Effect of elicitors on expression of MYB transcription factor genes in Selaginella bryopteris (L.) Baker By Anjani Kumar (M/PBG/286/BAC/2015-16)

Selaginella bryopteris is a medicinally important plant species enriched with flavonoids and biflavonoids and has been used for centuries in India as folklore medicine. Flavonoids are synthesized via the phenylpropanoid (PP) pathway and flavonoid (FL) pathway using phenylalanine as precursor. MYB genes play important roles in the regulation of these biosynthetic pathways. Despite immense potential for commercial exploitation of this plant for bioactive molecules particularly flavonoids, it has been least studied on aspect of their biosynthesis. Molecular aspect of biosynthesis of flavonoids involving transcription factors, transporters and pathway genes is very important for overall understanding of regulation of biosynthesis of bioactive molecules. In this direction, the present research work was formulated to study the expression pattern of genes of MYB transcription factors with following objectives (1) to study the effect of elicitors on expression of MYB transcription factor genes and flavonoid content and (2) cloning and in silico analysis of putative MYB transcription factor gene(s). The primers of MYB genes for gene expression study were designed using sequences from NGS data of Selaginella available in our lab. Results showed that the elicitors (methyl jasmonate, MeJ, salicylic acid, SA and Na-nitropruside, SNP) affect the expression of MYB genes (SbMYB1, SbMYB2 and SbMYB3) in fronds at different time point after the treatment. SbMYB2 expression found to be increased after 6h of treatment of MeJ as compared to control in contrast to SbMYB1 and SbMYB3. A prominent expression of SbMYB1 and SbMYB2 in response to SA elicitation was found as their expression level increased from 3 to 6h as compared to control. While in response to SNP, SbMYB2 and SbMYB3 gene expression was also found to be higher in comparison to control. The effect of elicitation by MeJ, SA and SNP on the flavonoid production was also evident in the present study. The content of flavonoid in methanolic extract of MeJ treated sample was found to be increased (1.2 fold) after 3h but declined at 6h and 9h as compared to control. Similarly, the content of flavonoid in methanolic extract of SA treated sample was found to be higher (1.85 fold) than control at 3h and later declined at 6h and 9h.The flavonoid content in methanolic extract of SNP treated sample was higher (1.84 fold) at 3h, 6h (2.13 fold) and at 9h (1.42 fold) as compared to control. The correlation was established between the gene expression and flavonoid content in response to elicitors (MeJ, SA and SNP). SbMyb1 gene expression was positively correlated (0.89) with flavonoid content in response to SA and negatively correlated with SNP (-0.98). While SbMYB2 gene expression was positively correlated with content of flavonoid in response to MeJ (0.68), SA (0.82) and SNP (0.88). Out of three MYB genes selected from NGS data of S. bryopteris analysed for their gene expression, SbMYB 2 was found to be highly responsive to known elicitors (MeJ, SA, and SNP) of FL pathway. Hence, we focused on this SbMYB2 only and its full length CDS was cloned. SbMYB2 coding sequence contained 1281 bp nucleotides, encoding protein of 426 amino acids and various in silico analysis confirmed it to be a MYB family gene. In conclusion, in the present study a putative MYB gene (SbMYB2) responsive to known elicitors of flavonoid biosynthesis was identified and cloned that could be helpful in future study related to modulation of flavonoid biosynthesis. Ravi Shankar Singh Anjani Kumar Chairman, Advisory Committee M/PBG/286/BAC/2015-16 List of Symbols and Abbreviation Symbol Description /Abbreviation % Percentage a.a Amino acid A260 Absorbance at 260 nm A280 Absorbance at 280 nm bp Base pair cDNA Complementary deoxyribonucleic acid DEPC Diethyl pyrocarbonate ddH2O Double distilled water dATP Deoxy adenosine triphosphate dNTPs Deoxy ribonucleoside triphosphates DNA Deoxyribonucleic acid DTT Dithiothreitol EDTA Ethylenediaminetetra-acetic acid E. coli Escherichia coli h(s) Hour(s) IHBT Institute of himalayan bioresources technology IPTG Isopropylthio--D-galactoside LB Luria Bertani l micro liter MEP Mevalonate ml Milli liter min Minute mM Millimolar mRNA Messenger ribonucleic acid nm Nanometer nmoles Nanomoles nM Nanomolar OD Optical density PCR Polymerase chain reaction RNA Ribonucleic acid rRNA Ribosomal ribonucleic acid RNase Ribonuclease rpm Revolutions per minute RT Reverse Transcriptase Sec Second rRNA Ribosomal ribonucleic acid SDS Sodium dodecyl sulphate TAE Tris-acetate EDTA Taq Thermus aquaticus Tc Tetracycline UV Ultra voilet Wt Weight X-gal 5bromo-4-chloro-3-indolyl--D-galactoside