RESUMEN
The sucrose nonfermenting 1 (SNF1)-related protein kinase 2 (SnRK2) genes play central roles in plant stress signal transduction. In this study, 8 SnRK2 genes were identified from the tea plant genome database and named CsSnRK2.1-8. Phylogenetic analysis showed that the CsSnRK2 genes were classifiable into three groups, similar to those of Arabidopsis thaliana, Oryza sativa and maize. The coding sequences (CDSs) of all CsSnRK2s were separated by eight introns, and their exon-intron organizations exhibited high similarity to those of other plants. The fluorescence of GFP fused with CsSnRK2.3 was detected in only the cytoplasm, while the rest of the proteins showed GFP signal in both the nucleus and the cytoplasm. The results of the expression patterns of the CsSnRK2 genes showed that CsSnRK2s were differentially induced by salt, polyethylene glycol (PEG) and abscisic acid (ABA) stress. Interestingly, The expression of CsSnRK2.3 was inhibited by ABA, suggesting the complicated roles of CsSnRK2s in the ABA signal transduction pathway. Some CsSnRK2 gene pairs showed significant expression change correlations under stresses, indicating that CsSnRK2s might exhibit synergistic effects of signal regulation in response to various stresses. In summary, this comprehensive analysis will facilitate further studies of the SnRK2 family of Camellia sinensis and provide useful information for the functional validation of CsSnRK2s.
Asunto(s)
Camellia sinensis/enzimología , Camellia sinensis/genética , Genoma de Planta , Familia de Multigenes , Proteínas Serina-Treonina Quinasas/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia Conservada/genética , Exones/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Redes Reguladoras de Genes , Genes de Plantas , Intrones/genética , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Estrés Fisiológico/genética , Fracciones Subcelulares/metabolismoRESUMEN
Tea leaves contain abundant flavan-3-ols, which include dihydroxylated and trihydroxylated catechins. Flavonoid 3'-hydroxylase (F3'H: EC 1.14.13.21) is one of the enzymes in the establishment of the hydroxylation pattern. A gene encoding F3'H, designated as CsF3'H, was isolated from Camellia sinensis with a homology-based cloning technique and deposited in the GenBank (GenBank ID: KT180309). Bioinformatic analysis revealed that CsF3'H was highly homologous with the characterized F3'Hs from other plant species. Four conserved cytochrome P450-featured motifs and three F3'H-specific conserved motifs were discovered in the protein sequence of CsF3'H. Enzymatic analysis of the heterologously expressed CsF3'H in yeast demonstrated that tea F3'H catalyzed the 3'-hydroxylation of naringenin, dihydrokaempferol and kaempferol. Apparent Km values for these substrates were 17.08, 143.64 and 68.06 µM, and their apparent Vmax values were 0.98, 0.19 and 0.44 pM·min(-1), respectively. Transcription level of CsF3'H in the new shoots, during tea seed germination was measured, along with that of other key genes for flavonoid biosynthesis using real-time PCR technique. The changes in 3',4'-flavan-3-ols, 3',4',5'-flavan-3-ols and flavan-3-ols, were consistent with the expression level of CsF3'H and other related genes in the leaves. In the study of nitrogen supply for the tea plant growth, our results showed the expression level of CsF3'H and all other tested genes increased in response to nitrogen depletion after 12 days of treatment, in agreement with a corresponding increase in 3',4'-catechins, 3',4',5'-catechins and flavan 3-ols content in the leaves. All these results suggest the importance of CsF3'H in the biosynthesis of 3',4'-catechins, 3',4',5'-catechins and flavan 3-ols in tea leaves.
