Cloning, characterization, expression, and feeding response of thyrotropin receptor in largemouth bass (Micropterus salmoides).

Thyrotropin receptor (TSHR) is a G-protein-coupled receptor that regulates the synthesis, storage, and secretion of thyroid hormones in the thyroid tissue. The aims of the present study were to characterize the full-length TSHR cDNA in largemouth bass (Micropterus salmoides), and to determine the TSHR gene transcription levels in different tissues. In addition, the response of TSHR transcription levels to daily feeding in thyroid tissue was investigated. The results showed that the full-length cDNA sequence was 2743 bp with an open reading frame of 2340 bp encoding a 779-amino acid peptide. BLAST analysis indicated that the amino acid sequence displayed 58.4-90.2% identity and 5.6-125.8 divergence, compared with other known fish species. The most abundant TSHR transcription levels were found in the spleen, head kidney, and kidney. Feeding did not affect the transcription level of TSHR in thyroid tissue over the course of the day. Thus, the current study suggests that there was no relationship between daily nutritional status and TSHR transcription level in the thyroid tissue of largemouth bass. The spleen, head kidney, and kidney exhibited the most abundant TSHR transcription levels.

Analysis of QTLs for panicle exsertion and its relationship with yield and yield-related traits in rice (Oryza sativa L.).

Panicle exsertion (PE) is an important morphological trait that is closely associated with spikelet fertility and grain yield. To understand the genetic basis of PE and its relationships with yield and yield-related traits, a recombinant inbred population consisting of 240 lines derived from a cross between an Indica cultivar 'Kasalath' and a Japonica germplasm 'TD70', was studied over two years. PE was significantly correlated with plant height, heading date (HD), panicle length (PL), and panicle characteristics such as primary branch number, spikelet number per panicle, and spikelet density, but showed poor correlation with yield components. Based on linkage mapping of 141 SSR markers, a total of 38 quantitative trait loci (QTLs) were located for 12 investigated traits, with the contribution varying from 6.51 to 8.61%. Among these, four QTL clusters were identified on chromosomes 1, 2, 3, and 6, suggesting the existence of pleiotropic alleles. In some intervals, two loci for PE were collocated with several traits, which is consistent with the correlations observed with phenotypic variations. The PE QTLs with 'Kasalath' alleles and without pleiotropic effects would be valuable for the improvement of PE in 'TD70' and in other rice varieties.

Development and characterization of Rift Valley fever virus-like particles.

Rift Valley fever (RVF) is an acute, febrile zoonotic disease that is caused by the RVF virus (RVFV) and spread by arthropod vectors. RVF is currently prevalent in Africa and the Arabian Peninsula, and causes substantial economic losses. Furthermore, this disease poses a serious threat to animal and human health in regions worldwide, making it a serious public health concern. However, RVFV vaccines for human use are still unavailable, and hence there is an urgent need for novel efficient vaccines against RVFV. Vaccine preparation techniques have become a crucial factor in developing new vaccines. In the current study, the N and G protein genes of RVFV were inserted into the pFastBacDual baculovirus expression vector downstream of the pP10 and pPH promoters. The resultant recombinant vector, pFastBacDual-S-M, was transfected into Sf9 insect cells by lipofection. The recombinant baculovirus, named rBac-N-G, was retrieved and infected into Sf9 insect cells to generate RVFV virus-like particles (VLPs). Using polyclonal antibodies against RVFV proteins in immunofluorescence and western blot analyses, we positively identified the presence of the RVFV proteins in VLP preparations. Sucrose density gradient centrifugation and transmission electron microscopy revealed that the morphology of the RVFV VLPs was consistent with previous reports of RVFV virions. This study describes a technique for efficient production of RVFV VLPs, and has laid the foundation for future VLP-based RVFV vaccines.

Haplotypes of qGL3 and their roles in grain size regulation with GS3 alleles in rice.

Grain size is an important trait that directly influences rice yield. The qGL3 and GS3 genes are two putative regulators that play a role in grain size determination. A single rare nucleotide substitution (C→A) at position 1092 in exon 10 of qGL3 might be responsible for variations in grain size. However, little is known about the haplotype variations of qGL3 and their interactions with GS3 during the regulation of grain length and grain weight. In this study, qGL3 haplotype variations were examined in 61 Indica varieties, and the effects of qGL3 and GS3 on grain trait variation in 110 lines were evaluated. Six qGL3 haplotypes were identified, and qGL3-2 was a major haplotype in Indica varieties. Moreover, qGL3-6, a reported key single nucleotide polymorphism, was validated. Our results showed that the mutants qgl3 and gs3 (loss-of-function mutation types of qGL3 and GS3, respectively) had significant effects on grain length and grain weight. However, no significant effects associated with differences in the regulation of grain thickness were observed. The genetic effects of qgl3 on grain phenotypes were stronger than those of gs3. In addition to increased grain length, qgl3 had an evident role in grain width increases. In contrast, gs3 played an opposite role in grain width regulation. These results provided novel insights into grain size control and the functions of qgl3 and gs3 in rice yield improvement.

Verification and evaluation of grain QTLs using RILs from TD70 x Kasalath in rice.

Grain size is an important trait that directly influences the yield of rice. Validation and evaluation of grain genes is important in rice genetic studies and for breeding. In a population of 240 recombinant inbred lines (RILs) derived from a cross between an extra-large grain japonica variety TD70 and a small grain indica variety Kasalath, we mapped 19 QTLs controlling grain traits. These QTLs included six cloned grain genes, namely, GW2, GS3, qSW5, qGL3, GS5, and GW8. All of the alleles with the optimal effects on grain size came from TD70, the variety with extra-large grains. To verify these gene loci, we cloned and sequenced GW2, GS3, GW5 (qSW5), qGL3, GS5, GW8, and TGW6 in TD70 and Kasalath, and found several functional polymorphisms in the sequences of the genes. New functional markers for the cloned genes were designed to identify parents and RILs. The contributions of these polymorphisms to the improvement in rice grain size traits were evaluated. Our results indicate that at least six functional polymorphisms have additive effects on grain shape and that one non-functional polymorphism in TGW6 affects grain shape in TD70. The newly designed markers will be useful in further studies to identify functional grain genes. Our findings provide insight into the control of grain size in rice, and they will be of value for improving rice grain yield.

Cloning and sequence analysis of the coding sequence of ß-actin cDNA from the Chinese alligator and suitable internal reference primers from the ß-actin gene.

ß-Actin is an essential component of the cytoskeleton and is stably expressed in various tissues of animals, thus, it is commonly used as an internal reference for gene expression studies. In this study, a 1731-bp fragment of ß-actin cDNA from Alligator sinensis was obtained using the homology cloning technique. Sequence analysis showed that this fragment contained the complete coding sequence of the ß-actin gene (1128 bp), encoding 375 amino acids. The amino acid sequence of ß-actin is highly conserved and its nucleotide sequence is slightly variable. Multiple alignment analyses showed that the nucleotide sequence of the ß-actin gene from A. sinensis is very similar to sequences from birds, with 94-95% identity. Ten pairs of primers with different product sizes and different annealing temperatures were screened by PCR amplification, agarose gel electrophoresis, and DNA sequencing, and could be used as internal reference primers in gene expression studies. This study expands our knowledge of ß-actin gene phylogenetic evolution and provides a basis for quantitative gene expression studies in A. sinensis.

Molecular cloning and characterization of a novel Y-box gene from Sepiella maindroni.

Y-box proteins are a family of highly conserved nucleic acid binding proteins that interact with genome and transcription product to modulate the transcriptional and translational processes. In the present study, a complete mRNA of Y-box binding protein (designated SmYB) was obtained from Sepiella maindroni by amplification of flanking sequences. The full size of SmYB cDNA was 1502 bp, including 99 bp at the 5ꞌ untranslated region (UTR), a 3ꞌ UTR of 821 bp with a poly (A) tail, and an open reading frame of 582 bp, encoding a polypeptide of 193 amino acids with the predicted molecular weight of 16.48 kDa. The conserved cold-shock domain and two known RNA binding motifs identified in SmYB strongly suggested that SmYB was a new member of Y-box proteins. Quantitative real-time PCR was performed to examine the expression of SmYB mRNA in various tissues, embryos, and its temporal expression in liver after cold shock. The mRNA transcript of SmYB was detected in all examined tissues, with the highest expression level in testis and ovary. SmYB was abundant in early developmental stages of S. maindroni embryos but diminished in the late post-embryonic development. In addition, cold-shock treatment upregulated the transcription of SmYB mRNA in liver. These results demonstrated that SmYB is involved in embryonic development of S. maindroni and its tolerance to acute low temperatures.

Ulinastatin promotes T lymphocyte apoptosis in rats with severe acute pancreatitis via mitochondrial pathways.

We explored the influence of ulinastatin on apoptosis of T lymphocytes in rats with severe acute pancreatitis (SAP) and the effect of ulinastatin on mitochondrial apoptosis pathways in spleen lymphocytes. Thirty-six Wistar rats were randomly divided into three groups (N = 12): a sham operated group, a SAP group, and an ulinastatin-treated SAP group. The SAP model was established by injecting 5% sodium taurocholate into the intrapancreatobiliary duct. Study rats were sacrificed after 24 h, and splenic lymphocytes were then collected. CD4(+) and CD8(+) T lymphocytes were labeled by direct immune fluorescence assays; the percentage of apoptotic cells, mitochondrial membrane potential levels, and mitochondria permeability transition pore opening levels were measured by flow cytometry. In the ulinastatin-treated SAP group, the ratio of CD4(+)/CD8(+) T lymphocytes was significantly higher than that in the SAP group, and the apoptosis percentage of CD4(+) T lymphocytes was significantly decreased. The percentage of lymphocytes with an abnormal opening of the mitochondrial permeability transition pore and lymphocytes with decreased mitochondrial membrane potential in the ulinastatin-treated SAP group were significantly lower than that in the SAP group. Ulinastatin can directly enhance immunological function and attenuate immune suppression in SAP rats through inhibiting the apoptosis of CD4(+) T lymphocytes. These study findings demonstrate that therapeutic effects may occur through inhibiting the apoptosis induced by mitochondrial signaling pathways.

Direct DNA extraction method of an obligate parasitic fungus from infected plant tissue.

Powdery mildew and rust fungi are obligate parasites that cannot live without host organisms. They are difficult to culture in synthetic medium in the laboratory. Genomic DNA extraction is one of the basic molecular techniques used to study the genetic structure of populations. In this study, 2 different DNA extraction methods, Chelex-100 and cetyltrimethylammonium bromide (CTAB), were used to extract DNA from euonymus powdery mildew and Puccinia striiformis f. sp Tritici. Polymerase chain reaction was carried out with a race-specific-marker rDNA-internal transcribed spacer sequence. Both DNA extraction methods were compared and analyzed. The results showed that both Chelex-100 and CTAB were effective for extracting genomic DNA from infected plant tissue. However, less DNA was required for the Chelex-100 method than for the CTAB method, and the Chelex-100 method involved fewer steps, was simpler and safer, and did not require organic solvents compared to the CTAB method. DNA quality was evaluated by polymerase chain reaction, and the results showed that genomic DNA extracted using the Chelex-100 method was better than that using CTAB method, and was sufficient for studying the genetic structure of population.

Screening of genes related to ovarian development in the swimming crab, Portunus trituberculatus, by suppression subtractive hybridization.

The swimming crab, Portunus trituberculatus, is an important marine animal and is widely cultured in China. In the present study, suppression subtractive hybridization was applied to identify the differentially expressed genes in the ovaries of mature and immature P. trituberculatus. One hundred and seventy six expressed sequence tag (ESTs) were identified, of which 100 were down-regulated, and 76 up-regulated. BLAST analysis identified 51 unigenes, of which 27 were down-regulated, and 24 up-regulated. Quantitative real-time reverse transcriptase polymerase chain reaction results indicated that the SSH technique is valuable in screening genes related to ovarian development. Genes identified in this study encoded proteins corresponding to a wide range of functions and included immune response protein, transcription initiation factor, metabolic proteins, chromosome, histone h3, ovarian development-related protein, and vitellogenin. In addition, 64 metabolic pathways were annotated in differentially expressed ESTs by using the Kyoto Encyclopedia of Genes and Genomes pathway. Four annotated pathways (oxidative phosphorylation, carbon metabolism, fatty acid degradation, and protein digestion and absorption) appeared to be involved in ovarian development. In ontology analysis, 5.83% of the cellular process genes in reverse subtraction cDNA library are involved in reproduction, and 5.88% involved in developmental process. In up-regulated genes, myosin II-expressed polehole-like protein; histone h3; ovigerous-hair stripping substance; peritrophin 48; and ovarian development-related protein appeared to be involved in ovarian development. Identification of differentially expressed genes in the mature and immature ovary of the swimming crab provides new insights for further studies on the mechanism underlying ovarian development in this species.

Full-length cDNA cloning and structural characterization of preproinsulin in Alligator sinensis.

Insulin is an important endocrine hormone that plays a critical physiological role in regulating metabolism and glucostasis in vertebrates. In this study, the complete cDNA of Alligator sinensis preproinsulin gene was cloned for the first time by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends methods; the amino acid sequence encoded and protein structure were analyzed. The full-length of preproinsulin cDNA sequence consists of 528 base pairs (bp), comprising a 34-bp 5'-untranslated region, a 170-bp 3'-untranslated region and an open reading frame that is 324 bp in length. The open reading frame encodes a 107-amino acid preproinsulin with a molecular weight of approximately 12,153.8 Da, theoretical isoelectric point of 5.68, aliphatic index of 92.06, and grand average of hydropathicity of -0.157, from which a signal peptide, a B-chain, a C-peptide, and an A-chain are derived. Online analysis suggested that the deduced preproinsulin amino acid sequence contains a transmembrane region, and that it has a signal peptide whose cleavage site occurs between alanine 24 and alanine 25. Comparative analysis of preproinsulin amino acid sequences indicated that the A-chain and B-chain sequences of preproinsulins are highly conserved between reptiles and birds, and that the preproinsulin amino acid sequence of Alligator sinensis shares 89% similarity to that of Chelonia mydas, but low similarity of 48-63% to those of mammals and fishes. The phylogenetic tree constructed using the neighbor-joining method revealed that preproinsulin of Alligator sinensis had high homology with reptiles and birds, such as Chelonia mydas, Gallus gallus, and Columba livia.

mRNA expression profiles of heat shock proteins of wild and salinity-tolerant swimming crabs, Portunus trituberculatus, subjected to low salinity stress.

Challenged by the low salinity, 4 parts per thousand (4 ppt), for 72h, the survivals of swimming crabs (Portunus trituberculatus) were collected as the screened group (SG, tolerant to low salinity). Aiming at identifying the mechanism of low salinity tolerance, quantitative real-time PCR was employed to investigate the expression profiles of 4 HSP genes (HSP60, HSP70, HSP90-1, HSP90-2) in the hepatopancreas of wild (WG) and screened (SG) groups of P. trituberculatus exposed to low salinity (4 ppt). The results showed that 3 of the candidate genes (HSP60, HSP70, HSP90-1) exhibited similarly downregulated expression profiles in the first 3 h (P < 0.05), which became upregulated from 3 h to 72 h after being subjected to low salinity conditions. In contrast, the expression profile of the HSP90-2 gene was upregulated during the first 6 h for the WG, and during the first 12 h for the SG, after which it became downregulated. HSP90-1 and HSP90-2 were highly expressed at 12 h after low salinity challenge in the SG, but not the WG. The response of these 2 genes to salinity stress indicates their suitability as biomarkers to differentiate SG from WG crabs. The results indicate that HSP genes are involved in the adaptation of crabs to low salinity exposure, and that different HSPs have diverse functions in response to low salinity stress in P. trituberculatus. In addition, HSP expression in SG indicates that this group is more tolerant to low salinity conditions compared to WG.

Predictive role of RRM1 and BRCA1 mRNA expression on the clinical outcome of advanced non-small cell lung cancer.

This study aimed to evaluate the association between RRM1 and BRCA1 expressions and the therapeutic efficacy of platinum-based chemotherapy in non-small cell lung cancer patients in terms of their response and prognosis. In total, 377 patients agreed to participate in our study, and all of them received platinum-based combination chemotherapy between January 2008 and January 2009. The relative cDNA quantitation for RRM1 and BRCA1 was conducted using a fluorescence-based, real-time detection method, using ß-actin as a reference gene. The average age of the 377 patients was 64.6 years (range: 25.5-86.4 years), including 269 men and 108 women. Patients with high RRM1 expression benefited more from a platinum-containing regimen, and patients with high BRCA1 expression showed a high response rate to a platinum-containing regimen and reduced disease progression. Patients with high RRM1 expression were associated with a longer progression-free survival (PFS) and overall survival (OS) than those with low expression, and the hazard ratios (HRs) (95% confidence interval (CI)) were 0.67 (0.32-0.91) and 0.54 (0.30-0.95), respectively. Patients with high BRCA1 expression showed longer PFS and OS compared to those with low expression, and the HRs (95%CI) were 0.54 (0.30-0.95) and 0.62 (0.32-0.93), respectively. These results could be used in personalized chemotherapy decisions and to increase the response rate and prolonged survival, and could encourage exploration of the predictive value of other genes.

Development of polymorphic expressed sequence tag-single sequence repeat markers in the common Chinese cuttlefish, Sepiella maindroni.

The common Chinese cuttlefish (Sepiella maindroni) is one of the popular edible cephalopod consumed across Asia. To facilitate the population genetic investigation of this species, we developed fourteen polymorphic microsatellite makers from expressed sequence tags of S. maindroni. The number of alleles at each locus ranged from 6 to 10 with an average of 7.9 alleles per locus. The ranges of observed and expected heterozygosity were from 0.615 to 0.962 and 0.685 to 0.888, respectively. Four loci were found deviated significantly from Hardy-Weinberg equilibrium. The polymorphism information content ranged from 0.638 to 0.833. These polymorphic microsatellite loci will be helpful for the population genetic, genetic linkage map, and other genetic studies of S. maindroni.