MOLECULAR MECHANISM OF METHIONINE DIFFERENTIATION IN HIGH AND LOW METHIONINE MAIZE LINES

Abstract

Since maize is primary food stuff for humans and livestock, its amino acid balance is important for proper nutrition. Methionine, an essential amino acid and a primary source of sulfur, is lacking in maize endosperm. Several maize populations were developed through breeding with enhanced methionine content compared with normal maize populations. BS31HM (high methionine) and BS31LM (low methionine) maize were among such populations created by the selection from the highest or lowest methionine content population from original BS31 maize. Candidate gene approach was adopted to determine the difference between the two populations at of transcript level of the selected genes in the endosperm. The selected genes i.e. 15-kDa β-zein, 16-kDa γ-zein, 19-kDa α- zeinB1, 27-kDa γ-zein, 22-kDa α-zein and 18-kDa δ-zein were responsible for coding of endosperm storage proteins when analyzed through real time RT-PCR. Similarly, expression level relative to the high population (2-ΔΔct) values were also calculated for BS31HM and BS31LM respectively. The p-values were determined by student’s t-test at confidence level of 95%. The expression of 18-kDa δ-gene, 15-kDa β-gene and 16-kDa γ- gene were found to be significant (p<0.05) in high methionine maize population when compared with low methionine maize population. Non significant (p>0.05) differences in the expression level of 27-kDa γ-gene, 22-kDa α-gene and 19-kDa α-gene were observed in both (HM) and (LM) maize populations. From these results it can be concluded that all zein genes did not show expression equally in high methionine and low methionine maize populations. For selection of high and low methionine or other limiting amino acid levels in maize and feed samples auxotrophic strains could be a useful tool to identify the limiting amino acids. The bacterial assays for amino acid assessment are based on the nutritional requirements of the strain, relative concentration and availability of the amino acid in the liquid media, which shows a precise growth response to added amino acids. In the present study we tested triple auxotrophic E. coli strain with the objectives to identify whether this strain can be used to identify all the three limiting amino acids in maize samples. The triple auxotrophic strain was checked on M9 media for nutritional requirement to identify whether it is auxotrophic for methionine, lysine and tryptophan or only for one or two amino acids. The data indicated that triple strain was deficient for all the three amino acids. For further confirmation another experiment was conducted on different corn samples with different level of amino acids. Analysis of variance indicated that the probability of the observed variation due to chance was 0.0329 or less. The data revealed that growth of the triple auxotrophic strain was limited by lysine in most of the corn samples, but samples with extremely high lysine levels may cause growth to be limited by other amino acids. Further experiments should be conducted to optimize triple strain for identification of the all three limiting amino acids in maize.