Cloneing Of Rubisco And PEPC From Sugarcane And Their Expression Under Different Nitrogen Fertilization And Sugarcane Cultivars

Sugarcane (Saccharrum spp.) is an important C4 crop contributing to over 90% of sugar production in China. In recent years, the sugarcane industry is facing a lot of challenges restricting its development, among which over-fertilization is one of the most important concerns. In China, nitrogen over-fertilization is very serious which has led to not only low nitrogen utilization efficiency and high sugarcane production cost, but also heavier occurrence of pests and diseases, and even environment pollution. The Rubisco and PEPC are the key enzymes catalyzing photosynthesis in C4 plant, accounting for the large amount of soluble protein in leaf. Nitrogen can affect the photosynthesis by impacting the content and enzymatic characteristics. Photosynthesis is closely related with nitrogen utilization, and it is important to study the mechanism of interaction between photosynthesis and nitrogen utilization to improve the nitrogen utilization efficiency in sugarcane production.In this study the full length CDS sequences of three genes encoding two key enzymes catalyzing photosynthesis in sugarcane were cloned from cultivar GT11 using PCR technology, and the prokaryotic expression and protein purification were done. The expression characteristics of the three genes were detected in three sugarcane cultivars (GT11, ROC22 and GXB9) holding different nitrogen utilization efficiency grown under three different nitrogen fertilization levels, and the photosynthesis was measured at the same time. The results are as follows.1. The full length CDS sequences of genes rbcS, rbcL and pepc encoding the enzymes Rubisco and C4-PEPC catalyzing carbon dioxide assimilaton in sugarcane were cloned from cultivar GT11, and their prokaryotic expressions were successfully detected in Rosetta. The purified fusion proteins SSU and LSU were obtained and further purified, which can be used to make monoclonal antibodies.2. The genes expressions were analyzed with real-time PCR and Western blotting. The results showed that the expressions of three genes rbcS, rbcL and pepc were the most constant and highest in sugarcane cultivar GXB9 with high nitrogen utilization efficiency than the other two cultivars not only at transcript but also in protein content, and the most fluctuated in GT11 with the lowest nitrogen utilization efficiency under different application rates of nitrogen fertilizer. In addition, it seems that different nitrogen fertilization rates impacted the photosynthesis through affecting the expression of rbcS in sugarcane.3. The results of Western blotting showed that the protein LSU degradation happened in October, which may be the reason why the photosynthesis rate reduced when sugarcane entered maturing stage.4. Under the condition of this study, different rates of nitrogen fertilization had significant effect on the photosynthesis of sugarcane cultivars. Although the net photosynthetic rate showed little difference in the middle and high nitrogen fertilization treatments, but significantly lower in the low nitrogen treatment with no nitrogen fertilization. The results of real-time PCR and Western blotting showed that nitrogen mainly affectted the expression of gene rbcS to regulate the amount of Rubisco holoenzyme to impact the photosynthesis in sugarcane.