Cloning of down-regulated genes under cold stress and identification of important genes related to cold tolerance in zebrafish (Danio rerio).

Low-temperature stress poses a significant risk to the survival of both cultivated and wild fish populations. Existing studies have found that the pre-acclimation of fishes to moderate cold stress can stimulate the activation of acclimation pathways, thereby enhancing their tolerance to cold stress. The fitness of fish relies heavily on appropriately controlled transcriptional reactions to environmental changes. Despite previous characterization of gene expression profiles in various fish species during cold acclimation, the specific genes responsible for essential functions in this process remain largely unknown, particularly the down-regulated genes induced by cold acclimation. To investigate the genes involved in cold acclimation, this study employed real-time quantitative PCR (RT-qPCR), molecular cloning, microinjection techniques, and cold stress experiments to determine the genes that play an essential part in cold acclimation. Consequently, 18 genes were discovered to be down-regulated in larval zebrafish experiencing cold stress. All 18 genes successfully detected overexpression in zebrafish at 96 and 126 hpf (fold change ≥3), which declined with the growth of zebrafish. Following microinjection, it was observed that her8a, cyp51, lss, txnipb, and bhlha9 had an adverse impact on the survival rate of zebrafish larvae under cold stress. These genes have been identified to play significant roles in various biological processes. For instance, bhlha9 has been found to be involved in both limb development and temperature sensing and her8a has been implicated in neural development. Additionally, cyp51 and lss have been identified as participants in the cholesterol synthesis pathway. Txnipb has been reported to induce cell apoptosis, thereby potentially influencing the survival rate of zebrafish larvae under cold stress. These findings offered crucial data for the analysis of molecular processes related to cold tolerance and the development of cold-resistant fish breeding.

A novel hypoxic lncRNA, LOC110520012 sponges miR-206-y to regulate angiogenesis and liver cell proliferation in rainbow trout (Oncorhynchus mykiss) by targeting vegfaa.

Long non-coding RNA (lncRNA) is a novel emerging type of competitive endogenous RNA (ceRNA) that performs key functions in multiple biological processes. However, little is known about the roles of lncRNA under hypoxia stress in fish. Here, vascular endothelial growth factor-Aa (vegfaa) was cloned in rainbow trout (Oncorhynchus mykiss), with the complete cDNA sequence of 2914 bp, encoding 218 amino acids. The molecular weight of the protein was approximately 25.33 kDa, and contained PDGF and VEGF_C domains. Time-course and spatial expression patterns revealed that LOC110520012 was a key regulator of rainbow trout in response to hypoxia stress, and LOC110520012, miR-206-y and vegfaa exhibited a ceRNA regulatory relationship in liver, gill, muscle and rainbow trout liver cells treated with acute hypoxia. Subsequently, the targeting relationship of LOC110520012 and vegfaa with miR-206-y was confirmed by dual-luciferase reporter analysis, and overexpression of LOC110520012 mediated the inhibition of miR-206-y expression in rainbow trout liver cells, while the opposite results were obtained after LOC110520012 silencing with siRNA. We also proved that vegfaa was a target of miR-206-y in vitro and in vivo, and the vegfaa expression and anti-proliferative effect on rainbow trout liver cells regulated by miR-206-y mimics could be reversed by LOC110520012. These results suggested that LOC110520012 can positively regulate vegfaa expression by sponging miR-206-y under hypoxia stress in rainbow trout, which facilitate in-depth understanding of the molecular mechanisms of fish adaptation and tolerance to hypoxia.

Functional Characterization of F3H Gene and Optimization of Dihydrokaempferol Biosynthesis in Saccharomyces cerevisiae.

The 1092 bp F3H gene from Trapa bispinosa Roxb., which was named TbF3H, was cloned and it encodes 363 amino acids. Bioinformatic and phylogenetic tree analyses revealed the high homology of TbF3H with flavanone 3-hydroxylase from other plants. A functional analysis showed that TbF3H of Trapa bispinosa Roxb. encoded a functional flavanone 3-hydroxylase; it catalyzed the formation of dihydrokaempferol (DHK) from naringenin in S. cerevisiae. The promoter strengths were compared by fluorescence microscopy and flow cytometry detection of the fluorescence intensity of the reporter genes initiated by each constitutive promoter (FITC), and DHK production reached 216.7 mg/L by the promoter adjustment strategy and the optimization of fermentation conditions. The results presented in this study will contribute to elucidating DHK biosynthesis in Trapa bispinosa Roxb.

PI3KR1 and AKT1 in largemouth bass (Micropterus salmoides): molecular cloning, characterization, and its involvement in the alleviation of hepatic glycogen deposition caused by insulin inclusion in vitro.

In this study, the full-length cDNA sequences of the phosphatidylinositol-3-kinase p85 alpha (PI3KR1) and serine/threonine kinase 1 (AKT1) genes in largemouth bass (Micropterus salmoides) were obtained using the rapid amplification of cDNA ends (RACE) method. Sequence analysis revealed that the cloned sequences of PI3KR1 and AKT1 are 4170 bp and 3672 bp in length, with open reading frames (ORFs) of 1389 bp and 1422 bp encoding 462 and 473 amino acids, respectively. Sequence alignment and evolutionary tree analysis indicated their close relationship to other teleosts, especially those with similar feeding habits. Tissue distribution demonstrated widespread distribution of both genes in various tissues, with the highest abundance in the liver. Further results found that the upregulation of the expression of p-PI3KR1, p-AKT1, p-FoxO1, and GLUT2 proteins by insulin, while suppressing the expression of the total FoxO1 protein, effectively triggers a significant activation of the PI3KR1-AKT1 insulin signaling pathway. Meanwhile, the mRNA levels of the key glycolytic genes, including glucokinase (gk), pyruvate kinase (pk), and phosphofructokinase liver type (pfkl), have been enhanced evidently. In contrast, the expression of gluconeogenic genes such as phosphoenolpyruvate carboxykinase (pepck), glucose-6-phosphatase catalytic subunit (g6pc), and fructose-1,6-bisphosphatase-1 (fbp1) has been notably down-regulated. In addition, insulin treatment promoted the phosphorylation of glycogen phosphorylase (PYGL) and the dephosphorylation of glycogen synthase (GS), and the glycogen content in the insulin-treated group was remarkably reduced compared to the control group. Overall, our study indicates that the activation of PI3KR1-AKT1 insulin signaling pathway represses the hepatic glycogen deposition via the regulation of glycolysis and gluconeogenesis, which provides some new insights into nutritional strategy to effectively regulate the glucose metabolism in carnivorous fish.

Competitive inhibition of the classical complement pathway using exogenous single-chain C1q recognition proteins.

Complement component C1q is a protein complex of the innate immune system with well-characterized binding partners that constitutes part of the classical complement pathway. In addition, C1q was recently described in the central nervous system as having a role in synapse elimination both in the healthy brain and in neurodegenerative diseases. However, the molecular mechanism of C1q-associated synapse phagocytosis is still unclear. Here, we designed monomer and multimer protein constructs, which comprised the globular interaction recognition parts of mouse C1q (globular part of C1q [gC1q]) as single-chain molecules (sc-gC1q proteins) lacking the collagen-like effector region. These molecules, which can competitively inhibit the function of C1q, were expressed in an Escherichia coli expression system, and their structure and capabilities to bind known complement pathway activators were validated by mass spectrometry, analytical size-exclusion chromatography, analytical ultracentrifugation, CD spectroscopy, and ELISA. We further characterized the interactions between these molecules and immunoglobulins and neuronal pentraxins using surface plasmon resonance spectroscopy. We demonstrated that sc-gC1qs potently inhibited the function of C1q. Furthermore, these sc-gC1qs competed with C1q in binding to the embryonal neuronal cell membrane. We conclude that the application of sc-gC1qs can reveal neuronal localization and functions of C1q in assays in vivo and might serve as a basis for engineering inhibitors for therapeutic purposes.

Analysis of aquaporins in northern grasses reveal functional importance of Puccinellia nuttalliana PIP2;2 in salt tolerance.

To better understand the roles of aquaporins in salt tolerance, we cloned PIP2;1, PIP2;2, PIP2;3, PIP1;1, PIP1;3, and TIP1;1 aquaporins from three northern grasses varying is salt tolerance including the halophytic grass Puccinellia nuttalliana, moderately salt tolerant Poa juncifolia, and relatively salt sensitive Poa pratensis. We analysed aquaporin expression in roots by exposing the plants to 0 and 150 mM for 6 days in hydroponic culture. NaCl treatment upregulated several PIP transcripts in P. nuttalliana while decreasing PnuTIP1;1. The PnuPIP2;2 transcripts increased by about six-fold in P. nuttalliana, two-fold in Poa juncifolia, and did not change in Poa pratensis. The NaCl treatment enhanced the rate of water transport in yeast expressing PnuPIP2;2 by 56% compared with control. PnuPIP2,2 expression also resulted in a higher Na+ uptake in yeast cells compared with an empty vector suggesting that PnuPIP2;2 may have both water and ion transporting functions. Structural analysis revealed that the transport properties of PnuPIP2;2 could be affected by its unique pore characteristics, which include a combination of hourglass, cylindrical, and increasing diameter conical entrance shape with pore hydropathy of -0.22.

Molecular cloning, expression, and functional analysis of a putative lectin from the pearl oyster (Pinctada fucata, Gould 1850).

Marine lectins are a group of proteins that possess specific carbohydrate recognition and binding domains. They exhibit various activities, including antimicrobial, antitumor, antiviral, and immunomodulatory effects. In this study, a novel galectin-binding lectin gene named PFL-96 (GenBank: OQ561753.1) was cloned from Pinctada fucata. The PFL-96 gene has an open reading frame of 324 base pairs (bp) and encodes a protein comprising 107 amino acids. The protein has a molecular weight of 11.95 kDa and an isoelectric point of 9.27. It contains an N-terminal signal peptide and a galactose-binding lectin domain. The sequence identity to lectin proteins from fish, echinoderms, coelenterates, and shellfish ranges from 31.90 to 40.00 %. In the phylogenetic analysis, it was found that the PFL-96 protein is closely related to the lectin from Pteria penguin. The PFL-96 recombinant protein exhibited coagulation activity on 2 % rabbit red blood cells at a concentration of ≥8 µg/mL. Additionally, it showed significant hemolytic activity at a concentration of ≥32 µg/mL. The PFL-96 recombinant protein exhibited significant antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Candida albicans, and Vibrio alginolyticus, with minimum inhibitory concentrations (MIC) of 4, 8, 16, and 16 µg/mL, respectively. The minimum bactericidal concentrations (MBC) were determined to be 8, 16, 32, and 32 µg/mL, respectively. Furthermore, the PFL-96 recombinant protein exhibited inhibitory effects on the proliferation of Hela tumor cells, HepG2 tumor cells, and C666-1 tumor cells, with IC50 values of 7.962, 8.007, and 9.502 µg/mL, respectively. These findings suggest that the recombinant protein PFL-96 exhibits significant bioactivity in vitro, contributing to a better understanding of the active compounds found in P. fucata. The present study establishes a fundamental basis for further investigation into the mechanism of action and structural optimization of the recombinant protein PFL-96. The aim is to develop potential candidates for antibacterial and anti-tumor agents.

Construing recombinant ZFP160 from Aspergillus terreus as pterin deaminase enzyme.

Pterin deaminase stands as a metalloenzyme and exhibits both antitumor and anticancer activities. Therefore, this study aimed to explore the molecular function of zinc finger protein-160 (zfp160) from Aspergillus terreus with its enzyme mechanism in detail. Subsequently, preliminary molecular docking studies on zfp160 from A. terreus were done. Next, the cloning and expression of zfp160 protein were carried out. Following, protein expression was induced and purified through nickel NTA column with imidazole gradient elution. Through the Mascot search engine tool, the expressed protein of MALDI-TOF was confirmed by 32 kDa bands of SDS-PAGE. Furthermore, its enzymatic characterization and biochemical categorization were also explored. The optimum conditions for enzyme were determined to be pH 8, temperature 35°C, km 50 µm with folic acid as substrate, and Vmax of 24.16 (IU/mL). Further, in silico analysis tried to explore the interactions and binding affinity of various substrates to the modeled pterin deaminase from A. terreus. Our results revealed the binding mode of different substrate molecules with pterin deaminase using the approximate scoring functions that possibly correlate with actual experimental binding affinities. Following this, molecular dynamic simulations provided the in-depth knowledge on deciphering functional mechanisms of pterin deaminase over other drugs.

Phylogeny of Alisma (Alismataceae) revisited: implications for polyploid evolution and species delimitation.

Alisma L. is a genus of aquatic and wetland plants belonging to family Alismataceae. At present, it is thought to contain ten species. Variation in ploidy level is known in the genus, with diploids, tetraploids and hexaploids recorded. Previous molecular phylogenetic studies of Alisma have generated a robust backbone that reveals important aspects of the evolutionary history of this cosmopolitan genus, yet questions remain unresolved about the formation of the polyploid taxa and the taxonomy of one particularly challenging, widely distributed species complex. Here we directly sequenced, or cloned and sequenced, nuclear DNA (nrITS and phyA) and chloroplast DNA (matK, ndhF, psbA-trnH and rbcL) of multiple samples of six putative species and two varieties, and conducted molecular phylogenetic analyses. Alisma canaliculatum and its two varieties known in East Asia and A. rariflorum endemic to Japan possess closely related but heterogeneous genomes, strongly indicating that the two species were generated from two diploid progenitors, and are possibly siblings of one another. This evolutionary event may have occurred in Japan. Alisma canaliculatum var. canaliculatum is segregated into two types, each of which are geographically slightly differentiated in Japan. We reconstructed a single phylogeny based on the multi-locus data using Homologizer and then applied species delimitation analysis (STACEY). This allowed us to discern A. orientale as apparently endemic to the Southeast Asian Massif and distinct from the widespread A. plantago-aquatica. The former species was most likely formed through parapatric speciation at the southern edge of the distribution of the latter.

Discovery of α-Linolenic Acid 16(S)-Lipoxygenase: Cucumber (Cucumis sativus L.) Vegetative Lipoxygenase 3.

The GC-MS profiling of the endogenous oxylipins (Me/TMS) from cucumber (Cucumis sativus L.) leaves, flowers, and fruit peels revealed a remarkable abundance of 16-hydroxy-9,12,14-octadecatrienoic acid (16-HOT). Incubations of homogenates from these organs with α-linolenic acid yielded 16(S)-hydroperoxide (16-HPOT) as a predominant product. Targeted proteomic analyses of these tissues revealed the presence of several highly homologous isoforms of the putative "9S-lipoxygenase type 6". One of these isoenzymes (CsLOX3, an 877 amino acid polypeptide) was prepared by heterologous expression in E. coli and exhibited 16(S)- and 13(S)-lipoxygenase activity toward α-linolenic and linoleic acids, respectively. Furthermore, α-linolenate was a preferred substrate. The molecular structures of 16(S)-HOT and 16(S)-HPOT (Me or Me/TMS) were unequivocally confirmed by the mass spectral data, 1H-NMR, 2D 1H-1H-COSY, TOCSY, HMBC, and HSQC spectra, as well as enantiomeric HPLC analyses. Thus, the vegetative CsLOX3, biosynthesizing 16(S)-HPOT, is the first 16(S)-LOX and ω3-LOX ever discovered. Eicosapentaenoic and hexadecatrienoic acids were also specifically transformed to the corresponding ω3(S)-hydroperoxides by CsLOX3.

Gene Characterization of Nocturnin Paralogues in Goldfish: Full Coding Sequences, Structure, Phylogeny and Tissue Expression.

The aim of this work is the full characterization of all the nocturnin (noc) paralogues expressed in a teleost, the goldfish. An in silico analysis of the evolutive origin of noc in Osteichthyes is performed, including the splicing variants and new paralogues appearing after teleostean 3R genomic duplication and the cyprinine 4Rc. After sequencing the full-length mRNA of goldfish, we obtained two isoforms for noc-a (noc-aa and noc-ab) with two splice variants (I and II), and only one for noc-b (noc-bb) with two transcripts (II and III). Using the splicing variant II, the prediction of the secondary and tertiary structures renders a well-conserved 3D distribution of four α-helices and nine ß-sheets in the three noc isoforms. A synteny analysis based on the localization of noc genes in the patrilineal or matrilineal subgenomes and a phylogenetic tree of protein sequences were accomplished to stablish a classification and a long-lasting nomenclature of noc in goldfish, and valid to be extrapolated to allotetraploid Cyprininae. Finally, both goldfish and zebrafish showed a broad tissue expression of all the noc paralogues. Moreover, the enriched expression of specific paralogues in some tissues argues in favour of neo- or subfunctionalization.

A Novel Antimicrobial Peptide, Dermaseptin-SS1, with Anti-Proliferative Activity, Isolated from the Skin Secretion of Phyllomedusa tarsius.

The emergence of multidrug-resistant bacteria has severely increased the burden on the global health system, and such pathogenic infections are considered a great threat to human well-being. Antimicrobial peptides, due to their potent antimicrobial activity and low possibility of inducing resistance, are increasingly attracting great interest. Herein, a novel dermaseptin peptide, named Dermaseptin-SS1 (SS1), was identified from a skin-secretion-derived cDNA library of the South/Central American tarsier leaf frog, Phyllomedusa tarsius, using a 'shotgun' cloning strategy. The chemically synthesized peptide SS1 was found to be broadly effective against Gram-negative bacteria with low haemolytic activity in vitro. A designed synthetic analogue of SS1, named peptide 14V5K, showed lower salt sensitivity and more rapid bacteria killing compared to SS1. Both peptides employed a membrane-targeting mechanism to kill Escherichia coli. The antiproliferative activity of SS1 and its analogues against lung cancer cell lines was found to be significant.

Molecular characterisation and expression profile of the PRLR gene during goose ovarian follicle development.

1. Although PRL-PRLR signalling plays important roles in regulating avian reproduction, there is a paucity of information regarding the functional significance of PRLR in goose ovarian follicle development.2. The full-length 2,496 bp coding sequence of PRLR was obtained from Sichuan White goose (Anser cygnoides) for the first time and was seen to encode a polypeptide containing 831 amino acids. Goose PRLR shares similar sequence characteristics and conserved functional domains to other avian species and was phylogenetically clustered into the avian clade.3. The qPCR results suggested that the mRNA levels of PRLR significantly increased in primary follicles during weeks 3 to 4 of age and were higher in secondary- than in primordial follicles at week 5 post-hatching, which suggested that the PRLR-mediated signalling could be involved in regulation of early folliculogenesis.4. The PRLR mRNA was expressed at the highest levels in the prehierarchical 8-10 mm granulosa layers throughout goose ovarian follicle development, indicating a role for PRLR in the process of follicle selection.5. PRLR mRNA was differentially expressed in the three cohorts of in vitro cultured granulosa cells harvested from different sized goose ovarian follicles, which suggested that PRLR was involved in regulating granulosa cell functions depending on the stage of follicle development. These data provide novel insights into the role of PRLR during goose ovarian follicle development, although the underlying mechanisms await further investigations.

Molecular cloning and functional characterization of MhHEC2-like genes in Malus halliana reveals it enhances Fe (iron) deficiency tolerance.

The basic helix-loop-helix (bHLH) family, as one of the largest families of transcription factors (TFs) in plants, plays crucial roles in regulating growth, development, and abiotic stress responses. However, studies on the association of the bHLH genes with apple iron (Fe) deficiency are limited. Here, multiple bHLH genes that responded to Fe deficiency were selected for quantitative real-time PCR in Malus halliana. The results showed that the expression of HEC2-like gene exerted more values compared to other genes under Fe deficiency stress, but the mechanism by which it regulates Fe deficiency stress is unclear. Subsequently, MhHEC2-like gene (ID: 103,455,961) was cloned from M. halliana for functional identification. We found that both transgenic Arabidopsis thaliana and tobacco displayed less chlorosis and more robust growth than wild-type (WT) controls under Fe deficiency stress. At the same time, the overexpressed apple calli grew prominently larger and better under Fe deficiency compared to the wild type. On the other hand, physiological index measurements revealed that overexpressed MhHEC2-like gene enhanced tolerance to Fe deficiency stress in A. thaliana and tobacco by promoting the synthesis of photosynthetic pigments, improving antioxidant enzyme activity, and enhancing Fe reduction, and strengthened tolerance to Fe deficiency stress in apple calli by reducing pH, boosting Fe reduction, and increasing antioxidant enzyme activity. To sum up, the overexpression of MhHEC2-like gene strengthened tolerance to Fe deficiency stress in Arabidopsis thaliana, tobacco, and apple calli.

The transcription factor LaMYC4 from lavender regulates volatile Terpenoid biosynthesis.

BACKGROUND: The basic helix-loop-helix (bHLH) transcription factors (TFs), as one of the largest families of TFs, are essential regulators of plant terpenoid biosynthesis and response to stresses. Lavender has more than 75 volatile terpenoids, yet few TFs have been identified to be involved in the terpenoid biosynthesis. RESULTS: Based on RNA-Seq, reverse transcription-quantitative polymerase chain reaction, and transgenic technology, this study characterized the stress-responsive transcription factor LaMYC4 regulates terpenoid biosynthesis. Methyl jasmonate (MeJA) treatment increased volatile terpenoid emission, and the differentially expressed gene LaMYC4 was isolated. LaMYC4 expression level was higher in leaf than in other tissues. The expression of LaMYC4 decreased during flower development. The promoter of LaMYC4 contained hormone and stress-responsive regulatory elements and was responsive to various treatments, including UV, MeJA treatment, drought, low temperature, Pseudomonas syringae infection, and NaCl treatment. LaMYC4 overexpression increased the levels of sesquiterpenoids, including caryophyllenes, in Arabidopsis and tobacco plants. Furthermore, the expression of crucial node genes involved in terpenoid biosynthesis and glandular trichome number and size increased in transgenic tobacco. CONCLUSIONS: We have shown that the stress-responsive MYC TF LaMYC4 from 'Jingxun 2' lavender regulates volatile terpenoid synthesis. This study is the first to describe the cloning of LaMYC4, and the results help understand the role of LaMYC4 in terpenoid biosynthesis.

cDNA cloning, prokaryotic expression, and functional analysis of squalene synthase (SQS) in Camellia vietnamensis Huang.

Camellia vietnamensis Huang, which belongs to Camellia oleifera, is a traditional Chinese medicinal plant widely planted on Hainan Island. Tea saponin is an important functional component of C. vietnamensis, and squalene is the precursor substance that controls its formation. Squalene synthase (SQS: EC 2.5.1.21) synthesizes squalene from 2 molecules of farnesyl pyrophosphate (FPP). In this study, 1683 bp of the C. vietnamensis SQS gene, designated as CvSQS, was cloned and encoded 414 amino acids. Bioinformatics and phylogenetic tree analysis revealed the high homology of CvSQS with squalene synthases from other plants. For soluble proteins, the carboxy-terminal deleted CvSQS was obtained for expression in Escherichia coli Transetta (DE3), and the recombinant protein with a weight of 42.5 kDa was detected using SDS-PAGE and Western blot. After an enzymatic reaction, the presence of squalene in the product was analyzed using GC-MS detection, which indicated that CvSQS had catalytic activity. The tissue specificity of CvSQS and its presence in seeds at various ripening stages was detected by q-RT PCR. CvSQS had the highest transcriptional level in leaves, followed by seeds, roots, and flowers; the amount of CvSQS in the seeds was highest in September. The identification and functional characterization of CvSQS is essential for further studies on the regulation mechanism of tea saponin in C. vietnamensis.

CD4-1 and CD8α T lymphocytes subsets in spotted sea bass (Lateolabrax maculatus) and comparison on antigenicity of T lymphocytes subsets in other three marine fish species.

CD4 and CD8 molecules play an important role in the identification of T lymphocytes, and diverse among fish species. In this study, CD4-1 and CD8α gene of spotted sea bass (Lateolabrax maculatus) were cloned, polyclonal antibodies against CD4-1 (CD4-1 pAbs) and CD8α (CD8α pAbs) were produced, respectively. And the variations in CD4-1+ and CD8α+ T-lymphocytes in spotted sea bass and the cross-reactivity with leukocytes in pearl gentian grouper (Epinephelus fuscoguttatus x E. lanceolatus), schlegel's black rockfish (Sebastes schlegelii) and flounder (Paralichthys olivaceus) were investigated using CD4-1 pAbs and CD8α pAbs. The results showed that CD4-1 molecule ORF was 1413 bp and CD8α was 690 bp, both molecules are transmembrane glycoproteins with high amino acid homology to grouper. The CD4-1 pAbs specifically recognized both the CD4-1 recombinant and natural proteins, as does the CD8α pAbs to CD8α molecule, and no cross-reactivity between the two antibodies. CD4-1+ and CD8α+ T lymphocytes were detected in peripheral blood, spleen and head kidney leukocytes in spotted sea bass. In cross-reactivity assay with other three fish, CD4-1 pAbs could recognize the lymphocytes from pearl gentian grouper and schlegel's black rockfish, both with highest proportions in the spleen leukocytes, 5.3 ± 0.4% and 2.6 ± 0.3%, respectively, and CD8α pAbs could only recognize the lymphocytes in pearl gentian grouper, and no cross-reactivities to lymphocytes of flounder. These data suggested that the CD4-1 and CD8α molecules varied by fish species in the genes features and antigenicity, which might result in the diversities of T lymphocytes subpopulations. This will be a key to elucidating the classification and evolution of T lymphocytes in fish.

Molecular cloning and characterization of an alpha-amylase inhibitor (TkAAI) gene from Trichosanthes kirilowii Maxim.

Trichosanthes kirilowii Maxim taxonomically belongs to the Cucurbitaceae family and Trichosanthes genus. Its whole fruit, fruit peel, seed and root are widely used in traditional Chinese medicines. A ribosome-inactivating protein with RNA N-glycosidase activity called Trichosanthrip was isolated and purified from the seeds of T. kirilowii in our recent previous research. To further explore the biological functions of Trichosanthrip, the cDNA of T. kirilowii alpha-amylase inhibitor (TkAAI) was cloned through rapid-amplification of cDNA ends and its sequence was analyzed. Also, the heterologous protein was expressed in Escherichia coli and its alpha-amylase activity was further measured under optimized conditions. The full-length cDNA of TkAAI was 613 bp. The speculated open reading frame sequence encoded 141 amino acids with a molecular weight of 16.14 kDa. Phylogenetic analysis demonstrated that the Alpha-Amylase Inhibitors Seed Storage domain sequence of TkAAI revealed significant evolutionary homology with the 2S albumin derived from the other plants in the Cucurbitaceae group. In addition, TkAAI was assembled into pET28a with eGFP to generate a prokaryotic expression vector and was induced to express in E. coli. The TkAAI-eGFP infusion protein was proven to exhibit alpha-amylase inhibitory activity against porcine pancreatic amylase in a suitable reaction system. Analysis of gene expression patterns proved that the relative expression level of TkAAI in seeds is highest. The results presented here forecasted that the TkAAI might play a crucial role during the development of T. kirilowii seeds and provided fundamental insights into the possibility of T. kirilowii derived medicine to treat diabetes related diseases.

RNAi-Mediated Knockdown of Calreticulin3a Impairs Pollen Tube Growth in Petunia.

Pollen tube growth depends on several complex processes, including exo/endocytosis, cell wall biogenesis, intracellular transport, and cell signaling. Our previous results provided evidence that calreticulin (CRT)-a prominent calcium (Ca2+)-buffering molecular chaperone in the endoplasmic reticulum (ER) lumen-is involved in pollen tube formation and function. We previously cloned and characterized the CRT gene belonging to the CRT1/2 subgroup from Petunia hybrida (PhCRT1/2), and found that post-transcriptional silencing of PhCRT1/2 expression strongly impaired pollen tube growth in vitro. Here, we report cloning of a new PhCRT3a homolog; we identified the full-length cDNA sequence and described its molecular characteristics and phylogenetic relationships to other plant CRT3 genes. Using an RNA interference (RNAi) strategy, we found that knockdown of PhCRT3a gene expression caused numerous defects in the morphology and ultrastructure of cultivated pollen tubes, including disorganization of the actin cytoskeleton and loss of cytoplasmic zonation. Elongation of siPhCRT3a pollen tubes was disrupted, and some of them ruptured. Our present data provide the first evidence that PhCRT3a expression is required for normal pollen tube growth. Thus, we discuss relationships between diverse CRT isoforms in several interdependent processes driving the apical growth of the pollen tube, including actomyosin-dependent cytoplasmic streaming, organelle positioning, vesicle trafficking, and cell wall biogenesis.

Molecular Cloning, Expression, and Functional Analysis of Glycosyltransferase (TbUGGT) Gene from Trapa bispinosa Roxb.

Trapa bispinosa Roxb. is an economical crop for medicine and food. Its roots, stems, leaves, and pulp have medicinal applications, and its shell is rich in active ingredients and is considered to have a high medicinal value. One of the main functional components of the Trapa bispinosa Roxb. shell is 1-galloyl-beta-D-glucose (ßG), which can be used in medical treatment and is also an essential substrate for synthesizing the anticancer drug beta-penta-o-Galloyl-glucosen (PGG). Furthermore, gallate 1-beta-glucosyltransferase (EC 2.4.1.136) has been found to catalyze gallic acid (GA) and uridine diphosphate glucose (UDPG) to synthesize ßG. In our previous study, significant differences in ßG content were observed in different tissues of Trapa bispinosa Roxb. In this study, Trapa bispinosa Roxb. was used to clone 1500 bp of the UGGT gene, which was named TbUGGT, to encode 499 amino acids. According to the specificity of the endogenous expression of foreign genes in Escherichia coli, the adaptation codon of the cloned original genes was optimized for improved expression. Bioinformatic and phylogenetic tree analyses revealed the high homology of TbUGGT with squalene synthases from other plants. The TbUGGT gene was constructed into a PET-28a expression vector and then transferred into Escherichia coli Transsetta (DE3) for expression. The recombinant protein had a molecular weight of 55 kDa and was detected using SDS-PAGE. The proteins were purified using multiple fermentation cultures to simulate the intracellular environment, and a substrate was added for in vitro reaction. After the enzymatic reaction, the levels of ßG in the product were analyzed using HPLC and LC-MS, indicating the catalytic activity of TbUGGT. The cloning and functional analysis of TbUGGT may lay the foundation for further study on the complete synthesis of ßG in E. coli.