| The study was undertaken on four boll worm resistant transgenic cotton cultivars, which were Sikang 1 (small-boll, conventional cultivar) and Siza 3 (big-boll, hybrid cultivar) from China, DP410B (small-boll, conventional cultivar)and Daiza 1 (big-boll, hybrid cultivar) from USA,during the 2008 and 2009 growing seasons at the Jiangsu Provincial Key Laboratory of Crops Genetics and physiology, Yangzhou University. Pot and field experiments were conducted to investigate the effects of combination stress of temperature and humidity, boll size, growth substances GA3 and DPC on Bt protein expression and metabolism.The main results were as follows:1. Effect of combination stress of temperature and humidity on the leaf Bt protein content and the recovery of the leaf Bt protein content after combined stress stopped for different cultivars at three growth periods.The difference of leaf Bt protein content between the stress treatments and control became larger gradually following cotton growing periods. The reductive extent of the leaf Bt protein was low at peak square period, middle at peak flowing period, high at peak boll period under four combined stress, which was high temperature-humidity, low temperature-humidity, high temperature with low humidity and low temperature with high humidity. The leaf Bt protein reduced 43.2% to 51.3% at 48 hours under low temperature-humidity stress at peak boll period. Under high humidity(95%RH)or low humidity(50%RH), the Bt protein content was less affected in 24 hour stress and then decreased gradually from 24 to 48 hour under high temperature (37℃) . The leaf Bt protein content also decreased under low temperature (17℃), and the values were lower than that under high temperature after 48 hour stress. This result suggested that the reduction extent of the leaf Bt content was greater under low temperature stress than that under high temperature stress at the three growing periods, there was similar effect on the leaf Bt protein content between low humidity and high humidity stress when the temperature maintained at 37℃or 17℃. The leaf Bt protein content of the hybrid cultivars were less affected than those of conventional cultivars at peak square and peak flower periods under combined stress. The extent of Bt protein decrease was similar between hybrids and conventional varieties at peak boll period. The cultivars whose Bt gene from the United States were less affected under low temperature stress.The recovery ability of the leaf Bt protein contents were different after the stress stopped at three growth stages. The restoation effect was highest at peak square period, and lowest at peak boll period. At peak square period, the leaf Bt protein content can recovered rapidly again and the value of that was even more than the levels before the stress after the stress stopped for 96 hours. The longer the stress lasted, the higher the leaf Bt protein content after the stress stopped for 96 hours. The Bt protein recovery content was higher under high temperature-humidity stress than that under low temperature with high humidity after the stress stopped, There were similar effects for the increase of the leaf Bt protein after the stress stopped between the high temperature with low humidity and the low temperature-humidity stress at peak square period. For example, the leaf Bt protein content increased 18.4% to31.7% and 6.8% to14.3% respectively than that without stress after high and low temperature stress with 95%RH stress stopped for 96 hours which lasted 48 hours. At peak flowering period, the Bt protein recovery content was also higher than that without stress after the combined stress stopped for 96 hours which lasted 24 hours, the values increased 8.6% to 16.2% than that without stress. Under high or low humidity, there was on different of the recovery Bt protein content between high temperature stress and low temperature stress. However, the leaf protein contents could not recovered to the level before stress after the stress stopped for 96 hours which lasted 48 hours, At peak boll period, the leaf Bt protein content continued to decrease after combined stress stopped during 96 hour. The leaf Bt protein content decreased slightly after the combination stress of the temperature and humidity stopped which lasted 24 hours, however, the content reduced significantly after the combined stress stopped which lasted for 48 hours. Under the same humidity stress, the reduction extent of Bt content of high temperature stress was more than than of low temperature after these stresses stopped for 96 hours which lasted 24 hours. For example, the leaf Bt content reduced by 33.1% and 19.2%, respectively after high temperature-humidity and low temperature with high humidity stress stopped for 96 hours for Sikang 1 when the stress treatments lasted 48 hours.There were different in the recovery ability of Bt proteins content for 4 varieties after the stress stopped. The leaf Bt protein content of the hybrid varieties enhanced more rapidly again than that of the conventional varieties at peak square period and peak flowering period, there was weakest recovery effect of the leaf Bt protein content after high temperature with low humidity stress at peak boll period.2. Effects of the combination stress of temperature and humidity on the leaf nitrogen metabolism for different Bt cultivars at three growth periods.The leaf GPT activities, soluble protein content was less affected within 24 hour stress and decreased slightly from 24 to 48 hour stress under high temperature associated with high humidity or low humidity stress at peak square period. Free amino acid content also increased gradually, and total N content decreased slightly in 48 hours under high temperature stress. In comparison to the high temperature stress, GPT and GS activities, soluble protein content decreased significantly under low temperature, and the content of free amino acid and total N decreased, the total N content was lower significantly than control after 36 hours stress. In contrast, GS activities increased for 48 hours stress under the combination stress of temperature and humidity, The GPT and GS activities difference was no significant between high and low humidity treatments with the same temperature.The leaf GPT and GS activities, soluble protein and total nitrogen content also less affected within 24 hours and then decreased gradually during 24 to 48 hours under high temperature associated with high humidity or low humidity stress at peak flowering period, and the rising rate of free amino acid content was higher than that at peak square period; The leaf GPT and GS activities decreased gradually during 24 to 48 hours under low temperature stress. The difference of the leaf nitrogen metabolism index was big between high temperature and low temperature stress during 12 to 36 hours, and the largest difference was observed at 24 hours. Nitrogen metabolism intensity was lower with low humidity stress than that with high humidity associated with high or low temperature.The enzyme of GPT and GS activities, the content of soluble protein and total nitrogen was slowly declined within 24 hours, and then reduced rapidly under high temperature with low or high humidity at peak boll period. In contrast to high temperature, GPT and GS activities, the content of soluble protein and total nitrogen was rapidly declined within 24 hours, and then slowly decreased under low temperature stress with low or high humidity. The Protease activity, free amino acid content increased and soluble protein content decreased at high or low temperature, especially at high temperature stress. The difference of leaf nitrogen metabolism index between high humidity and low humidity stress was small within 24 hours, and became bigger during 24 to 48 hours under high temperature stress. Nitrogen metabolism intensity was lower under low humidity stress, and was lowest under low temperature-humidity stress.The nitrogen metabolism intensity of the hybrids was higher than that of the conventional varieties under combined stress at peak square period and peak flowering period. The correlation between GPT activities, GS activities, total N, soluble protein content and leaf Bt protein content was positive at both peak flower period and peak boll period. There was a negative correlation between protease activity and soluble protein content, and positive correlation between protease activity and free amino acid at three periods.3. Effects of combination stress of the temperature and humidity on the leaf amino acids content.There was a variation of the content of eight major amino acids of Bt protein under both high temperature with low humidity stress and low temperature-humidity stress. Acidic amino acids showed most obvious changes under these stresses which was consisted of Glutamic Acid and Aspartic Acid. Acidic amino acids fluctuated within 48 hour, and increased slightly in peak square period, but significantly increased under high or low temperature with low humidity stress at both peak flower period and peak boll period, especially at high temperature with low humidity stress. There existed negative correlation between total content of eight major amino acids of Bt protein and Bt protein content, the correlation coefficients were -7918**,-8537** respectively under high temperature stress with low humidity stresses and -5703,-7806** under low temperature-humidity stress respectively at peak flower period andpeak boll period .4. Effects of combination stress of temperature and humidity on the leaf glucose metabolism for different Bt cultivars at three growth periods.The leaf Inv activities decreased, but the SPS activities, the contents of sucrose and soluble sugar increased combination stress of temperature and humidity, especially under low temperature associated with high or low humidity stress at peak square period. The leaf SPS activities and the content of sucrose and soluble sugar enhanced under high temperature stress, while the leaf Inv, SPS activities,the content of sucrose and soluble sugar increased under low temperature stress in the same humidity condition. The leaf Inv activities and soluble sugar content under low temperature was higher than that under high temperature stress at peak flowering period. There was no different of glucose metabolism between high humidity and low humidity associated with high or low temperature treatments at both peak square period and peak flower period. The Inv activities,SPS activities, sucrose and soluble sugar content increased significantly under high or low temperature stress,especially under high temperature stress at peak boll period. The leaf glucose metabolism intensity of low humidity was higher than that of high humidity under high temperature, however, the difference of leaf glucose metabolism was small between high humidity and low humidity treatments at 17℃.The ratio of C/N increased under combined stresses at three periods, which was smallest at peak square period and largest at peak boll period. The longer stress time lasted, the higher C/N ratio was. Under high or low humidity, the value of C/N ratio was higher under low temperature stress than under high temperature stress at both peak square period and peak flower period, but lower at peak boll period. The correlation between C/N ratio and leaf Bt protein content was negative, and the correlation coefficients were -0.5192,-0.7601* and -0.9158**, respectively at peak square period, peak flower period and peak boll period.5. Effects of the boll size on Bt protein expression and nitrogen metabolism.The hybrid cultivars were bigger in boll volume and boll shell volume, higher boll weight,boll shell weight and 100-seeds weight than that of the conventional cultivars. However, the insecticidal protein contents were lower in the boll shell and cotton seed. There were significant negative relationship between the boll volume, boll shell volume, boll weight, boll shell weight and the boll shell Bt protein contents, the correlation coefficient was -0.704*for boll volume, -0.739*for boll shell volume, -0.706* for boll weight and, -0.675* for boll shell weight. There were also very significant negative relationship between 100-seed weight and the cotton seed insecticidal protein content (r=-0.869**), Under artificial regulation, the boll and cotton seed insecticidal protein contents increased with the boll volume,boll shell volume,boll weight,boll shell dry weight and 100-seed weight decreased, the opposite results was observed with the boll volume,boll shell volume,boll weight,boll shell dry weight and 100-seed weight increased. The nitrogen metabolism showed that the content of total nitrogen, amino acid, soluble protein and the enzyme activities of GPT, GS were higher when the boll volume, boll shell volume, boll weight, boll shell dry weight, 100-seed weight were smaller, the opposite results was observed when the boll volume, boll shell volume, boll weight, boll shell dry weight, 100-seed weight were bigger.6. Effects of GA3and DPC on the boll Bt protein expression and nitrogen metabolismIn order to study the plant growth regulators on the effect of boll Bt protein content and nitrogen metabolism, GA3 and DPC were used to spray the bolls of the two Bt cotton cultivars under field condition, the results showed that DPC inhibited the development of bolls, decreased boll size and fresh weight of boll shell at 10 DPA, and was no significant difference between control at 30 DPA; while GA3 promoted the development of bolls, increased boll size and fresh weight of boll shell at both 10 DPA and 30DPA. DPC increased Bt protein contents of the bolls at both 10 DPA and 30DPA; GA3 decreased the Bt protein contents of the boll shell and the seeds at 10 DPA, and enhanced the contents at 30 DPA significantly respectively.The boll with DPC treatment had higher total nitrogen, free amino acid and soluble content, greater for GS, GPT and GOT activity at both 10 DPA and 30DPA, while the boll with GA3 treatment at 30 DPA. There were similar results for two Bt cultivars with different genotype. There were significant positive correlation between the Bt toxin content and the activity of GS, GPT and GOT. The correlation coefficients were 0.7786**,0.7325**andr3=0.7023**.. The results suggested that spraying GA3 and DPC could change boll nitrogen metabolism intensity and the expression of the Bt protein. |
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