The Cloning And Functional Verification Of A Key Waterlogging Tolerance Gene ZnCOMT In Nicaragua Teosinte

Waterlogging largely affected the growth and yield of maize in China. With long-term directional selection and domestication, the cultivated maize variety has lost its tolerance to waterlogging. Fortunately, wild relative’s teosinte which has lived in severe environment for a long history possesses the tolerance to waterlogging, has excellent traits like stain-resistant. Therefore, the discovery and use of teosinte stain-resistant genetic resources for breeding new varieties of maize has important significance. In this reseach, the Nicaragua teosinte TZ04was studied, using homology-based cloning methods to clone the key genes ZnCOMT, through analysis by real-time fluorescence quantitative PCR and method of agrobacterium mediated successfully into tobacco (Nicotiana tabacum) W38for functional research.1. Clone and analysis of caffeic acid-O-methyl transferase ZnCOMTTo compare the homology COMT gene sequences of sugar cane, rice, maize and other gramineae plants, we designed degenerate primers and obtained gene fragment about1183bp (Submitted to GenBank, ID:JX462676) by several rounds of PCR. Within the fragment, amino acids coding region is about1095bp, encoding364amino acids, with90%similarity of sugar cane, sorghum and rice was81%, wheat by75%. Compared with Mo17,and B73, each found2,3different amino acids, Results showed that the amino acid sequence similarity is high, but the different amino acids are located in methylation functional site area, with different physicochemical properties. We find that the amino acids translated by ZnCOMT sequence is composed of two subunits analysised by Swiss-Model, and the different three-dimensional structure regions from that of B73and Mo17exist in the random coil parts. MEGA5.2analysis results show that the genetic relationship between TZ04and maize was the closest among11different gramineae plants.2. The relative quantitative expression of caffeic acid-O-methyl transferase ZnCOMTThe RNA was extracted from roots of TZ04, which were under different type waterlogging conditions, for quantitative analysis. The results showed that there was a peak after3h of soaking (1.57times Oh processing). With the extension of time, relative expression levels began to decrease, and reached lowest point at12h (0.43times Oh processing). With time going by, the level of ZnCOMT expression began to rise, reaching the highest value at2d (1.76times Oh processing), relative expression levels at4d,8d compared with Oh treatment, remaining at a high level. Showing that plant roots under waterlogging condition for a long time, the level of expression of ZnCOMT in root still have relatively high expression level and also further corroboration that ZnCOMT participation physiological metabolic regulation of expression. The expression level of gene should be based on its caffeic acid-O-methyl, the abundance as well as the G/S-lignin content determination.3. Transgene of caffeic acid-O-methyl transferase ZnCOMT and T1generation seedThe ZnCOMT-CPB expression vector plasmid was builded and then transformed into Agrobacterium C58. And the leaf of Tobacco W38was infected by Agrobacterium as mentioned previously by agrobacterium-mediated for screening and differentiation. Several rounds of PCR in the regenerated plants were tested to determine the positive plants, and the plants showed positively were grown in an appropriate environment, at the same time non-transgenic was in control. To transgenic plants grew better than non-transgenic plants when transplanted in soil with saturated moisture or dry soil. The results preliminarily indicated that ZnCOMT was involved in regulating the tolerance of waterlogging. Now, T1generation seeds of the transgenic tobacco plants been acquired.