Genome-wide identification and expression profiling of duplicated flavonoid 3'-hydroxylase gene family in Carthamus tinctorius L.

Flavonoid 3′-hydroxylase (F3’H) enzyme is essential in determining the flavonoids B-ring hydroxylation pattern. It is mainly implicated in the biosynthetic pathway of cyaniding-based anthocyanins, flavonols, and flavan-3-ols. However, the evolution and regulatory mechanism of these important flavonoid hydroxylases have not been systematically investigated in safflower (Carthamus tinctorius L.). In this study, we identified 22 duplicatedCtF3'H-encoding genes from safflower through genome-wide prediction and conservation analysis. Phylogenetic analysis revealed the pattern of conservation and divergence of CtF3'Hs encoding proteins and their homologs from different plant species. The distribution of conserved protein motifs and cis-regulatory units suggested several structural components that could be crucial in deciphering the final function of CtF3'H proteins. Furthermore, the results of RNA-seq and qRT-PCR assay in different flowering tissues suggested differential expression level of CtF3’H genes during flower development. Based on the unique homology of CtF3’H5 with flavonoid 3’ hydroxylases from other plant species, further validation of CtF3’H5 was carried out. The transient expression of CtF3’H5 in onion epidermal cells implied that the subcellular localization of the fusion construct containing CtF3’H5 and GFP was predominantly detected in the plasma membrane. Similarly, the prokaryotic expression and western blot hybridization of CtF3’H5 demonstrated the detection of a stable 50.3kD target protein. However, more efforts are needed to further extend the functional validation of CtF3’H5 in safflower. This study provides a fundamental gateway for future functional studies and understanding the genetic evolution of F3'Hs in plants.

The induction of the synthesis of interferon, CD4 and CD8 t lymphocytes in blood and spleen of mice after oral vaccination with the “early” protein HPV16 E2

Very high contents of the interferon, CD4 and CD8 T lymphocytes were found in blood and spleen cells after the oral vaccination of mice with the “early” protein of high-risk oncogenic papillomavirus HPV16 E2 by using immunoassays analyses: ELISPOT for the detection of the content of interferon, Western-blot hybridization and enzyme immunoassays for analyses of СD4 and CD8 T lymphocytes. Both blood cells and splenocytes showed high content of alive mononuclear cells during ELISA and ELISPOT analyses that had very characteristic features for T lymphocytes. The ratio of CD4/CD8 was very close to 1 after the quanttitive determination by the enzyme immunoassays with appropriate antibodies. It was concluded that the “early” protein HPV16 E2 participated in the activation of the immune system during pathogenesis and neoplasia.