responsible for the height in coconut, which is different from other plants because no mutation was present in CnGA20ox1 gene of dwarf type coconut.     

Electroporation-mediated RNA interference reveals a role of the multicopper oxidase 2 gene in dragonfly cuticular pigmentation

Dragonflies are colorful insects, and recent RNA sequencing studies have identified a number of candidate genes potentially involved in their color pattern formation and color vision. However, functional aspects of such genes have not been assessed due to the lack of molecular genetic tools applicable to dragonflies. We established an electroporation-mediated RNA interference (RNAi) procedure using the tiny dragonfly Nannophya pygmaea Rambur, 1842 (Odonata: Libellulidae) that targets the multicopper oxidase 2 gene (MCO2; also known as laccase2 gene) responsible for cuticular pigmentation in many insects. RNA sequencing of N. pygmaea and genomic survey of the dragonfly Ladona fulva identified four multicopper oxidase family genes: MCO1, MCO2, MCO3 and multicopper oxidase-related protein gene (MCORP). In N. pygmaea, MCO2 was specifically expressed around the cuticular pigmentation period, whereas MCO1 was constantly expressed. MCORP was expressed at adult stages, and MCO3 was scarcely expressed. When we applied in vivo electroporation, final instar larvae injected with MCO2 small interfering RNA became adults with patchy unpigmented regions. RNAi without in vivo electroporation did not affect cuticular pigmentation, suggesting that dragonflies do not show a systemic RNAi response. These results indicate that MCO2 is required for cuticular pigmentation across diverse insects, and highlight the usefulness of the electroporation-mediated RNAi method in dragonflies.     

Distinct Histone Modifications Modulate <Emphasis Type="Italic">DEFB1</Emphasis> Expression in Human Vaginal Keratinocytes in Response to <Emphasis Type="Italic">Lactobacillus</Emphasis> spp.

Vaginal commensal lactobacilli are considered to contribute significantly to the control of vaginal microbiota by competing with other microflora for adherence to the vaginal epithelium and by producing antimicrobial compounds. However, the molecular mechanisms of symbiotic prokaryotic-eukaryotic communication in the vaginal ecosystem remain poorly understood. Here, we showed that both DNA methylation and histone modifications were associated with expression of the DEFB1 gene, which encodes the antimicrobial peptide human β-defensin-1, in vaginal keratinocyte VK2/E6E7 cells. We investigated whether exposure to Lactobacillus gasseri and Lactobacillus reuteri would trigger the epigenetic modulation of DEFB1 expression in VK2/E6E7 cells in a bacterial species-dependent manner. While enhanced expression of DEFB1 was observed when VK2/E6E7 cells were exposed to L. gasseri, treatment with L. reuteri resulted in reduced DEFB1 expression. Moreover, L. gasseri stimulated the recruitment of active histone marks and, in contrast, L. reuteri led to the decrease of active histone marks at the DEFB1 promoter. It was remarkable that distinct histone modifications within the same promoter region of DEFB1 were mediated by L. gasseri and L. reuteri. Therefore, our study suggested that one of the underlying mechanisms of DEFB1 expression in the vaginal ecosystem might be associated with the epigenetic crosstalk between individual Lactobacillus spp. and vaginal keratinocytes.     

Construction of heterologous gene expression cassettes for the development of recombinant <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

Gene-expression cassettes for the construction of recombinant Clostridium beijerinckii were developed as potential tools for metabolic engineering of C. beijerinckii. Gene expression cassettes containing ColE1 origin and pAMB origin along with the erythromycin resistance gene were constructed, in which promoters from Escherichia coli, Lactococcus lactis, Ralstonia eutropha, C. acetobutylicum, and C. beijerinckii are examined as potential promoters in C. beijerinckii. Zymogram analysis of the cell extracts and comparison of lipase activities of the recombinant C. beijerinckii strains expressing Pseudomonas fluorescens tliA gene suggested that the tliA gene was functionally expressed by all the examined promoters with different expression level. Also, recombinant C. beijerinckii expressing C. beijerinckii secondary alcohol dehydrogenase by the constructed expression cassettes successfully produced 2-propanol from glucose. The best promoter for TliA expression was the R. eutropha phaP promoter while that for 2-propanol production was the putative C. beijerinckii pta promoter. Gene expression cassettes developed in this study may be useful tools for the construction of recombinant C. beijerinckii strains as host strains for the valuable chemicals and fuels from renewable resources.