Molecular Cloning Of 10 Transaminases And Functional Characterization Of 2 Alanine Transaminases In The White-Backed Planthopper, Sogatella Furcifera

Transaminases catalyze the reversible transfer of an amino group from an amino acid to an a-keto acid. The white-backed planthopper, Sogatella furcifera sucks the phloem sap to obtain nutrition. Phloem sap has an unbalanced amino acid composition, some essential amino acids are deficient. Rice planthoppers reportedly harbored yeast-like symbionts that may compensate for the unbalanced amino acid composition. Transaminases play important roles in amino acid metabolism.Based on the S. furcifera transcriptome data, some genes encoding transaminases were found and 10 full-length cDNAs were obtained. The enzyme activities of Sf9 cell-expressed two alanine transaminase proteins were tested in vitro.1. Cloning of genes encoding transminasesBased on the S. furcifera transcriptome data,20 fragments encoding alanine transaminase (EC 2.6.1.2), aspartate transaminase (EC 2.6.1.1), acetylornithine transaminase (EC 2.6.1.11), branched chain aminotransferase (EC 2.6.1.42), phosphoserine aminotransferase (EC 2.6.1.52), kynurenine-oxoglutarate transaminase (EC 2.6.1.7), glutamine fructose-6-phosphate amidotransferase (EC 2.6.1.16), alanine-glyoxylate transaminase (EC 2.6.1.44) and aromatic-amino-acid transaminase (EC 2.6.1.57) were cloned. Using RACE method, the full length cDNAs of 2 alanine transaminases,3 kynurenine-oxoglutarate transaminases,2 aspartate transaminases,1 branched chain aminotransferase,1 acetylornithine transaminase and 1 alanine-glyoxylate transaminase were obtained. Similarity analysis, phylogenetic analysis and codon usage bias suggested that some originated from symbiont, and the remaining fragments come from S. furcifera. Symbiont is implied to function in the transmination of Val, Leu, Ile, Arg, Ser, Tyr, Phe and Trp. S. furcifera and symbiont enzymes may work collaboratively to catalyze the transmination of Ala, Glu, Asp and Gly.2. Similarity analysis and expression profiles of two alanine transaminase and one branched chain aminotransferase genesConserved domain analysis revealed that the two putative alanine transaminases (SfylsALT and SfALTl) contain the complete domain of transaminase subgroup I, and have 10 converved amino acid residues involved in pyridoxal 5’-phosphate binding. SfylsALT showed a high expression peak at the fifth-instar nymphs, followed by those in male adults, female adults, third and fourth instars. In addition, the expression levels of SfylsALT were low and formed a trough in the newly-molted neonates and second-instar nymphs. In contrast, SfALT1was highly expressed in male adults, followed by fourth-and first-instar nymphs, and was lowly expressed in fifth-, third-, second-instar nymphs, and in female adults. The spatial mRNA levels of SfylsALT and SfALT1were also measured in sexually mature female adults. SfylsALT clearly had a high transcript level in the abdomen in which YLSs were located. Only trace amounts of transcripts were detected in the heads, wings, legs and thoraces. In contrast, SfALTl was highly expressed in the heads, wings, legs, thoraxes and abdomens.Moreover, conserved domain analysis revealed that the putative branched chain aminotransferase (SfylsBCAT1) contain the complete domain of transaminase subgroup III, and have converved amino acid residues involved in homodimer interface and pyridoxal5’-phosphate binding. SfylsBCAT1 was highly expressed in fifth-instar nymphs, and it was lowly expressed in second-instar nymphs, and it was equivalently expressed in others.3. Expression of two alanine transaminase and one branched chain aminotransferase genes in Sf9 cell line and ALT activity of recombinant proteinsThe expression plasmid vectors including complete ORFs of egfp, SfylsALT, SfALT1 and SfylsBCAT1 genes were constructed and successfully transformed to Escherichia coli DH10Bac cells. The recombinant proteins were successfully expressed in Sf9 cell line, and were detected by western blotting with anti-6×His-tag antibody.ALT activities in the enzyme preparations from blank Sf9 cells, the cells expressed egfp, SfylsALT, and SfALT1 were 8.3±1.2,10.15±2.0,139.88±2.4 and 506.41±5.2 nmol/min/ml, respectively. ANOVA analysis revealed that the ALT activities in the enzyme preparation from SfALTl-and SfylsALT-expressed cells were significantly higher than those in blank cells and egfp-expressed cells.