Ultrastructure Observation And Nitrogen Regulation On Photosynthesis Of Sugar Beet Leaves (Beta Vulgaris L.) Senescence, Rubisco Antibody Preparation

In this paper, in order to study senescence mechanism of sugar beet leaves, ultrastructure observation and changes of photosynthetic characteristics such as photosynthetic rate (Pn), chlorophyll content, Rubisco carboxylation activity and soluble protein content those characteristics involved in both cell ultrastructure and physiological biochemistry fields, in the functional leaves (10th,15th and 20th true leaf) on different sampling dates under five nitrogen levels(0,60,120,180 and 240 kg/hm2), of two sugar beet varieties (Tianyan 7 and Tianyan 8) were investigated. Based on that below, we provided a reasonable theoretical basis for extending leaf photosynthetic function period and increasing the photosynthetic efficiency by Nitrogen levels regulation.Meanwhile, we purificated the Rubisco (1,5-ribulose bisphosphate carboxylase/ oxygenase) (EC 4.1.1.39) of the sugar beet mature leaves by using three-steps method, ammonium sulfate precipitation, gel filtration and ion chromatography and, measured the whole enzyme molecular weight and subunit numbers and molecular weight; through immunization experiment we prepared the Rubisco polyclonal antibody, and did some basic work for the study in-depth on Rubisco of sugar beet leaves in the future.The results were as followed:As the leaves getting senescence, there were series of irreversible cell ultrastructure changes, those were:decreasing of the chloroplast numbers, chloroplast membrane defects, cracking; the outflow of chloroplast stroma within, dissolved; mitochondrial membrane damage even dissolved and membrane system damaged, the two varieties showed the same. There were certain differences between the two varieties of the chloroplast numbers and chloroplast size in a single field of vision, but not significant.During the growth process of function leaves, Photosynthetic rate (Pn), Transpiration rate (Tr), Stomatal conductance (Gs) and other characteristics of photosynthetic all presented single peak curve, which first increased and then decreased, the peak time of approximately the same. Those three leaves of two varieties showed this same trend, but Tianyan 8 overall slightly higher than Tianyan 7. Photosynthetic rate (Pn) with Chlorophyll content, Rubisco carboxylation activity, soluble protein content and other physiological indicators have reached a significant positive correlation. Photosynthetic characteristics under different nitrogen levels have a significant effect:in the N0-N120 interval, increased with the rise of nitrogen levels; but more than N120, the changes of various indicators was not significant, even decreased.In this trial, root yield and sugar yield reached highest at N180 or N120 of the two varieties. photosynthetic rate (Pn), chlorophyll content, Rubisco carboxylation activity, soluble protein content those photosynthetic characteristics and root yield were significantly positive correlation or very, those were the most influence factors of beet yield and sugar yield.Identified the methods of Rubisco extraction and purification of sugar beet leaves and prepared pure enzyme, purification factor reached 7.7 folds; Native-PAGE shown as one band and purity above 90%, measured full enzyme molecular weight as about 540kDa; SDS-PAGE shown as two bands, which meant two subunits in existence, the molecular weight were about 50kDa and 14kDa.Prepared Rubisco polyclonal antibody through immunization experiment with white rabbits, then detected by double immunodiffusion experiment, antibody titers shown over 1:32, met the requirements of general experimental technique.