Validation of Housekeeping Genes for Gene Expression Analysis in Iwagaki Oyster(Crassostrea nippona)Under Salinity Stress by Quantitative Real-Time PCR

Hypo-salinity can reduce the immunological reaction in Crassostrea nippona,even lead to massive mortality.It is important to understand the molecular mechanism of oyster defense system,while quantitative real-time PCR can be employed in the study.However,the accuracy of quantitative real-time PCR relies on the use of suitable reference genes.In this study,the expression stability of 14 candidate reference genes including traditional housekeeping genes EF1A,TUB,TUA,GAPDH,RO21,as well as new candidate reference genes RPL5,RPL8,RPS27,RPL14,RPL4,CO3,RPS8,RPS4,CYTB in different tissues of C.nippona under salinity stress has been validated by quantitative real-time PCR.Ribosomal protein genes selected through expression analysis of transcriptome data from C.nippona generally were more stable than traditional reference genes.According to the geNorm analysis,RPL4 and RPS4 could be used as internal controls for studying gene expression in C.nippona with real-time PCR under salinity stress.

Resistance identification of bivalent fungi-resistant genes transformed soybean to Phytophthora sojae

Soybean is one of the most important sources of edible oil and proteins in the world. However, it suffers from many kinds of fungal diseases which is a major limiting factor in soybean production. The fungal disease can be effectively controlled by breeding plant cultivars with genetic transformation. In this study, the resistance to Phytophthora sojae of five bivalent transgenic soybean lines was identified using the hypocotyls inoculation technique. The lines were the T2 of the transgenic soybean which were transformed with kidney bean chitinase gene and barley ribosome inactivating protein gene, and were positive by Southern Blot analysis. The resistance difference was studied through comparing the death percentage of transgenic soybean with the control. The results showed that four lines were more resistant to P sojae, whereas other one had no significant difference in comparison with the control. These transgenic soybean lines with enhanced resistance to P sojae will be useful in soybean resistance breeding.

Molecular characterization and m RNA expression of ribosomal protein L8 in Rana nigromaculata during development and under exposure to hormones

Like Xenopus laeuis, some species of the Rang genus are also used to study endocrine disrupting chemicals （EDCs）. Although ribosomal protein L8 （rp18） is the most-used reference gene for analyzing gene expression by quantitative reverse transcription polymerase chain reaction in Rang, its suitability as the reference gene has never been validated in any species of the Rana genus. We characterized rp18 cDNA in Rana nigromaculata, a promising native species in East Asia for assaying endocrine disrupting effects. We found that the rp18 cDNA consisted of 919 bp and encoded 257 amino acids, exhibiting high identities of amino acid sequence with known rp18 in other Rana species. Then, we examined the stability of mRNA expression during development. Compared with elongation factor 1 alpha 1, another common housekeeping gene, neither stage-specific nor tissue-specific expression of the rp18 gene was found in all tissues examined （brain, liver, intestine, tail, testis and ovary） during R. nigromaculata development. Finally, we investigated rp18 expression under exposure to hormones. No change in rp18 mRNA expression was found under exposure to thyroid hormone （T4） and estrogen （estradiol）, whereas expression of the corresponding biomarker genes was induced. Our results show that rp18 is an appropriate reference gene for analyzing gene expression by quantitative reverse transcription polymerase chain reaction for assaying EDCs using R. nigromaculata, and might also provide support for using rp18 as a reference gene in other Rang species due to the high conservation of rp18 among the Rana genus.

RIBOSOMAL PROTEIN S12 GENE MUTATION AFFECTING THE EXPRESSION OF BACTERIOPHAGE LAMBDA N GENE

Previous results in this laboratory indicated that in ribosomal protein S12 strepto-mycin-dependent mutants of Bacillus subtilis, the burst size of bacteriophage φ105 was decreased, and the protein synthesis was inhibited, while the DNA and