Yan Shui

In-depth transcriptome analysis of the red swamp crayfish Procambarus clarkii.

The red swamp crayfish Procambarus clarkii is a highly adaptable, tolerant, and fecund freshwater crayfish that inhabits a wide range of aquatic environments. It is an important crustacean model organism that is used in many research fields, including animal behavior, environmental stress and toxicity, and studies of viral infection. Despite its widespread use, knowledge of the crayfish genome is very limited and insufficient for meaningful research. This is the use of next-generation sequencing techniques to analyze the crayfish transcriptome. A total of 324.97 million raw reads of 100 base pairs were generated, and a total of 88,463 transcripts were assembled de novo using Trinity software, producing 55,278 non-redundant transcripts. Comparison of digital gene expression between four different tissues revealed differentially expressed genes, in which more overexpressed genes were found in the hepatopancreas than in other tissues, and more underexpressed genes were found in the testis and the ovary than in other tissues. Gene ontology (GO) and KEGG enrichment analysis of differentially expressed genes revealed that metabolite- and immune-related pathway genes were enriched in the hepatopancreas, and DNA replication-related pathway genes were enriched in the ovary and the testis, which is consistent with the important role of the hepatopancreas in metabolism, immunity, and the stress response, and with that of the ovary and the testis in reproduction. It was also found that 14 vitellogenin transcripts were highly expressed specifically in the hepatopancreas, and 6 transcripts were highly expressed specifically in the ovary, but no vitellogenin transcripts were highly expressed in both the hepatopancreas and the ovary. These results provide new insight into the role of vitellogenin in crustaceans. In addition, 243,764 SNP sites and 43,205 microsatellite sequences were identified in the sequencing data. We believe that our results provide an important genome resource for the crayfish.

Molecular characterization of cyclin B and its expression profile during oogenesis in the red swamp crayfish, Procambarus clarkii (Girard, 1852) (Decapoda, Astacidea)

The maturation promoting factor (MPF), a complex of CDC2 (CDK1) and cyclin B, is a key regulator of controlling the G2/M phase transition in the meiotic maturation of the oocyte in multi-cellular organisms. In this study, full-length cDNA of cyclin B (Pc-cyclin B) from the red swamp crayfish, Procambarus clarkii , was cloned using the degenerate RT-PCR and RACE methods. The cDNA of Pc-cyclin B is 2595 bp in length and encoding a protein of 402 amino acids, with a calculated molecular mass of 45.75 kDa. Six potential cytoplasmic polyadenylation elements (CPE) as well as one signal sequences (AATAAA) were found in the 3′-UTR location. Semi-quantitative RT-PCR analysis revealed that the amount of cyclin B mRNA was highest in the ovary, followed by the heart ( P < 0 . 05 ); and also significantly higher in the pre-vitellogenesis (pre-Vt) and primary-vitellogenesis (pVt) stages, while low in the tertiary-vitellogenesis and GVBD stages ( P < 0 . 05 ), suggesting that differential expression of Pc-cyclin B is closely related to oogonial proliferation (mitosis) and oocyte meiotic maturation in this species of crayfish.

Establishment of a markerless multiple-gene deletion method based on Cre/loxP mutant system for Bacillus pumilus

In this study, we established a Cre/loxP mutant recombination system (Cre/lox71‐66 system) for markerless gene deletion to facilitate our follow‐up rational genetic engineering to the strain Bacillus pumilus W3. This modified method uses two mutant loxP sites, which after recombination creates a double‐mutant loxP site that is poorly recognized by Cre recombinase, facilitating multiple gene deletions in a single genetic background. Two selected genes, cotA and sigF, were continuously knocked out and verified at different levels using this method. This method is simple and efficient and can be easily implemented for multiple gene deletions in B. pumilus.

The hepatopancreas and ovary are the sites of vitellogenin synthesis in female red swamp crayfish ( Procambarus clarkii (Girard, 1852)) (Decapoda: Astacoidea: Cambaridae)

Vitellogenin (Vg) is the precursor of vitellin, the major yolk protein. The site of Vg synthesis in crustaceans remains controversial. This study investigated the synthesis site of Vg in female red swamp crayfish Procambarus clarkii (Girard, 1852). Results revealed that the mRNA of Vg was detected in the tissues of hepatopancreas of P. clarkii . The mRNA expression level of Vg increased in the previtellogenic stage and peaked in the vitellogenic stage in the hepatopancreas and ovary; the mRNA expression level of Vg in the hepatopancreas was approximately 20-25-fold of that of the ovary in the same ovarian stage. Eyestalk ablation could upregulate the mRNA expression level of Vg in both organs. Our RNAi experiment verified that the hepatopancreas and ovary are the Vg synthesis sites in female P. clarkii .