Gene Cloning and Tissue-Specific Expression of G Protein β Subunit in Microplitis mediator (Hymenoptera: Braconidae)

A gene encoding a novel G protein β subunit of β1 subclass, GβMmed was isolated from Microplitis mediator (Hymenoptera: Braconidae). The full-length sequence of GβMmed is 1 119 bp, the cDNA contains a 1 023 bp open reading frame that encodes a protein with 340 amino acids, and the predicted molecular weight of GβMmed is 37.23 kDa and isoelectric point is 5.86. By the quantitative real-time RT-PCR method, the tissue-specific expression and quantitative changes in the developmental expression profile of GβMmed were detected. It was found that GβMmed was abundantly expressed in M. mediator antennae, head (without antennae), thorax, abdomen, legs and the wings, and especially at high levels in abdomen. In antennae, expression varied through 1st day before emergence to 5-d-old adults, and had equal expression levels detected in females and males in total. In head, GβMmed expresses while initially high in females, and have another peaked in stage 4 and 1st day, in males showed a peak of GβMmed expression prior to emergence and relatively low levels after emergence. In female abdomen GβMmed expression levels have two peaks in stage 1 and the 5th d, but just have one peak in male abdomen in stage 1. In all other tissues expression was low and stable.     

Effects of lead stress on Vg expression in the beet armyworm over five successive generations

Heavy metals have been found to be endocrine disruptors in invertebrates.Lead is one of the most widespread elements of contamination,but there has been no research about the effects of lead stress on vitellogenin(Vg) gene expression in insects exposed to lead over multiple generations.In this paper,the effects of different concentrations of lead(0,0.3,4.8 and 76.8 mg kg~(–1)) on the expression of Vg in the beet armyworm over five successive generations were studied.The results showed that lead stress had significant effects on Vg expression in a dose-dependent manner.For females at the larval and adult stages,as lead concentration increased,Vg expression was significantly inhibited; for males at these two developmental stages,Vg expression was induced and increased as lead concentration increased.In addition,with the increase over stressed generations,inhibited effects for females and induced effects for males at the larval and adult stages became increasingly more obvious.However,at the pupal stage,Vg expression in the two genders was different from that at the larval and adult stages.The results indicate that lead stress can upregulate Vg expression in males which should be a useful indicator for environmental risk assessment.     

Role of TRP channels and HSPs in thermal stress response in the aphid parasitoid Aphelinus asychis(Hymenoptera:Aphelinidae)

Aphelinus asychis is an important aphid endoparasitoid. Under field and greenhouse conditions, high temperature is one of the factors limiting the application of A. asychis for biological pest control. To explore the potential role of transient receptor potential(TRP) channels and heat shock proteins(HSPs) in this process, we identified 11 genes encoding TRP channels and nine genes encoding HSPs. Three proteins(AasyTRPA5, AasyPyrexia, AasyPainless) that belong to transient receptor potential ankyrin(TRPA) subfamily and nine HSPs are involved in the response to high temperature. We also investigated the survival of A. asychis and the response of the identified TRP channels and HSPs to high temperature. The results showed that the maximum temperature that allowed A. asychis survival was approximately 41℃; females had higher survival rates than that of the males at 40 and 41℃. Short-term heat-shock resulted in increased expression of Aasyshsp in males, and Aasyhsp40, Aasyhsp68, Aasyhsp70-4, Aasyhsp70-5 and Aasyhsp90 were upregulated and then downregulated, whereas Aasyhsp70-3 was upregulated at 41℃. Moreover, Aasyhsp40 and Aasyhsp90 showed higher expression levels in females, while Aasyshsp and Aasyhsp70-3 presented opposite expression patterns. At temperature above 35℃, expression of AasyPyrexia in females was significant higher than that in males, whereas AasyPainless and AasyTRPA5 presented higher expression in males at 40 and 41℃, respectively. Altogether, these results indicate that protection against thermal stress in A. asychis is coordinated by TRP channels and HSPs. These findings provide a basis for understanding the potential mechanism of A. asychis in response to high temperatures.     

Functional Analysis of the Phosphoenolpyruvate Carboxylase on the Lipid Accumulation of Peanut（Arachis hypogaea L.） Seeds

Phosphoenolpyruvate carboxylase(PEPC;EC 4.1.1.31) catalyses phosphoenolpyruvate(PEP) to yield oxaloacetate,which is involved in protein biosynthesis.Pyruvate kinase(PK;EC 2.7.1.40) catalyzes PEP to yield pyruvate,which is involved in fatty acid synthesis.In this study,five PEPC genes(AhPEPC1,AhPEPC2,AhPEPC3,AhPEPC4,and AhPEPC5) from peanut have been cloned.Using a quantitative real-time RT-PCR approach,the expression pattern of each gene was monitored during the seed development of four peanut varieties(E11,Hebeigaoyou,Naihan 1,and Huayu 26).It was found that these five genes shared similar expression behaviors over the developmental stages of E11 with high expression levels at 30 and 40 d after pegging(DAP);whereas these five genes showed irregular expression patterns during the seed development of Hebeigaoyou.In Naihan 1 and Huayu 26,the expression levels of the five genes remained relatively high in the first stage.The PEPC activity was monitored during the seed development of four peanut varieties and seed oil content was also characterized during whole period of seed development.The PEPC activity followed the oil accumulation pattern during the early stages of development but they showed a significantly negative correlation thereafter.These results suggested that PEPC may play an important role in lipid accumulation during the seed development of four peanut varieties tested.     

Expression of Inflammatory Factors in Bovine Reproductive Tract During Follicular and Luteal Phases

In order to improve reproduvtive efficieny and understand reproduvtive defense mechanism, the oviduct, uterine horn and uterine body of bovine were used to detect the changes of inflammatory factors and the relationship between estrogen and progesterone receptor protein during estrous cycle by real-time PCR and Elisa method. The results showed that interleukin-4(IL-4), interleukin-6(IL-6), interleukin-10(IL-10), interleukin-1α(IL-1α) and interleukin-1β(IL-1β) were expressed in cow oviduct, uterine horn and uterine body. In the follicular phase and the luteal phase, m RNA expression of five inflammatory factors in the uterine horn and uterine body was higher than that in the oviduct. In the follicular phase, IL-10 was highly expressed in the uterine horn and uterine body, IL-4 was highly expressed in the uterine horn, uterine body and oviduct. Additionally, in the luteal phase, IL-6 and IL-1β were highly expressed in the uterine horn, uterine body and oviduct, and the highest expression of IL-1β was observed in the uterine horn. The levels of Estrogen Receptor(ERα) protein in the oviduct, uterine horn and uterine body significantly increased in the follicular phase. The levels of Progesterone Receptor(PR) protein in the same portions of the reproductive tract in the luteal phase were significantly higher than those in the follicular phase. IL-4 and IL-10 in the cow reproductive tract might play a major role in the follicular phase, while IL-6 and IL-1β might play a major role in the luteal phase. The expression of five inflammatory factors was not directly regulated by ERα and PR.     

Hoxc13 Expression Pattern in Cashmere Goat Skin During Hair Follicle Development

Hoxc13 has an important role in controlling hair formation. In this study, we examine the Hoxc13 RNA expression pattern of skin during embryo development. The result indicated that changes of the Hoxe13 gene expression and thickness of skin have a similar trend during hair follicle morphogenesis. In interpreting these results, we investigated whether the regulation motifs is in Hoxc13 intron, which is a 5.4 kb fragment. To blast with other mammals, we found a very conservative region in all mammal animals and two regions in livestock, such as cow, sheep, horse, dog, and so on, which are not in other Hox genes. We have examined putative pre-miRNA in this region, providing an entry point for elucidating currently unknown mechanisms that are required for regulating quantitative levels of Hoxc13 gene expression.     

Selection of reference genes for RT-qPCR analysis of Phenacoccus solenopsis (Hemiptera: Pseudococcidae) sex-dimorphic development

Mealybugs, such as Phenacoccus solenopsis, are highly sexually dimorphic. Winged adult males present such remarkable morphological differences from females that, to the untrained eye, conspecific adults of both sexes of P. solenopsis may be considered as two different insect species. A method to investigate sex-dimorphic mechanisms is by evaluating gene expression using RT-qPCR. However, the accuracy and consistency of this technique depend on the reference gene(s) selected. In this study, we analyzed the expression of 10 candidate reference genes in male and female P. solenopsis at different development stages, using common algorithms including the ?Ct method, NormFinder, geNorm, BestKeeper, and a web-based analysis tool, RefFinder. The results showed that EF1-β, RP-L32 and RP-18 S were selected as the most stable genes by both the ?Ct method and NormFinder; TUB-α was the most stable gene identified by BestKeeper; and RP-L40 and RP-L32 were the most stable genes ranked by geNorm. RefFinder, a comprehensive analysis software, ranked the ten genes and determined EF1-β and RP-L32 as the most suitable reference genes for the various developmental stages in male and female P. solenopsis. Furthermore, the two most suitable reference genes were validated by examining expression of the juvenile hormone acid O-methytransferase(JHAMT) gene. Results of the validation portion of the study showed that JHAMT expression was sex-biased towards males and exhibited a dynamic and classic expression pattern among the P. solenopsis developmental stages. The results can help further our knowledge on the molecular mechanisms underlying sexual dimorphic development in P. solenopsis.     

Comparative Studies of Substrate and Inhibitor Specificity of Glutathione S-Transferases in Six Tissues of Oxya chinensis （Thunberg） （Orthoptera： Acrididae）

Specific activity, substrate specificity, and kinetic parameters （Km and Vmax） of glutathione S-transferases （GSTs） towards three substrates, 1-chloro-2,4-dinitrobenzene （CDNB）, 1,2-dichloro-4-nitrobenzene （DCNB）, and p-nitrobenzene chloride （pNBC） were investigated in six tissues （foregut, midgut, hindgut, fat body, hemolymph, and muscle） of Oxya chinensis. In addition, the inhibition in vitro （ethacrynic acid, and Cibacron Blue 3GA） of Oxya chinensis in the six tissues was also investigated. Glutathione S-transferase activity was detected in all the six tissues examined. The rank order of GST activities towards CDNB was fat body 〉 midgut 〉 hindgut 〉 muscle 〉 foregut 〉 hemolymph both in females and males. Glutathione S-transferase activities in the fat body in females and males were 1.3- to 10.4-fold and 1.1- to 10.0- fold higher than those in the other tissues. The rank order of GST activities towards the other substrates changed slightly. From these results, it was inferred that GSTs in the fat body and midgut played important roles in detoxifying xenobiotics including insecticides and plant allelochemicals in O. chinensis. In the three substrates examined, CDNB seemed to be the best substrate, followed by pNBC and DCNB. The kinetic parameters of GSTs were different among the six tissues. This suggested that GSTs in different tissues have various affinities and catalytic efficiency to substrates. In vitro inhibition study showed that the median inhibition concentration （IC50） values of the two inhibitors to GSTs from the six tissues were different. The results suggested that the two inhibitors have different inhibition potency to GSTs from the different tissues. The observed changes in kinetic parameters and inhibition in vitro among the six tissues of the insect might suggest that the number and structure of isoenzymes and their rate of expression varied for the different tissues.     

Expansin Genes Expressed Differentially in Peduncle Elongation of Near- Isogenic Wheat Rht Lines

The introduction of reduced height （Rht） genes Rht-Blb and Rht-Dlb led to impressive increases in wheat （Triticum aestivum L.） yields during the Green Revolution. In the present study, the dynamic elongation of peduncle in a set ofnear-isogenic lines （NILs） carrying different Rht alleles （Rht-Blb, Rht-Dlb, Rht-Blc, Rht-Dlb＋Rht-Blb, and Rht-Dlb＋Rht-Blc） were investigated. The reduction of the final length of peduncle in NILs was dependent mainly on the elongation rate, which was reduced by Rht genes, during rapid elongation phase. Resin sections showed that Rht genes strongly reduced the cell extension in peduncle. The expression of expansin genes, which mediate cell wall loosening and leading to cell expansion, were analysed by using real- time quantitative PCR （qPCR）. Among the 23 possible wheat expansin genes, 17 were expressed in the peduncle. The spatial distribution of expression was further analysed for five expansins that showed high expression levels in the peduncles of Rht lines. Compared to wild type plants, the incorporation of Rht-Dlb allele decreased about 37 and 80% of the expression levels of ExpA 7 and ExpA3 in elongation zone, respectively. The presence Rht-Blc dwarfing genes, however, produced 53% reduction in the expression level of ExpA7, and seriously decreased about 70% of ExpB9 expression. Although the expression levels of five genes exhibited variability among the lines, an expansin gene, ExpB2, showed its expression level highly associated with the cell elongation rate in peduncle of different Rht lines.     

Construction and Characterization of a cDNA Library from the Pulp of Coconut （Cocos nucifera L.）

To investigate the gene expression profile of endosperm development, a cDNA library was constructed and characterized from the pulp of coconut at different developmental stages. The constructed cDNA library incorporated approximately 1 × 10^7 clones in total, and the size of the insertion fragments ranged from 800 to 2 000 bp. Sequencing results of 100 randomly picked clones showed that the recombination rate was 96%. In subsequent sequence analysis, 41 clones （41%） were homologous to known function proteins, and 23 clones showed high amino acid identity （more than 80%） with the corresponding genes of different plants. Semi-quantitative RT-PCR indicated that oleosin and globulin genes are pulpspecific expression, and have differential expression level in different developmental stage. Clone 29, recognized as homologous to KIAA1239 protein （Homo sapiens）, was observed to occur nine times, indicating that this gene may be over-expressed during the endosperm development stage. However, the homologous protein was found only in mammals, and the detailed function is still unknown. Elucidation of the functional characterization of these genes will be carried out immediately.     

Cloning,expression, and polymorphism of the ECI1 gene in various pig breeds

The enzyme Δ~3,Δ~2-dienoyl-CoA isomerase(ECI1) plays a crucial role in the mitochondrial β-oxidation of fatty acids with a double-bond in odd and even positions. The ECI1 gene might be a qualified candidate for studies pertaining to lipid deposition and meat quality in swine. In the present study, ECI1 cDNA of the Tibetan pig was obtained by in silico cloning and verified by PCR analysis. Single-nucleotide polymorphisms(SNPs) of ECI1 were screened by PCR-sequencing and genotypes of those SNPs were tested by PCR-restriction fragment length polymorphism(PCR-RFLP) in Diannan small-ear pigs(DSP, n=40), Tibetan pigs(TP, n=60) and Yorkshire pigs(YP, n=30). The expression levels of ECI1 were analyzed by real-time quantitative PCR and Western blotting in tissues of the liver, backfat, and longissimus dorsi(LD) muscle of DSP(n=8), TP(n=8) and YP(n=8). Single factor linear correlation analysis was applied separately for each breed to evaluate correlations between ECI1 gene expression in the LD muscle and intramuscular fat(IMF) content. We obtained an ECI1 gene length of 1 401 bp from the cDNA that contained a full coding region of 909 bp. Three novel SNPs(g.42425337GA; g.42424666AG; and g.42422755AG) were detected, and only g.42424666AG exhibited three genotypes among the three breeds. The ECI1 expression levels in the LD muscle of DSP and TP were significantly higher than that of YP(P0.05). Moreover, TP had the highest ECI1 expression in backfat(P0.01), and a positive correlation was observed between gene expression and IMF content. The results suggest that differences in ECI1 gene expression might be related to lipid deposition and meat quality in pig.     

Effects of Initial Infestation Levels of Callosobruchus maculatus (F.)(Coleoptera: Chrysomelidae) on Cowpea and Use of Nicotiana tabacum L.Aqueous Extract as Grain Protectant

This study determined the effects of initial infestation of cowpea seeds(Ife brown variety) with different insect densities(0, 2, 4 and 6 pairs per 50 g seeds) of Callosobruchus maculatus(F.) and evaluated the effects of aqueous leaf extract of Nicotiana tabacum L. on C. maculatus in the laboratory. It was observed that adult beetle population increased significantly(p0.05) with increase in insect density. The increase in population of beetles and corresponding weight loss of the seeds in different levels of infestation showed that the cowpea variety was susceptible to beetle infestation, emergence and survival of progeny. Significantly more adults emerged on higher infestation compared to lower and no infestation. In Nigeria, Nicotiana tabacum L. is a locally available plant, with known insecticidal properties. The plant leaf extract was easily extracted with water and confirmed its effectiveness as a protective agent for stored cowpea seeds. Experiment was conducted to assess the effects of aqueous extracts of N. tabacum at 0, 0.1, 0.2 and 0.3 m L · 50 g~(-1) cowpea seeds on C. maculatus. Data was recorded and showed varying levels of effectiveness against C. maculatus. Result showed that seed appearance was dependent on levels of insect population, while N. tabacum aqueous extract exerted effects on survival of C. maculatus. Aqueous leaf extract of N. tabacum probably contained some insecticidal properties which might have significantly conferred beetle mortality and reduced beetle emergence leading to a decrease in seed weight loss.     

The Pig Lhx8 Gene： cDNA Cloning, Bioinformatic Analysis and Expression Level in Tissues and Preimplantation Embryos

Evidence has shown in mouse that Lhx8 is a critical factor for maintenance and differentiation of the oocyte during early oogenesis. In the current paper, attempts were made to clone and characterize a gene encoding Lhx8 from pig. Rapid amplification of cDNA ends （RACE） gave rise to a full-length of Lhx8 which contained 1 681 bp nucleotides, with a complete open reading frame of 885 bp, encoding a 295 amino acid polypeptide. Homology search and sequence multialignment demonstrated that the deduced pig Lhx8 protein sequence shared a high identity with Lhx8 from other mammals, including several highly conservative motifs and amino acids. The phylogenetic tree of the LIM superfamily proteins has been constructed to reveal the evolutionary relationship of various species. RT-PCR analysis showed that the Lhx8 gene was expressed in gonad and immunity tissues. In preimplantation embryos, Lhx8 mRNA expression profiling using realtime PCR revealed that its mRNA levels were highest in 4-cell stage embryos and gradually decreased until the blastocyst stage.     

Cloning and Expressing of a Gene Encoding Cytosolic Copper/Zinc Superoxide Dismutase in the Upland Cotton

In this study, a gene encoding a superoxide dismutase （SOD） was cloned from senescent leaves of cotton （Gossypium hirsutum）, and its expressing profile was analyzed. The gene was cloned by rapid amplification of cDNA ends （RACE） method. Northern blotting was used to show the profile of the gene expression, and the enzyme activity was mensurated by NBT deoxidization method in different growth periods. The full length of a gene of cytosolic copper/zinc superoxide dismutase （Cu/Zn-SOD） was isolated from cotton （GenBank Accession Number： DQ445093）. The sequence of cDNA contained 682 bp, the opening reading frame 456 bp, and encoded polypeptide 152 amino acids with the predicted molecular mass of 15.03 kD and theoretical pI of 6.09. The amino acid sequence was similar with the other plants from 82 to 87%. Southern blotting showed that the gene had different number of copies in different cotton species. Northern blotting suggested that the gene had different expression in different tissues and development stages. The enzyme activity was the highest in peak flowering stage. The cotton cytosolic （Cu/Zn-SOD） had lower copies in the upland cotton. The copper/zinc superoxide dismutase mRNA expressing level showed regular changing in the whole development stages; it was lower in the former stages, higher in latter stages and the highest at the peak flowering stage. The curve of the copper/zinc superoxide dismutase mRNA expressing level was consistent with that of the Cu/Zn-SOD enzyme activity. The copper/zinc superoxide dismutase mRNA expressing levels of different organs showed that the gene was higher in the root, leaf, and lower in the flower.     

The Susceptibilities of Oxya chinensis （Orthoptera： Acridoidea） to Malathion and Comparison of the Esterase Properties from Three Collected Populations in Tianjin Area,China

The susceptibilities of Oxya chinensis to malathion were studied in three populations collected from outskirt of Tianjin, China, using bioassays and biochemical analysis. Populations were chosen to represent different exposure to insecticides： BDG （Beidagang; low exposure）, BD （Baodi; high exposure previously but low exposure now）, and JN （Jinnan; high exposure）. The results showed that the LD50 values of BD and JN populations were 3.95- and 12.02-fold and 3.64- and 10.07- fold higher than that of BDG population in females and males, respectively. The LD50 values in females were higher than those in males. The results of biochemical analysis indicated that the esterase （EST） activities in JN population were higher than those in BD and BDG populations. They showed that when α-NA, α-NB, and α-NA were used as substrates, females＇ EST activities of JN population were 1.11-, 1.30-, and 1.14-fold and 1.39-, 1.59-, and 1.54-fold higher than those of BD and BDG populations, respectively. When α-NA, α-NB, and β-NA were used as substrates, males＇ EST activities of JN population were 1.13-, 1.12-, and 1,00-fold and 1.20-, 1.14-, and 1.07-fold higher than those of BD and BDG populations, respectively. The results also showed that the specific activities of the females were higher than those of the males in the BD and JN populations, whereas the specific activities of the males were higher than those of the females in the BDG population. The results of bioassay were consistent with those of biochemical analysis. Thus, it was inferred that the elevated ESTs activities might play an important role in conferring the differences of susceptibility of O. chinensis to malathion in the three collected populations. Enzyme kinetic studies indicated that the Km and Vmax values were different among the three collected populations and between the females and the males. The observed changes in the kinetic parameters might be explained by differential expression patterns of isozymes so that the insect esterases have different affinities and maximum velocities toward the same substrate.