Effects Of Nitrogen Fertilizer On The Expression Of NifH Gene In The Leaves Of Different Sugarcane Varieties

Sugarcane is an important crop that can produce sugar and energy,and it has long growth cycle that requireda large amount offertilizer.Nitrogen fertilizer is one of the important factors in the growth ofsugarcane.A large amount ofnitrogen fertilizer put into use in sugarcane planting,while ensuring the production of sugarcane,the soil and the environment also caused great damage.The study of utilization nitrogen fixation capacity of sugarcane has great significance to sugarcane production and environmental protection.The interaction between sugarcane and azotobacteria is a complex unity,and many factors affect the biological nitrogen fixation of nitrogen fixing bacteria.The nifH gene encodes a nitrogenase ferritin,which is thought to be the most conserved functional gene in nitrogen-fixing microorganisms.In this study,sugarcane grown in field was used as material,and the effects of nitrogen fertilizer on nifH expression in different sugarcane varieties were analyzed by RNA and protein levels,that want to reveal the effect of nitrogen fertilizer on the growth of nitrogen-fixing bacteria in sugarcane.The main results are as follows:1.Using the ROC22 sugarcane’s leaves and stems as materials which grow in field,three pairs of nifH universal primers UedaR/UedaF,ZehrF/ZehrR and PolyF/PolyR were screened,and find that PolyF/PolyR primers was the best one to amplify the nifH gene of sugarcane leaves;Western blot was use to detect nifH protein in ROC22 stem and leaf,and successfully was detected the nifH protein in leaves,but was not detected in the stem.2.The nifH gene was detected at both DNA level and RNA level by using PolyF/PolyR primers in the leaves of three sugarcane cultivars(GXB9,ROC22,GT11)that cultivated with nitrogen and non-nitrogen treatments in the fields.At the same time using I primers and P primers for nested PCR primers at the level of RNA,and the nifH gene amplified from sugarcane leaves was cloned and sequenced.Then it was found that although the two pairs of primers amplified the nifH gene sequence was had a great difference,the sequence alignment results in different samples was similar.The results showed that the treatment with nitrogen fertilizer reduced the diversity of endophytic diazotroph nifH gene expression in sugarcane.At the same time,it found that there was a great difference in the nifH sequences between with nitrogen fertilizertreatment and withoutnitrogen fertilizer treatment.Nitrogen fertilizer treatment affected the expression of endogenous nitrogen fixation nifH gene and the type of nitrogen-fixing bacteria.3.The relative expression quantity of nifH were studied by Western blot in three sugarcane cultivars leaves with nitrogen fertilizer treatment and without nitrogen fertilizer treatment on the four stages.The results showed that the relative expression of nifH protein in sugarcane leaves was lower in wihtout nitrogen fertilizer treatment thanwith nitrogen fertilizer treatment,and found that the relative expression level of sugarcane was higher in the first two periods.4.The nitrogenase activity of leaves in GXB9,ROC22 and GT11 were studied by acetylene reduction method at four stages,with nitrogen fertilizer treatment and without nitrogen fertilizer treatment.The results showed that the activity of nitrogenase in the first period was higher under no nitrogen treatment,and the other period was higher under nitrogen a treatment.