ISSR Analysis Of Tobacco Germplasm And Physiological Genetics And Differential Proteomics In Response To Nitrogen Nutrition

Nitrogen nutrition plays an essential role in the growth and quality formation of tobacco, which has become an interesting subject in tobacco breeding and cultivation, and thus more attention is given by foregin and domestic experts in this field. This paper analyzed the genetic diversity of 96 tobacco germplasms with ISSR markers, and used 36 tobacco varieties that displayed significant genetic difference to analyze the genotype difference of response to three different nitrogen supplying levels, and to investigate the biological character markers of low nitrogen tolerence. Furthermore, the evaluation system of low nitrogen tolerance, uptake mechanism of nitrogen, physiological inheritance and different expression and adjustment of proteins were detected using water cultivation experiments under different nitrogen supplying levels. The major results were as follows:The genetic diversity among tobacco species revealed by ISSR marker was much higher than that among common cultivated varieties. The order of genetic diversity index in different cultivated types was Cured tobacco > Suncured tobacco > Burley tobacco > Aromatic tobacco. Viewed from genetic evolution, genetic relationship between cured tobacco and suncured tobacco was the closest, but that between aromatic tobacco and Nicotiniana rustica was the furthest. No genetic differentiation was detected between Chinese cultivated varieties and foregin cultivated varieties. The big crisis exists due to narrow genetic background within common cultivated varieties, germplasm innovation with distant genetic background is quitely urgent. UPGMA analysis based on ISSR marker showed that all cultivated cultivars were clustered into one big group, which were different from Nicotiniana rustica and wild types. Morover, the cultured cultivars can be further grouped into eight small groups.The genotypic difference of response to three nitrogen supplying levels was significant, and the difference under low nitrogen treatment was the most significant, which could work as nitrogen supplying level to measure nitrogen effificency. The difference of leave weight per plant was the biggest among investigated agricultural traits. The relationship analysis showed that leave weight per plant, area of the biggest leaf, stem thickness, and biomass worked as evaluation system of low nitrogen tolerance. Among 36 varieties tested, Yongding 400, Jinyan 6, Hong-hua-da-jin-yuan, G 80 and NC 82 showed the highest nitrogen efficiency under low nitrogen level; Yunyan 85, Nc89, Cuibiyihao, Hulizhong and Delhi76 revealed higher nitrogen efficiency; De-gold, Baihua G28, K399, G28, Southern Rhodesia, Hick55 and K326 displayed the lowest nitrogen efficiency.Evaluation of physiological characteristics under different nitrogen supply levels showed that root nitrogen content, chlorophyll content, light energy acquisition ability, light quantum yield, and lightchemical efficiency worked as evaluation indexes under low nitrogen tolerance. On the other hand, compared with nitrogen utilization efficiency, nitrogen uptake efficiency was of higher contribution in tobacco nitrogen efficiency. Comparision of differenct tobacco varieties such as K326 with Cuibi 1 in above ground growth and root nitrogen uptake dynamics showed that higher growth vigor of above ground part was the inherent determinant of high uptake efficiency of nitrogen, that higher Vmax, lower Km, and lower Cmin was the performance of high uptake efficiency of nitrogen in root. The dynamics indexes of nitrogen uptake (Vmax, Km, Cmin) changed with the nitrogen supplying levels, but the variation trend had genotypic difference, and this difference was closely related with genotypic difference of response to different nitrogen supplying levels.Additive, dominance, additive×environment interaction, and dominance×environment interaction effects played remarkable roles in major traits conferring nitrogen absorption and utilization. The nitrogen contents of above ground part of plant and root were mainly controlled by additive effect. Dominance effect played major role in dry weight in above ground part, in root, in nitrogen accumulation of over ground part of plant and root, total nitrogen absortion of per plant. Additive×environment interaction effect appeared in all traits conferring nitrogen absorption and utilization, which showed the interaction of genotypes and nitrogen levels was very important, and suggested we should pay more attention to selecting traits correlated to nitrogen utilization in special nitrogen suppling level when using heterosis. Chlorophyll content, photosynthetic parameter and chlorphyll fluorescence parameter were mainly controlled by dominance and environment interaction effect. Viewed from genetic correlation between different traits, phenotypic and genetic correlation existed between traits conferring nitrogen uptake and utilization. Genetic correlation could be further divided into additive genetic correlation, dominance genetic correlation, additive and environment interaction, and dominance and environment interaction. In addition, there were also significant genetic correlation beteween traits conferring nitrogen uptake and utilization, and photosynthesis physiological parameter, and chlorphyll fluorescence parameter, respectively.Under low nitrogen stress, 648 and 523 protein spots for leaf and root were well isolated, respectively. 67 protein spots were differentially expressed for leaf in high and low nitrogen supplying levels, and 47 protein spots were differentially expressed for root in high and low nitrogen supplying levels, which indicated the expression of some proteins had been changed due to low nitrogen stress. The function of 21 proteins was identified by MALDI TOF/TOF^TM. The expression profiles of protein after low nitrogen stress were as follows: proteins related to protein degradation, carbon metabolism, biotic and abiotic stresses were up-regulated; nitrogen hunger increased the requirement of root cells to molecular chaperones; proteins in leave conferring protective enzymes and photosynthesis were down-regulated.