Molecular characterization and expression analysis of the ß-actin gene from the ridgetail white prawn Exopalaemon carinicauda.

Actin is a highly conserved protein that is found in all eukaryotic cells, and has been widely used as an internal control gene in gene expression studies. In this study, we cloned an actin gene (named Ecß-actin) from Exopalaemon carinicauda and determined its expression levels. The full-length cDNA of Ecß-actin was 1335 bp long, comprising a 1131-bp ORF encoding 376 amino acids, a 65-bp 5'-UTR, and a 139-bp 3'-UTR with a poly(A) tail. The A + T content was approximately 79% in the 3'-UTR of the Ecß-actin mRNA. The 3'-UTR contained two repeats of the AUUUA motif. The putative protein Ecß-actin showed high identity (97-99%) with other actins from various species. Phylogenetic analysis revealed that Ecß-actin belongs to Crustacea, although it formed a singleton sub-branch that was located a short distance from crabs and other shrimp species. Ecß- actin expression was detected in the hepatopancreas, ovary, muscle, gill, stomach, and hemocytes, and was strongly expressed in the hemocytes and ovary of E. carinicauda. Ecß-actin mRNA expression varied during ovarian development, with high levels observed at stages I and V. Therefore, caution should be taken when using the Ecß-actin gene as an endogenous control gene.

Isolation, partial characterization, and cloning of an extracellular chitinase from the entomopathogenic fungus Verticillium lecanii.

The entomopathogenic fungus Verticillium lecanii is a well-known biocontrol agent of fungal phytopathogens, as well as insect pests. A 42-kDa chitinase belonging to family 18 of the glycosyl hydrolases was isolated and partially characterized. Chitinase was purified using successive column chromatography on phenyl-sepharose, DEAE-sepharose, and CM-sepharose. The enzyme showed the highest activity at 40°C and pH 4.6. Enzyme activity was strongly activated in the presence of Mg(2+). The purified enzyme showed inhibitory activity of spore germination against several plant pathogens, particularly Fusarium moniliforme. The genomic DNA and cDNA sequences were resolved by polymerase chain reaction amplification and sequencing. Protein modeling and comparative investigation of different chitinase amino acids showed that chitinases are conserved in parasitic fungi.

Modification of vectors for functional genomic analysis in plants.

Simple, efficient, and economical recombinant plant binary expression vectors for deciphering large-scale functional genomic research in plants and promoting crop improvement by genetically engineering and biotechnology is in great demand. In this research, using the pCHF3, pCAMBIA1301, pCAMBIA3300, pCAMBIA3301 vectors, we successfully constructed general plant binary expression vectors carrying CaMV35S and Arabidopsis rd29A promoters mediating multiple cloning sites ( MCS: SacI, KpnI, SmaI, BamHI, XbaI, SalI, and PstI). Meanwhile, a series of applicative binary expression vectors that can be utilized for subcellular localization were constructed by fusion of the MCS and eGFP. Subsequently, the recombinant vectors were successfully transferred into Arabidopsis thaliana and Nicotiana benthamiana for further investigation of functional elements in these plant binary expression vectors. Our results demonstrated that this system was a convenient and versatile vector system for phenotypic, functional, subcellular localization, and promoter activity analysis, and it provided a relatively high-efficiency and reliable platform for researchers in vector construction and may facilitate large-scale functional genomics analysis in plants.

Isolation and characterization of polymorphic microsatellite loci in the ridgetail white prawn Exopalaemon carinicauda.

The ridgetail white prawn Exopalaemon carinicauda is one of the major economic shrimp species cultured in China. In this study, 30 microsatellite loci were isolated and characterized for the ridgetail white prawn E. carinicauda using a microsatellite-enriched library. Polymorphisms were tested in 30 individuals from a single wild population. The number of alleles at each locus ranged from 2 to 14. The observed and expected heterozygosities varied from 0.1000 to 0.8000 and from 0.2299 to 0.9228, respectively. The PIC value ranged from 0.2002 to 0.8939. These new loci will be useful in the study of population genetic structure and genetic diversity in this species.

An optimized mini-preparation method to obtain high-quality genomic DNA from mature leaves of sunflower.

In order to investigate the mutation characteristics and to further examine the genetic variation of mutant sunflower (Helianthus annuus) obtained in plants grown from seeds exposed to space conditions, only the mature tissues such as leaf and flower could be used for DNA extraction after mutation characteristics were confirmed. The rich contents of polysaccharides, tannins, secondary metabolites, and polyphenolics made it difficult to isolate high-quality DNA from mature leaves of sunflower according to previous reports. Based on the comparison of the differences in previously reported protocols, a modified method for the extraction of high-quality DNA was developed. Using this protocol, the DNA isolated from sunflower was high in quality and suitable for restriction digestion (EcoRI, HindII, BamHI), random amplified polymorphic DNA study and further molecular research. Therefore, the modified protocol was suitable for investigating the genetic variation of sunflower using mature leaf DNA.

An optimized mini-preparation method toobtain high-quality genomic DNA from mature leaves of sunflower

In order to investigate the mutation characteristics and to further examine the genetic variation of mutant sunflower (Helianthus annuus) obtained in plants grown from seeds exposed to space conditions, only the mature tissues such as leaf and flower could be used for DNA extraction after mutation characteristics were confirmed. The rich contents of polysaccharides, tannins, secondary metabolites, and polyphenolics made it difficult to isolate high-quality DNA from mature leaves of sunflower according to previous reports. Based on the comparison of the differences in previously reported protocols, a modified method for the extraction of high-quality DNA was developed. Using this protocol, the DNA isolated from sunflower was high in quality and suitable for restriction digestion (EcoRI, HindII, BamHI), random amplified polymorphic DNA study and further molecular research. Therefore, the modified protocol was suitable for investigating the genetic variation of sunflower using mature leaf DNA.