| In C.hina,sugarcane is one of the most important sugar crops.Guangxi is the main sugarcane planting region,and the sugarcane cultivation area accounts for more than 60%of the total and about 70%sucrose annual output in China.In order to ensure the growth and improve the yield of sugarcane,a lot of nitrogenous fertilizer must be used during the sugarcane production,which not only greatly increased the production cost of sugarcane,also caused the serious energy and ecological crisis.Therefore,it is significant to improve the sugarcane yield,lower the production cost and protect the ecological environment if we excavate and utilize the the resource of nitrogen fixation in sugarcane.The interaction between Sugarcane and endogenous azotobacter is a complex biological process,the ability of the sugarcane nitrogen fixation is better or not is affected expect for the sugarcane genotypes and environmental factors,also associated with the species and genotypes of azotobacter.Three sugarcane varieties ROC22,GT21,B8 were took as the materials in this research.The different sugarcane varieties were incolated with the strain DX120E-gfp by taking root dip method.The fluorescence microscopy was used to observe the azotobacter and the ability of infection and colonization in sugarcane.The different period of sugarcane growth and key enzyme activity indicators were investigated at 60,90 and 120 d respecyively after inoculation.The qRT-PCRwas used to detect the nifH gene expression at different stage in different seasons with different organs.The main results were as follows:1.Three sugarcane varieties ROC22,GT21,B8 were took as the material.After inoculating 7 d,the body fluorescence microscope was used to abserve the inculated sugarcane leaves and roots and found that the azotobacter colonized in sugarcane root,root hair zone,lateral root,the wound of fracture in root and leaf transverse area.Meanwhile,the nifH gene was detected by PCR in the roots and leaves of three sugarcane varieties and the size of the segment about 360 bp was expanded.2.The qRT-polymerase chain reaction(PCR)was used after incoluting 60d,90d and 120d respecyively and the relative expression of nifH gene in sugarcane leaves and stems of was detected respecyively.Fluorescence quantitative PCR analysis showed the nifH gene relative expression that incoluted with azotobacter in three varieties of sugarcane stems and blades were higher than not incoluted.As the extension of inoculation time,nifH gene expression in sugarcane stems and blades gradually reduced.3.The different period of sugarcane growth and key enzyme activity indicators were investigated at 60,90 and 120 d respecyively after inoculation.Indoor pot experiment showed that compared with the treatment sugarcane that not inoculate with azotobacter,the sugarcane which inoculated with DX120E could effectively promote the growth of aerial part and underground part of three sugarcane varieties,the increase of plant height,chlorophyll content and there were differences among each index in three varieties.Compared to the sugarcane that not inoculate with azotobacter,the GS activity,NR activity and the nitrate nitrogen in the sugarcane was improved when inoculated with azotobacter.The general evaluation was that each physiological indicator of ROC22 and B8 were better than that of GT21 after inoculation.4.The root dip method was used to inoculate three sugarcane varieties in Spring and Autumn to detect the nifH gene expression in the stems and blades of sugarcane in different growth stages of sugarcane,such as tillering stage,elongation stage and maturation stage.The results showed that the nifH gene expression charaters was different in blades and stems of sugarcane in different seasons.The nifH gene expression in the blaeds and stems of the treatment sugarcane in autumn were higher than that of in Spring,and the nifH gene expression was higher at the elongation stage than that of maturation stage in the growth stages of sugarcane. |
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