Study On Molecular Character Of The Later Growth And Development Of Tobacco Between Neixiang And Huili

This paper was designed with the top leaves (the 18th leaf) of flue-cured tobacco (Nicotiana tabacum L.) between Henan and Sichuan for the materials. Sixty days after transplanting, we investigated the tobacco agronomic characters and collected the same position of the 18th leaves in order to detect chemical composition, related enzyme activity, related genes expression, and observations of tissue slice. We sampled after that every 10 days.Firstly, this paper discussed the differences in the three physicochemical properties: carbon metabolism, nitrogen metabolism and secondary metabolism of tobacco between Henan and Sichuan during 60-100 d after transplanting. Moreover, analyzed the differences of enzyme activity, genes expression related to the three metabolic, and analyzed the causes of the differences from molecular level. Secondly, this paper studied the differences on key genes (SPS, SS, lyc-β) related to sucrose and carotenoids synthetic of tobacco between Henan and Sichuan. This paper also studied the relation between these genes, temperature, and light to build a theoretical and technical basis to regulate these genes expression.In addition, this paper established Logistic model between tobacco DNA methylation levels and four meteorological factors (temperature, sunshine, rainfall, humidity) during the growth of Queshan tobacco, and researched the interact between meteorological factors during tobacco growth and DNA methylation, and explored meteorological factors’influence to tobacco physiology and biochemistry at the molecular level. In addition, this provides basis to further research the relation between the formation of characteristic leaf tobacco and meteorological factors. The main conclusions of this experiment were as follows:1. Total thickness of tobacco leaves both in Henan and Sichuan continuously increased after transplanting. The Neixiang tobacco growed fast in the early and slow in late. However, Huili’s growed slow at the earlier time and fast later. During 60-100 d after transplanting, both in Henan and Sichuan, soluble sugar content of tobacco presented single apex curve, 70 d after transplanting was the time that the peak value show, then 100 d after transplanting total soluble sugar content decreased to the lowest. Soluble sugar content of Huili tobacco was 1.5 times higher than Neixiang’s after cured. 2. Sixty-eighty days after transplanting, SPS activity of tobacco in Huili is higher than that of tobacco in Neixiang, however, at 90 d after transplanting, SPS activity of tobacco in Huili decreased rapidly to lower than that in Neixiang. 60-90 d after transplanting, the SS activity of tobacco in Huili is lower than that of Neixiang, and the SS activity in Neixiang decreased rapidly to lower than that of Huili at 90 d after transplanting. Between 60-100 d after transplanting, the activity of sucrose invertase in Huili is higher than Neixiang. During the period 60-100 d after tobacco transplanting, SPS expression of tobacco in Sichuan showed a gradual decline but the level is higher than that in Henan. During the same period, SS expression of tobacco in Sichuan is biased single peak changes, which at the 90 d reached a peak. The genes expression of tobacco had no significant difference in Henan and Sichuan.3. The leaf discs for temperature and light orthogonal experiment results in laboratory showed that the temperature and light treatment could decrease or raise the SPS and SS genes expression: only a proper temperature and light condition was conducive to the SPS expression. High temperature and light intensity conditions will be helpful for SS gene expression. Influence of the three factors on SPS and SS genes expression is in the order that (temperature×light intensity)> light intensity = temperature. Duncan’s test comparison results showed that the condition that the temperature interact light intensity achieves great influence on SPS and SS genes expression, which achieves significant levels (P < 0.05).4. SPS gene expression and Northern blot results showed that SPS gene expression levels of the tobacco in Sichuan were significantly higher than that of Henan. The result that SS gene hybridization bands brightness of Sichuan Tobacco was slightly higher than that of Henan revealed that SS gene expression in Sichuan tobacco leaves was slightly higher than that in Henan. In budding stage, SPS activity in Huili, Sichuan tobacco leaves is higher than that in Henan, however, SS activity in both regions had no much difference. In short, light and temperature may regulate the expression of lyc-βgene in tobacco, and cause the reasons for carotenoids contents in different ecological areas. Total soluble sugar content of Sichuan Huili tobacco was higher than in Henan Neixiang. The reason lies in the fact that SPS activity and gene expression of Sichuan Huili tobacco was higher than Hernan. However, SS activity and gene expression of tobacco were little difference between Sichuan and Henan.5. As tobacco was mature, nicotine content in two areas’tobacco both showed an increasing and content of total nitrogen and protein was decreasing. Variation of free amino acids was that it reached a peak on 70 d after transplanting, and then decreased gradually. During the periods 60-100 d after transplanting, NR activity and gene expression gradually decreased, and Huili’s always had higher levels than Neixiang’s. During the same period, GS gene expression of tobacco both Huili and Neixiang showed a gradual decline, however, Huili’s was higher than Neixiang’s. GDH gene expression in tobacco of the two areas showed a gradual increase tendency, moreover, Huili’s was higher than Neixiang’s. 6. At the budding age, in the cured upper leaves, the content of carotenoids in Sichuan tobacco was significantly higher than that in Henan. Results of semi-quantitative RT-PCR of Lyc-βgene and Northern blot show that the Lyc-βgene expression of Sichuan tobacco was significantly higher than Henan tobacco. Expression of Lyc-βgene was regulated by temperature and light and the influence effect is in the order that the light intensity> Temperature> Temperature×light intensity, furthermore, levels among the three factors had no significant difference (P < 0.05). Expression of Lyc-βgene under high light conditions is 1.60 times higher than that in low light. At the temperature of 30℃, its expression reaches the highest, followed by 25℃, the last 35℃. In the range of light time from 2 to 6 h, the expression extension of the gene increased with the extend of the light time, but would reduce when the light time exceeded 8 h.7. Single-factor effects analysis of Logistic model between meteorological factors and DNA methylation showed that the rise of the average maximum temperature would cause the greatest effect on DNA methylation increase. The extended total sunshine would cause the extension of DNA methylation in tobacco. DNA methylation relies little on mean relative humidity. The increase of total rainfall could reduce the total level of DNA methylation. Although the absolute value of the coefficient was relatively smaller, the value of total rainfall was large enough, which would also get great influence to DNA Methylation. Interaction analysis of two-factor showed that the average maximum temperature and average relative humidity, mean maximum temperature and total sunshine hours would cause a synergistic effect on tobacco DNA methylation; meanwhile, the total rainfall and average maximum temperature, total precipitation and total sunshine Hours, the total rainfall and average relative humidity would lead to antagonism to the DNA methylation. The mean relative humidity and total sunshine hours showed no apparent synergistic effect or antagonistic effect on the level of DNA methylation.