Ectopic and transient expression of VvDIR4 gene in Arabidopsis and grapes enhances resistance to anthracnose via affecting hormone signaling pathways and lignin production.

BACKGROUND: DIR (Dirigent) proteins play important roles in the biosynthesis of lignin and lignans and are involved in various processes such as plant growth, development, and stress responses. However, there is less information about VvDIR proteins in grapevine (Vitis vinifera L). RESULTS: In this study, we used bioinformatics methods to identify members of the DIR gene family in grapevine and identified 18 VvDIR genes in grapevine. These genes were classified into 5 subfamilies based on phylogenetic analysis. In promoter analysis, various plant hormones, stress, and light-responsive cis-elements were detected. Expression profiling of all genes following Colletotrichum gloeosporioides infection and phytohormones (salicylic acid (SA) and jasmonic acid (JA)) application suggested significant upregulation of 17 and 6 VvDIR genes, respectively. Further, we overexpressed the VvDIR4 gene in Arabidopsis thaliana and grapes for functional analysis. Ectopic expression of VvDIR4 in A. thaliana and transient expression in grapes increased resistance against C. gloeosporioides and C. higginsianum, respectively. Phenotypic observations showed small disease lesions in transgenic plants. Further, the expression patterns of genes having presumed roles in SA and JA signaling pathways were also influenced. Lignin contents were measured before and after C. higginsianum infection; the transgenic A. thaliana lines showed higher lignin content than wild-type, and a significant increase was observed after C. higginsianum infection. CONCLUSIONS: Based on the findings, we surmise that VvDIR4 is involved in hormonal and lignin synthesis pathways which regulate resistance against anthracnose. Our study provides novel insights into the function of VvDIR genes and new candidate genes for grapevine disease resistance breeding programs.

The transcription factor PbrbZIP52 positively affects pear pollen tube longevity by promoting callose synthesis.

In pear (Pyrus bretschneideri), pollen tube growth is critical for the double fertilization associated with seed setting, which in turn affects fruit yield. The normal deposition of callose mediates the polar growth of pollen tubes. However, the mechanism regulating callose synthesis in pollen tubes remains relatively uncharacterized. In this study, we revealed that the typical pear pollen tube lifecycle has a semi-growth duration (GD50) of 16.16 h under in vitro culture conditions. Moreover, callose plugs were deposited throughout the pollen tube lifecycle. The formation of callose plugs was inhibited by 2-deoxy-D-glucose, which also accelerated the senescence of pear pollen tubes. Additionally, PbrCalS1B.1, which encodes a plasma membrane-localized callose synthase, was expressed specifically in pollen tubes and restored the fertility of the Arabidopsis (Arabidopsis thaliana) cals5 mutant, in which callose synthesis is inhibited. However, this restoration of fertility was impaired by the transient silencing of PbrCalS1B.1, which restricts callose plug formation and shortens the pear pollen tube lifecycle. More specifically, PbrbZIP52 regulated PbrCalS1B.1 transcription by binding to promoter A-box elements to maintain the periodic formation of callose plugs and normal pollen tube growth, ultimately leading to double fertilization. This study confirmed that PbrbZIP52 positively affects pear pollen tube longevity by promoting callose synthesis. This finding may be useful for breeding high-yielding pear cultivars and stabilizing fruit setting in commercial orchards.

Theophylline Extracted from Fu Brick Tea Affects the Metabolism of Preadipocytes and Body Fat in Mice as a Pancreatic Lipase Inhibitor.

The dramatic increase in obesity is putting people under increasing pressure. Lipase inhibitors, as a kind of effective anti-obesity drug, have attracted more and more researchers' attention in recent years because of their advantages of acting on the intestinal tract and having no side effects on the central nervous system. In this study, lipase inhibitor Fu Brick Theophylline (FBT) was screened based on enzyme molecular dynamics, and the inhibition mechanism of lipase inhibitors on obesity was analyzed and discussed at the cellular level and animal model level. We found that FBT had high inhibition effects of lipase with an IC50 of 1.02~0.03 µg/mL. Firstly, the laboratory used 3T3-L1 proadipocytes as models, flow cytometry was used to detect the effects of FBT on the cycle, apoptosis and intracellular ROS activity of proadipocytes. To study the contents of triglyceride, total cholesterol, related metabolites and related gene and protein expression in adipocytes. The results showed that FBT could reduce ROS production and inflammatory factor mRNA expression during cell differentiation. Secondly, by establishing the animal model of high-fat feed ob nutritional obese mice, the morphological observation and gene expression analysis of body weight, fat rate, adipocyte and hepatocyte metabolism of FBT obese mice were further discussed. It was proven that FBT can effectively reduce the degree of fatty liver, prevent liver fibrosis and fat accumulation, and improve the damage of mitochondrial membrane structure. This study provides a theoretical basis for the screening and clinical treatment of lipase inhibitors.

SMRT and Illumina RNA-Seq Identifies Potential Candidate Genes Related to the Double Flower Phenotype and Unveils SsAP2 as a Key Regulator of the Double-Flower Trait in Sagittaria sagittifolia.

Double flowers are one of the important objectives of ornamental plant breeding. Sagittaria sagittifolia is an aquatic herb in the Alismataceae family that is widely used as an ornamental plant in gardens. However, the reference genome has not been published, and the molecular regulatory mechanism of flower formation remains unclear. In this study, single molecule real-time (SMRT) sequencing technology combined with Illumina RNA-Seq was used to perform a more comprehensive analysis of S. sagittifolia for the first time. We obtained high-quality full-length transcripts, including 53,422 complete open reading frames, and identified 5980 transcription factors that belonged to 67 families, with many MADS-box genes involved in flower formation being obtained. The transcription factors regulated by plant hormone signals played an important role in the development of double flowers. We also identified an AP2 orthologous gene, SsAP2, with a deletion of the binding site for miR172, that overexpressed SsAP2 in S. sagittifolia and exhibited a delayed flowering time and an increased number of petals. This study is the first report of a full-length transcriptome of S. sagittifolia. These reference transcripts will be valuable resources for the analysis of gene structures and sequences, which provide a theoretical basis for the molecular regulatory mechanism governing the formation of double flowers.

Genome-wide identification of ubiquitin proteasome subunits as superior reference genes for transcript normalization during receptacle development in strawberry cultivars.

BACKGROUND: Gene transcripts that show invariant abundance during development are ideal as reference genes (RGs) for accurate gene expression analyses, such as RNA blot analysis and reverse transcription-quantitative real time PCR (RT-qPCR) analyses. In a genome-wide analysis, we selected three "Commonly used" housekeeping genes (HKGs), fifteen "Traditional" HKGs, and nine novel genes as candidate RGs based on 80 publicly available transcriptome libraries that include data for receptacle development in eight strawberry cultivars. RESULTS: The results of the multifaceted assessment consistently revealed that expression of the novel RGs showed greater stability compared with that of the "Commonly used" and "Traditional" HKGs in transcriptome and RT-qPCR analyses. Notably, the majority of stably expressed genes were associated with the ubiquitin proteasome system. Among these, two 26 s proteasome subunits, RPT6A and RPN5A, showed superior expression stability and abundance, and are recommended as the optimal RGs combination for normalization of gene expression during strawberry receptacle development. CONCLUSION: These findings provide additional useful and reliable RGs as resources for the accurate study of gene expression during receptacle development in strawberry cultivars.

Logical modeling of thymus and natural killer lymphocyte differentiation.

Thymus (T) and natural killer (NK) lymphocytes are important barriers against diseases. Therefore, it is necessary to understand regulatory mechanisms related to the cell fate decisions involved in the production of these cells. Although some individual information related to T and NK lymphocyte cell fate decisions have been revealed, the related network and its dynamical characteristics still have not been well understood. By integrating individual information and comparing with experimental data, we construct a comprehensive regulatory network and a logical model related to T and NK lymphocyte differentiation. We aim to explore possible mechanisms of how each lineage differentiation is realized by systematically screening individual perturbations. When determining the perturbation strategies, the state transition can be used to identify the roles of specific genes in cell type selection and reprogramming. In agreement with experimental observations, the dynamics of the model correctly restates the cell differentiation processes from common lymphoid progenitors to CD4+ T cells, CD8+ T cells, and NK cells. Our analysis reveals that some specific perturbations can give rise to directional cell differentiation or reprogramming. We test our in silico results by using known experimental observations. The integrated network and the logical model presented here might be a good candidate for providing qualitative mechanisms of cell fate specification involved in T and NK lymphocyte cell fate decisions.

Identification and Characterization of Glucosyltransferase That Forms 1-Galloyl-ß-d-Glucogallin in Canarium album L., a Functional Fruit Rich in Hydrolysable Tannins.

Hydrolysable tannins (HTs) are useful secondary metabolites that are responsible for pharmacological activities and astringent taste, flavor, and quality in fruits. They are also the main polyphenols in Canarium album L. (Chinese olive) fruit, an interesting and functional fruit that has been cultivated for over 2000 years. The HT content of C. album fruit was 2.3-13 times higher than that of berries with a higher content of HT. 1-galloyl-ß-d-glucose (ßG) is the first intermediate and the key metabolite in the HT biosynthesis pathway. It is catalyzed by UDP-glucosyltransferases (UGTs), which are responsible for the glycosylation of gallic acid (GA) to form ßG. Here, we first reported 140 UGTs in C. album. Phylogenetic analysis clustered them into 14 phylogenetic groups (A, B, D-M, P, and Q), which are different from the 14 typical major groups (A~N) of Arabidopsis thaliana. Expression pattern and correlation analysis showed that UGT84A77 (Isoform0117852) was highly expressed and had a positive correlation with GA and ßG content. Prokaryotic expression showed that UGT84A77 could catalyze GA to form ßG. These results provide a theoretical basis on UGTs in C. album, which will be helpful for further functional research and availability on HTs and polyphenols.

RhoA/ROCK pathway mediates the effect of oestrogen on regulating epithelial-mesenchymal transition and proliferation in endometriosis.

Endometriosis is a benign gynaecological disease appearing with pelvic pain, rising dysmenorrhoea and infertility seriously impacting on 10% of reproductive-age females. This research attempts to demonstrate the function and molecular mechanism of RhoA/ROCK pathway on epithelial-mesenchymal transition (EMT) and proliferation in endometriosis. The expression of Rho family was abnormally changed in endometriotic lesions; in particular, RhoA and ROCK1/2 were significantly elevated. Overexpression of RhoA in human eutopic endometrial epithelial cells (eutopic EECs) enhanced the cell mobility, epithelial-mesenchymal transition (EMT) and proliferation, and RhoA knockdown exhibited the opposite function. Oestrogen up-regulated the RhoA activity and expression of RhoA and ROCK1/2. RhoA overexpression reinforced the effect of oestrogen on promoting EMT and proliferation, and RhoA knockdown impaired the effect of oestrogen. oestrogen receptor α (ERα) was involved with the regulation of oestrogen on EMT and proliferation and up-regulated RhoA activity and expression of RhoA and ROCK1/2. The function of ERα was modulated by the change in RhoA expression. Furthermore, phosphorylated ERK that was enhanced by oestrogen and ERα promoted the protein expression of RhoA/ROCK pathway. Endometriosis mouse model revealed that oestrogen enhanced the size and weight of endometriotic lesions. The expression of RhoA and phosphorylated ERK in mouse endometriotic lesions was significantly elevated by oestrogen. We conclude that abnormal activated RhoA/ROCK pathway in endometriosis is responsible for the function of oestrogen/ERα/ERK signalling, which promoted EMT and proliferation and resulted in the development of endometriosis.

Co-expression network analysis uncovers key candidate genes related to the regulation of volatile esters accumulation in Woodland strawberry.

MAIN CONCLUSION: FveERF (FvH4_5g04470.1), FveAP2 (FvH4_1g16370.1) and FveWRKY (FvH4_6g42870.1) might be involved in fruit maturation of strawberry. Overexpression of FveERF could activate the expression of AAT gene and ester accumulation. Volatile esters play an important role in the aroma of strawberry fruits, whose flavor is the result of a complex mixture of various esters. The accumulation of these volatiles is closely tied to changes in metabolism during fruit ripening. Acyltransferase (AAT) is recognized as having a significant effect in ester formation. However, there is little knowledge about the regulation network of AAT. Here, we collected the data of RNA-seq and headspace GC-MS at five time points during fruit maturation of Hawaii4 and Ruegen strawberry varieties. A total of 106 volatile compounds were identified in the fruit of woodland strawberries, including 58 esters, which occupied 41.09% (Hawaii4) or 33.40% (Ruegen) of total volatile concentration. Transcriptome analysis revealed eight transcription factors highly associated with AAT genes. Through the changes in esters and the weight co-expression network analysis (WGCNA), a detailed gene network was established. This demonstrated that ERF gene (FvH4_5g04470.1), AP2 gene (FvH4_1g16370.1) and one WRKY gene (FvH4_6g42870.1) might be involved in expression of AAT genes, especially ERF genes. Overexpression of FveERF (FvH4_5g04470.1) does activate expression of AAT genes and ester accumulation in fruits of strawberry. Our findings provide valuable clues to gain better insight into the ester formation process of numerous fruits.

Establishment of an immortalized stromal cell line derived from human Endometriotic lesion.

BACKGROUND: Endometriosis is a benign gynecological disease with obviously feature of estrogen-dependence and inflammatory response. The applications of primary endometriotic stromal cells in research of endometriosis are restricted for short life span, dedifferentiation of hormone and cytokine responsiveness. The objective of this study was to establish and characterize immortalized human endometriotic stromal cells (ihESCs). METHODS: The endometriotic samples were from a patient with ovarian endometriosis and the primary endometriotic stromal cells were isolated from the endometriotic tissues. The primary cells were infected by lentivirus to establish telomerase reverse transcriptase (hTERT)-induced immortalized cells. Quantification of mRNA and proteins was examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western Blot. CCK-8 assay and EdU labeling assay were assigned to assess the growth of ihESCs. Karyotype assay was performed to detect the chromosomes of ihESCs. Colony formation assay and nude mouse tumorigenicity assay were used to evaluate colony-formation and tumorigenesis abilities. RESULTS: ihESCs continuously overexpressed hTERT via infection of lentivirus and significant extended the life span reaching 31 passages. The morphology, proliferation and karyotype of ihESCs remained unchanged. The expression of epithelial-mesenchymal transition (EMT) markers, estrogen-metabolizing proteins and estrogen/progesterone receptors (ERs and PRs) were unaltered. Furthermore, the treatment of estrogen increased the proliferation and EMT of ihESCs. Lipopolysaccharides (LPS) and IL-1ß remarkably induced inflammatory response. The clonogenesis ability of ihESCs was consistent with primary cells, which were much lower than Ishikawa cells. In addition, nude mouse tumorigenicity assay demonstrated that ihESCs were unable to trigger tumor formation. CONCLUSION: This study established and characterized an immortalized endometriotic stromal cell line that exhibited longer life span and kept the cellular morphology and physiological function as the primary cells. The immortalized cells remained normal feedback to estrogen and inflammatory response. Moreover, the immortalized cells were not available with tumorigenic ability. Therefore, ihESCs would be serviceable as in vitro cell tool to investigate the pathogenesis of endometriosis.

Strong inhibitory activities and action modes of lipopeptides on lipase.

Lipopeptides have been reported to exhibit anti-obesity effects. In this study, we obtained a Bacillus velezensis strain FJAT-52631 that could coproduce iturins, fengycins, and surfactins. Results showed that the FJAT-52631 crude lipopeptide, purified fengycin, iturin, and surfactin standards exhibited strong inhibition activities against lipase with dose-dependence manners (half maximal inhibitory concentration (IC50) = 0.011, 0.005, 0.056, and 0.005 mg/mL, respectively). Moreover, fengycin and surfactin had the comparable activities with orlistat, but iturin not. It was revealed that the inhibition mechanism and type of the lipopeptides were reversible and competitive. The quenching mechanism of lipase was static and only one binding site between lipase and lipopoeptide was inferred from the fluorescence analysis. The docking analysis displayed that fengycin and surfactin could directly interact with the active amino acid residues (Ser or Asp) of lipase, but not with iturin. Our work suggests that the B. velezensis lipopeptides would have great potential to act as lipase inhibitors.

Exposure of helices α4 and α5 is required for insecticidal activity of Cry2Ab by promoting assembly of a prepore oligomeric structure.

Cry2Ab, a pore-forming toxin derived from Bacillus thuringiensis, is widely used as a bio-insecticide to control lepidopteran pests around the world. A previous study revealed that proteolytic activation of Cry2Ab by Plutella xylostella midgut juice was essential for its insecticidal activity against P. xylostella, although the exact molecular mechanism remained unknown. Here, we demonstrated for the first time that proteolysis of Cry2Ab uncovered an active region (the helices α4 and α5 in Domain I), which was required for the mode of action of Cry2Ab. Either the masking or the removal of helices α4 and α5 mediated the pesticidal activity of Cry2Ab. The exposure of helices α4 and α5 did not facilitate the binding of Cry2Ab to P. xylostella midgut receptors but did induce Cry2Ab monomer to aggregate and assemble a 250-kDa prepore oligomer. Site-directed mutagenesis assay was performed to generate Cry2Ab mutants site directed on the helices α4 and α5, and bioassays suggested that some Cry2Ab variants that could not form oligomers had significantly lowered their toxicities against P. xylostella. Taken together, our data highlight the importance of helices α4 and α5 in the mode of action of Cry2Ab and could lead to more detailed studies on the insecticidal activity of Cry2Ab.

Kinetic and computational molecular docking simulation study of novel kojic acid derivatives as anti-tyrosinase and antioxidant agents.

The novel kojic acid derivatives KAD1 and KAD2 have been demonstrated that they exhibited potent anti-melanogenesis activity in our previous report. In this study, we further study the inhibitory mechanism on mushroom tyrosinase. The inhibitory types of both KADs on diphenolase were classified as mixed type based on the results of the kinetic model. The interaction between KADs and tyrosinase was illustrated by fluorescence quenching, molecular docking and copper chelate activity. The KADs were also evaluated with respect to their antioxidant activities by DPPH and ABTS+ assays. The results showed that KADs have more potent antioxidant activities than kojic acid. Our study could provide new ideas for the development of new anti-tyrosinase and antioxidant agents.

Retinoic Acid Receptor α Knockdown Suppresses the Tumorigenicity of Esophageal Carcinoma via Wnt/ß-catenin Pathway.

BACKGROUND: Aberrant expression of retinoic acid receptor α (RARα) was correlated with diverse carcinomas such as acute promyelocytic leukemia and colorectal carcinoma. Nevertheless, the function and mechanism of RARα in esophageal carcinoma (EC) remain unclear. AIM: To investigate the expression of RARα in EC and its effect in the tumorigenesis of EC. METHODS AND RESULTS: In immunohistochemistry study, RARα was overexpressed in human EC tissues, and its overexpression was closely related to the pathological differentiation, lymph node metastasis, and clinical stages in EC patients. Functionally, RARα knockdown suppressed the proliferation and metastasis of EC cells through downregulating the expression of PCNA, Ki67, MMP7, and MMP9, as well as enhanced drug susceptibility of EC cells to 5-fluorouracil and cisplatin. Mechanistically, RARα knockdown inhibited the activity of Wnt/ß-catenin pathway through reducing the phosphorylation level of GSK3ß at Ser-9 and inducing phosphorylation level at Tyr-216, which resulted in downregulation of its downstream targets such as MMP7, MMP9, and P-gP. CONCLUSIONS: Our results demonstrated that RARα knockdown suppressed the tumorigenicity of EC via Wnt/ß-catenin pathway. RARα might be a potential molecular target for EC clinical therapy.

Crocodile choline from Crocodylus siamensis induces apoptosis of human gastric cancer.

Crocodile choline, an active compound isolated from Crocodylus siamensis, was found to exert potent anti-cancer activities against human gastric cancer cells in vitro and in vivo. Our study revealed that crocodile choline led to cell cycle arrest at the G2/M phase through attenuating the expressions of cyclins, Cyclin B1, and CDK-1. Furthermore, crocodile choline accelerated apoptosis through the mitochondrial apoptotic pathway with the decrease in mitochondrial membrane potential, the increase in reactive oxygen species production and Bax/Bcl-2 ratio, and the activation of caspase-3 along with the release of cytochrome c. In addition, this study, for the first time, shows that Notch pathway is remarkably deregulated by crocodile choline. The combination of crocodile choline and Notch1 short interfering RNA led to dramatically increased cytotoxicity than observed with either agent alone. Notch1 short interfering RNA sensitized and potentiated the capability of crocodile choline to suppress the cell progression and invasion of gastric cancer. Taken together, these data suggested that crocodile choline was a potent progression inhibitor of gastric cancer cells, which was correlated with mitochondrial apoptotic pathway and Notch pathway. Combining Notch1 inhibitors with crocodile choline might represent a novel approach for gastric cancer.

Effect of interleukin-1ß and lipoxin A4 in human endometriotic stromal cells: Proteomic analysis.

AIM: Lipoxin A4 (LXA4 ) can function as an endogenous 'breaking signal' in inflammation and plays an important role in the progression of endometriosis. The proteome responses to interleukin-1ß (IL-1ß) or LXA4 in human endometriotic stromal cells (ESC) are not well understood. METHODS: In this study, primary ESC were cultured from ovarian endometriosis tissue. Three groups were established: the control group; the IL-1ß stimulation group; and the IL-1ß and LXA4 incubation group. Proteins were assessed on 2-D polyacrylamide gel electrophoresis (2D-PAGE), and differentially expressed protein spots were further identified on matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MALDI-TOF-MS). Wound healing and transwell assays were performed to assess the migration and invasion of ESC after treatment. RESULTS: In total, 40 differentially expressed protein spots were identified successfully on MALDI-TOF-MS. The proteins identified were related to cell structure, metabolism, signal transduction, protein synthesis and membrane structure, processes that may be involved in the development of endometriosis. Vinculin and IL-4 were further analyzed on western blot and quantitative real-time polymerase chain reaction. Moreover, LXA4 could suppress the migration and invasion of ESC induced by IL-1ß. CONCLUSION: LXA4 may inhibit the progression of endometriosis partly by lowering or raising the effect of IL-1ß, mediated via some inflammation-related proteins (e.g. vinculin) and immune response-related protein (e.g. IL-4) in vitro.

Retinoid X receptor α enhances human cholangiocarcinoma growth through simultaneous activation of Wnt/ß-catenin and nuclear factor-κB pathways.

Retinoid X receptor α (RXRα) plays important roles in the malignancy of several cancers such as human prostate tumor, breast cancer, and thyroid tumor. However, its exact functions and molecular mechanisms in cholangiocarcinoma (CCA), a chemoresistant carcinoma with poor prognosis, remain unclear. In this study we found that RXRα was frequently overexpressed in human CCA tissues and CCA cell lines. Downregulation of RXRα led to decreased expression of mitosis-promoting factors including cyclin D1and cyclin E, and the proliferating cell nuclear antigen, as well as increased expression of cell cycle inhibitor p21, resulting in inhibition of CCA cell proliferation. Furthermore, RXRα knockdown attenuated the expression of cyclin D1 through suppression of Wnt/ß-catenin signaling. Retinoid X receptor α upregulated proliferating cell nuclear antigen expression through nuclear factor-κB (NF-κB) pathways, paralleled with downregulation of p21. Thus, the Wnt/ß-catenin and NF-κB pathways account for the inhibition of CCA cell growth induced by RXRα downregulation. Retinoid X receptor α plays an important role in proliferation of CCA through simultaneous activation of Wnt/ß-catenin and NF-κB pathways, indicating that RXRα might serve as a potential molecular target for CCA treatment.

Inactivation kinetics of formaldehyde on N-acetyl-ß-D-glucosaminidase from Nile tilapia (Oreochromis niloticus).

Formaldehyde is a widely used sanitizer in aquaculture in China, while the appropriate concentration is not available to be used effectively and without damage to tilapia much less to its reproductive function. N-acetyl-ß-D-glucosaminidase (EC 3.2.1.52, NAGase), hydrolyzing the oligomers of N-acetyl-ß-D-glucosamine into monomer, is proved to be correlated with reproduction of male animals. In this paper, NAGase from spermary of tilapia was chosen as the material to study the effects of formaldehyde on its activity in order to further investigate the effects of formaldehyde use on tilapia reproduction. The results showed the relationship between the residual enzyme activity and the concentration of formaldehyde was concentration dependent, and the IC50 value was estimated to be 3.2 ± 0.1 %. Appropriate concentration of formaldehyde leaded to competitive reversible inhibition on tilapia NAGase. Moreover, formaldehyde could reduce the thermal and pH stability of the enzyme. The inactivation kinetics of formaldehyde on the enzyme was studied using the kinetic method of substrate reaction. The inactivation model was setup, and the rate constants were determined. The results showed that the inactivation of formaldehyde on tilapia NAGase was a slow, reversible reaction with partially residual activity. The results will give some basis to determine the concentration of formaldehyde used in tilapia culture.

Characterization of a new cry2Ab gene of Bacillus thuringiensis with high insecticidal activity against Plutella xylostella L.

Bacillus thuringiensis (Bt) strain FJAT-12 was a novel Bt strain isolated by Agricultural Bio-Resources Institute, Fujian Academy of Agricultural Science. In this study, a new cry2Ab gene was cloned from Bt strain FJAT-12 and named as cry2Ab30 by Bt delta-endotoxin Nomenclature Committee. The sequencing results showed there were two mutations in conservative sites which led to two amino acids modification. Homology modeling indicated that the two changes were located in ß-sheet of Domain II. A prokaryotic expression vector pET30a-cry2Ab30 was constructed and the expressed protein was analyzed by western blot using Cry2Ab antibody. The expression conditions including IPTG concentration, revolution and temperature were optimized to get the highest expression level by SDS-PAGE and BandScan. The bioassay results also showed that the Cry2Ab30 toxin had high insecticidal activity against Plutella xylostella and the LC50 value was 0.0103 µg.mL(-1). The two mutations in ß-sheet of Domain II might contribute to insecticidal activity of Cry2Ab30 toxin against Plutella xylostella.

Identification of MYB Transcription Factor, a Regulator Related to Hydrolysable Tannin Synthesis in Canarium album L., and Functional Analysis of CaMYBR04.

Hydrolysed tannins (HTs) are polyphenols, which are related to the astringency, flavour, colour, stability, medicinal value and other characteristics of many fruits and vegetables. The biosynthetic mechanism of the majority of HTs remains unknown, and many biosynthetic pathways of HTs are speculative conclusions that have not been confirmed. The fruit of Canarium album L. (Chinese olive), which is notable for its pharmacological and edible properties, is rich in HTs. The fruit has a distinctive bitter and astringent taste when initially consumed, which mellows to a sweet sensation upon chewing. HTs serve as the primary material basis for the formation of the Chinese olive fruit's astringent quality and pharmacological effects. In this study, the fruit of C. album Changying was utilised as the research material. The objective of this study was to provide a theoretical basis for the quality control of Chinese olive fruit and the application and development of its medicinal value. In addition, the study aimed to identify and screen related MYB transcription factors involved in the synthesis of HTs in the fruit and to clarify the mechanism of MYBs in the process of synthesis and regulation of HTs in Chinese olive fruit. The principal findings were as follows. A total of 83 differentially expressed Chinese olive MYB transcription factors (CaMYBs) were identified, including 54 1R-MYBs (MYB-related), 25 2R-MYBs (R2R3-MYBs), 3 3R-MYBs, and 1 4R-MYB. Through trend analysis and correlation analysis, it was found that CaMYBR04 (Isoform0032534) exhibited a significantly higher expression (FPKM) than the other CaMYBs. The full-length cDNA sequence of CaMYBR04 was cloned and transformed into strawberry. The results demonstrated that CaMYBR04 significantly enhanced the fruit's hydrolysable tannin content. Consequently, this study elucidated the function of CaMYBR04, a regulator of the Chinese olive fruit hydrolysable tannin synthesis pathway, and established a theoretical foundation for the synthesis and regulation of fruit HTs.