Molecular cloning and characterization of a thermostable and halotolerant endo-β-1,4-glucanase from Microbulbifer sp. ALW1

The bacterium Microbulbifer sp. ALW1 was previously characterized with the capability to break down the cell wall of brown algae into fine pieces. The biological functions of strain ALW1 were yet to be elucidated. In this study, a gene, namely MaCel5A, was isolated from the ALW1 strain genome, encoding an endo-β-1,4-glucanase. MaCel5A was phylogenetically categorized under the glycoside hydrolase family GH5, with the highest identity to a putative cellulase of Microbulbifer thermotolerans. The recombinant MaCel5A protein purified from heterologous expression in E. coli exhibited maximum activity at 50 °C and pH 6.0, respectively, and functioned selectively toward carboxymethyl cellulose and barley β-glucan.

Recombinant MaCel5A demonstrated considerable tolerance to the exposure to high temperature up to 80 °C for 30 min retaining 49% residual activity. In addition, MaCel5A showed moderate stability against pH 5.0-11.0 and strong stability in the presence of nonionic surfactant. MaCel5A exhibited strong halo-stability and halotolerance. The activity of the enzyme increased about tenfold at 0.5 M NaCl, and about fivefold even at 4.0 M NaCl compared to the enzyme activity without the addition of salt. The two conserved glutamic acid residues in MaCel5A featured the typical catalytic acid/base and nucleophile machinery of glycoside hydrolases. These characteristics highlight the industrial application potential of MaCel5A.

Cloning and physical localization of male-biased repetitive DNA sequences in Spinacia oleracea (Amaranthaceae)

Spinach (Spinacia oleracea Linnaeus, 1753) is an ideal material for studying molecular mechanisms of early-stage sex chromosome evolution in dioecious plants. Degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) technique facilitates the retrotransposon-relevant studies by enriching specific repetitive DNA sequences from a micro-dissected single chromosome. We conducted genomic subtractive hybridization to screen sex-biased DNA sequences by using the DOP-PCR amplification products of micro-dissected spinach Y chromosome.

The screening yielded 55 male-biased DNA sequences with 30 576 bp in length, of which, 32 DNA sequences (12 049 bp) contained repeat DNA sequences, including LTR/Copia, LTR/Gypsy, simple repeats, and DNA/CMC-EnSpm. Among these repetitive DNA sequences, four DNA sequences that contained a fragment of Ty3-gypsy retrotransposons (SP73, SP75, SP76, and SP77) were selected as fluorescence probes to hybridization on male and female spinach karyotypes. Fluorescence in situ hybridization (FISH) signals of SP73 and SP75 were captured mostly on the centromeres and their surrounding area for each homolog. Hybridization signals primarily appeared near the putative centromeres for each homologous chromosome pair by using SP76 and SP77 probes for FISH, and sporadic signals existed on the long arms. Results can be served as a basis to study the function of repetitive DNA sequences in sex chromosome evolution in spinach.

Molecular cloning and functional characterization of TaIRI9 gene in wheat (Triticum aestivum L.)

The vernalization of wheat is one of the important factors that determine the planting region, introduction and cultivation techniques of wheat. However, the known vernalization genes (molecular marker) cannot precisely distinguish the vernalization requirement of winter wheat cultivars. Therefore, it is important to explore new vernalization genes and elucidate the mechanism of vernalization regulation. To explore the gene network in the vernalization pathway, we screened TaIRI9 (ice recrystallization inhibitor protein) gene associated with the expression profile of vernalization treatment of winter wheat Jing 841. Overexpression of TaIRI9 in wild type wheat resulted in reduced plant height, increased tiller number and delayed heading days.

After 4°C vernalization treatment for 30, 35, 45 or 50 days, TaIRI9 overexpression lines showed increased vernalization requirement and delayed heading time than wild type, indicating that TaIRI9 may affect vernalization process of wheat. In addition, the expression of the TaIRI9 genes were analyzed in winter Jing 841, strong winter wheat cultivar Xindong 18 and ten recombinant inbred lines (RILs, Hussar x Yanzhan1). The data showed that the expression of TaIRI9 was positively associated with the requirement of vernalization. These results indicated that TaIRI9 regulates heading and flowering time in wheat by promoting VRN2 and inhibiting flowering promoter VRN1 and VRN3 and may be involved in wheat vernalization regulation pathway. Bulked segregant CGT-Seq-facilitated map-based cloning of a powdery mildew resistance gene originating from wild emmer wheat (Triticum dicoccoides)

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a widely occurring foliar diseases of wheat worldwide. Wild emmer wheat (WEW, Triticum dicoccoides) (AABB, 2n=4x=28), the progenitor of the cultivated tetraploid and hexaploid wheat, is highly resistant to powdery mildew and many resistance alleles were identified in this wild species.

Cloning and characterization of a novel DNase gene from Trichogramma pretiosum

DNase is a powerful tool for a series of molecular biology applications. Developing a strategy for large-scale production of DNase with high purity and activity is critical for scientific research. In this study, a previously uncharacterized gene with nuclease activity was found in Trichogramma pretiosum genome. Pichia pastoris GS115 was preferred as the host to overcome the issues related to prokaryotic expression. Under the optimal conditions, the activity of T. pretiosum DNase (Tp-DNase) reached 1940 U/mL of culture supernatant in fed-batch fermentation. Using ion-exchange chromatography and adsorption chromatography, Tp-DNase was produced with a purity of > 99% and molecular weight of 45 kDa.

In vitro DNA degradation experiments showed that Tp-DNase could effectively degrade dsDNA, and its activity was slightly higher than that of bovine pancreas DNase I under the same conditions. Moreover, Tp-DNase can be used to eliminate nucleic acid contamination and improve the accuracy of nucleic acid detection.

Cloning, expression, and characterization of Baeyer-Villiger monooxygenases from eukaryotic Exophiala jeanselmei strain KUFI-6N

The fungus Exophiala jeanselmei strain KUFI-6N produces a unique cycloalkanone monooxygenase (ExCAMO) that displays an uncommon substrate spectrum of Baeyer-Villiger oxidation of 4-10-membered ring ketones. In this study, we aimed to identify and sequence the gene encoding ExCAMO from KUFI-6N and overexpress the gene in Escherichia coli. We found that the primary structure of ExCAMO is most closely related to the cycloalkanone monooxygenase from Cylindrocarpon radicicola ATCC 11011, with 54.2% amino acid identity. ExCAMO was functionally expressed in Escherichia coli and its substrate spectrum and kinetic parameters investigated.

Substrate profiling indicated that ExCAMO is unusual among known Baeyer-Villiger monooxygenases owing to its ability to accept a variety of substrates, including C4-C12 membered ring ketones. ExCAMO has high affinity and catalytic efficiency toward cycloalkanones, the highest being toward cyclohexanone. Five other genes encoding Baeyer-Villiger monooxygenases were also cloned and expressed in Escherichia coli.