Differential Expression Of Candidate Genes For Lignin Biosynthesis Under Drougth Stress In Maize

Drought is the most widespread abiotic constraints in maize production. The molecular mechanism is believed to be complex because drought tolerance is the result of cooperative interactions among multiple morphological, physiological and biochemical characters. Information of molecular genetic properties, cloning and differential expression of drought-tolerant candidate genes is useful to selection marker development of functional genes and their application to marker assisted selection to improve drought tolerance in maize.Drought-tolerant inbred line '81565' and sensitive line '200B' were treated by using 16% PEG to simulate drought stress at 7 leaf stage, with well-watering control. The top first fully open leaf was sampled at 17, 24 and 48 h after drought treatment for total protein extraction. The total protein samples were separated by two-dimensional electrophoresis of isoelectric focusing and SDS-polyacrylamide gel, scaned by GS-170 scan system and analysed by PDQuest-6.0 image analying software for their differential expression in drought tolerant and sensitive inbred lines under drought stress and well-watering control. More than 500 protein spots were identified from the consensus master map constructed from 12 scaned images. Fifty-eight of them were found in response to drought stress. The expression of spot 2506, 3507 and 4506 was induced under drought stress. The highest expression amount was found at 24 h of drought stress. These three protein spots were digested and used for measurement of mass to charge ratio and molecular weight by MALDI-TOF-MS. Alignment was conducted against Mascot and Ms-Fit databases and MOWSE scores were used for protein identification. The result showed that spots 2506, 3507 and 4506 had MOWSE scores of 66, 68 and 68 with cinnamyl alcohol dehydrogenase (CAD), cytochrome protein 96A8 (CYP96A8) and S-adenosyl-L-methionine synthase (SAMS), respectively.Because these three functional proteins are involved in lignin biosynthesis, we used real time PCR technique to analyse the mRNA differential expression of two key enzymes in lignin biosynthesis pathway, cinnamyl alcohol dehydrogenase (CAD) and caffeic acid 3-O-methyltransferase (COMT), in different inbred lines under drought stress. In drought-tolerant lines '81565', '200B' and '87-1', the expression of CAD and COMT showed an obvious picking up after the significant decrease at the early stage of drought stress. However, in sensitive line 'Dan340', this kind of picking up was not found after the decrease at the early stage.To analyse the phenotypic relationships between lignin biosynthesis and drought tolerance, leaf lignin content, anthesis-silking interval and per plant grain yield were investigated with inbred lines '81565', '87-1', '200B', '18-599', '18-599M' and 'ES40' of different drought tolerance under strong and moderate drought stress as well as well-watered control, and converted into drought tolerant coefficients for correlation analysis. Significant difference was found in the response of leaf lignin content of inbred lines with different drought tolerance to drought stress. Under drought stress, the leaf lignin content of highly tolerant inbred lines increased significantly with a high average drought coefficient, the leaf lignin content of intermediate tolerant inbred lines increased slightly, while the leaf lignin content of drought sensitive inbred lines matched with the well-watered control or decreased slightly. Under the strong and moderate drought stress, significant correlation coefficients (r = 0.848 and r = 0.889) were estimated between the average drought tolerant coefficient of leaf lignin content and the drought tolerant coefficients of per plant grain yield and anthesis-silking interval.As a conclusion, lignification of leaf can be speculated to be a kind of active response of maize to drought stress. The differential expression of the candidate genes to lignin biosynthesis is relative to drought tolerance of different inbred lines. Based on detail research on the relation of these genes to molecular genetic mechanism of drought tolerance, selection markers of functional genes can be developed according to the difference of single nucleotide polymorphism and expression regulation among different inbred lines, and applied to molecular assisted selection of drought tolerance breeding in maize.