摘要
本文以不同Bt基因来源不同基因型品种,即来源于我国的Bt基因常规棉品种泗抗1号(小铃型)和杂交种泗杂3号(大铃型);来源于美国的Bt基因常规棉品种DP410B(小铃型)和杂交种岱杂1号(大铃型)为试验材料,于2008-2009年在扬州大学农学院,采用盆栽和大田试验相结合,研究探明了温湿度逆境胁迫对叶片Bt蛋白表达量以及不同种质及人工调节的棉铃大小、生长物质GA3和缩节安(DPC)对棉铃虫首选取食的器官棉铃Bt蛋白表达量的影响及其相关生理机制。
主要研究结果如下:
1.温湿度互作胁迫对Bt棉3个生育期叶片Bt蛋白表达量及胁迫解除后Bt蛋白表达量恢复的影响。
随着棉花生育进程,温湿度胁迫下Bt蛋白含量下降幅度呈逐渐加大的趋势,3个生育期在高温高湿、高温低湿、低温高湿和低温低湿4种逆境下,盛蕾期叶片Bt蛋白下降不大,盛花期有一定程度下降,盛铃期则显著下降。盛铃期低温低湿胁迫48h后,Bt蛋白含量比对照下降43.2%～51.3%。在高湿(95%RH)或低湿(50%RH)条件下,高温(37℃)胁迫24h内叶片Bt蛋白下降幅度较低温(17℃)小,24h后随着胁迫时间的延长,Bt蛋白含量降幅明显增大,而低温胁迫24h内Bt蛋白含量下降快,24h后Bt蛋白含量降幅变缓。胁迫至48h,高温处理的叶片Bt蛋白含量或低于低温处理,或与低温相差不大。总体表现为,3个生育期胁迫相同时间,低温比高温对Bt蛋白的表达量影响大。在高温或低温条件下,高湿度和低湿度胁迫叶片Bt蛋白含量下降幅度表现为低湿度>高湿度。不同类型品种间,盛蕾和盛花期温湿度逆境胁迫后Bt棉杂交种Bt蛋白含量下降幅度小于常规品种。盛铃期杂交种和常规品种相比,逆境胁迫后下降幅度差异较小。美棉品种在低温胁迫下受到的影响较小。
不同生育期温湿度胁迫解除后,叶片Bt蛋白表达量的恢复程度不同,具体表现为盛蕾期>盛花期>盛铃期。盛蕾期温湿度胁迫24h或48h解除胁迫后,在96h内Bt蛋白表达量可以完全恢复甚至Bt蛋白恢复后含量超过未胁迫前水平,且胁迫时间越长,胁迫解除后Bt蛋白含量越高;高温高湿度和高温低湿度胁迫解除后叶片Bt蛋白含量恢复时间早,提高幅度分别大于相应的低温高湿度和低温低湿度处理,如高温高湿度胁迫48h解除胁迫96h后,叶片Bt蛋白含量比未胁迫前提高了18.4%～31.7%;低温高湿度胁迫48h解除胁迫96h后,叶片Bt蛋白含量比未胁迫前提高了6.8%～14.3%。盛花期温湿度胁迫24h解除胁迫后,不同品种叶片Bt蛋白恢复后含量同样超过了未胁迫前水平,但与盛蕾期相应处理相比,提高幅度较小,叶片Bt蛋白含量比未胁迫前仅提高了8.6%～16.2%,且在高湿或低湿条件下,高温和低温处理胁迫解除后叶片Bt蛋白恢复后含量差异较小;胁迫48h解除胁迫后,叶片Bt蛋白的表达量不能恢复到未胁迫前水平。盛铃期温湿度胁迫24h或48h解除胁迫后,不同品种叶片Bt蛋白表达量不但不能恢复,而且Bt蛋白含量随着恢复时间的延长而下降,其中胁迫24h解除胁迫后,高温高湿和高温低湿度处理的Bt蛋白降幅较小,但胁迫48h解除胁迫后Bt蛋白含量大幅度下降。随着解除胁迫时间的延长,Bt蛋白表达量的继续下降,下降程度高温处理明显大于低温处理,如高温高湿度和低温高湿度胁迫48h,解除胁迫后96h,泗杂3号Bt蛋白含量分别比对照下降了33.1%和19.2%。温湿度逆境胁迫解除后,不同品种Bt蛋白表达量的恢复水平不同。抗虫棉杂交种在盛蕾期和盛花期恢复能力强于常规品种,在盛铃期高温低湿度胁迫下Bt蛋白含量下降幅度大于常规品种。
2.温湿度胁迫对Bt棉3个生育期叶片氮代谢的影响。
盛蕾期在高湿或低湿条件下,高温胁迫对丙谷氨酸转氨酶(GPT)活性、可溶性蛋白含量和全氮含量影响较小,胁迫24h内处理与对照之间无明显差异,胁迫24h后稍有下降。高温处理下叶片游离氨基酸含量明显增加。与高温相比,低温胁迫显著降低GPT酶活性,游离氨基酸、可溶性蛋白和全氮含量也显著低于对照。高温和低温都提高了谷氨酰胺合成酶(GS)活性。在高温或低温条件下,高低湿度处理间氮代谢关键酶活性差异较小。
盛花期在高湿或低湿条件下,高温胁迫24h内对GPT、GS酶活性,可溶性蛋白和全氮含量影响较小,24h后4个品种以上酶活性和可溶性蛋白和全氮含量都随着胁迫时间的延长逐渐下降,高温胁迫提高了叶片游离氨基酸含量,且提高幅度要大于盛蕾期。低温胁迫12h后GPT、GS酶活性都随胁迫时间延长呈不断下降的趋势,在胁迫12-36h内,高温和低温处理的叶片GPT、GS酶活性、可溶性蛋白和全氮含量差异较大,又以胁迫至24h时差异最大。在高温或低温条件下,低湿度胁迫的氮代谢强度低。
盛铃期在高湿或低湿条件下,高温胁迫GPT、GS酶活性,可溶性蛋白和全氮含量在24h内呈缓慢下降的趋势,24h后快速下降;低温胁迫正好相反,24h前呈快速下降的特征,24h后降幅变缓。高、低温下均促进了叶片蛋白酶含量的提高,游离氨基酸含量大幅度提高,其中以高温处理更为明显。高温条件下,高湿度和低湿度胁迫处理在24h内对氮代谢影响差异较小,24h后差异逐渐变大,至48h低湿度处理叶片氮代谢强度显著低于高湿度处理。低温下一直以低湿度处理酶活性、可溶性蛋白和全N含量低。
盛蕾和盛花期都以杂交种叶片氮代谢强度受到温湿度胁迫的影响明显小于常规品种。相关分析表明,盛蕾期温湿度胁迫下叶片Bt蛋白含量与GPT酶活性呈显著正相关。盛花和盛铃期叶片Bt蛋白含量与GS、GPT酶活性呈显著或极显著正相关关系。3个生育期叶片Bt蛋白含量与全氮含量、可溶性蛋白含量都呈极显著正相关关系。在温湿度胁迫条件下,叶片蛋白酶活性与可溶性蛋白均呈负相关,与游离氨基酸含量均呈正相关关系。
3.温湿度胁迫对Bt棉3个生育期叶片氨基酸组分的影响。
高温低湿和低温低湿对叶片中Bt蛋白分子的八种主要氨基酸成分的含量在3个生育期有不同的影响,其中对酸性氨基酸(天冬氨酸和谷氨酸)含量影响最大。盛蕾期胁迫酸性氨基酸含量在48h内虽有所波动,但增加的幅度较小,盛花期和盛铃期逆境胁迫后酸性氨基酸含量明显增加,且增加幅度以高温下较大,盛铃期更为明显。盛花和盛铃期叶片Bt蛋白主要氨基酸组分总量与Bt蛋白含量呈显著或极显著负相关关系,高温低湿度胁迫下叶片Bt蛋白主要氨基酸组分的总量与Bt蛋白含量的相关系数分别为r1=-7918**,r2=-8537**;低温低湿度胁迫下叶片Bt蛋白主要氨基酸组分的总量与Bt蛋白含量的相关系数分别为r3=-5703,r4=-7806**。
4.温湿度胁迫对Bt棉3个生育期叶片糖代谢的影响。
盛蕾期在高湿或低湿条件下,高温和低温胁迫都降低了蔗糖酶(Inv)活性,提高了蔗糖磷酸合成酶(SPS)活性,增加蔗糖和可溶性总糖含量,且低温比高温影响更大。无论在高温还是在低温条件下,湿度对糖代谢影响较小。盛花期在高湿或低湿条件下,高温胁迫提高了SPS活性,增加了蔗糖和可溶性总糖含量;低温胁迫提高了叶片Inv、SPS活性,增加了蔗糖和可溶性总糖含量,其中Inv酶活性和可溶性总糖含量明显大于高温处理;在高温或低温条件下,高湿度和低湿度处理间叶片糖代谢差异较小。盛铃期在高湿或低湿条件下,高温或低温都显著提高了叶片Inv和SPS酶活性,蔗糖和可溶性总糖含量大幅度上升,且以高温胁迫效果更为明显;在高温条件下,低湿度胁迫的叶片糖代谢强度明显大于高湿度处理;低温条件下,高、低湿度间无显著性差异。
3个生育期温湿度逆境胁迫后叶片C/N比值增加,盛蕾期增加幅度小,盛铃期大;胁迫时间越长,C/N比值越高。在高湿或低湿条件下,盛蕾和盛花期低温胁迫对C/N比值影响较大;盛铃期以高温胁迫的C/N值更大。相关性分析表明在温湿度胁迫下,盛蕾期、盛花期和盛铃期叶片C/N值与Bt蛋白含量呈负相关关系,相关系数分别为r1=-0.5192,r2=-0.7601*,r3=-0.9158**。
5.不同棉铃大小的种质及其人工调节对棉铃Bt蛋白表达量及氮代谢的影响。
杂交种泗杂3号和岱杂1号在整个棉铃发育时期铃重、铃体积、铃壳体积、铃壳干重和子指都显著高于常规品种泗抗1号和DP410B;与之相反,铃壳和棉籽中Bt蛋白含量表现为常规品种Bt蛋白最高;杂交种最低。相关分析表明铃体积、铃壳体积、铃重与铃壳干重与铃壳Bt蛋白含量呈显著正相关关系,相关系数分别为r1= -0.704*,r2=-0.739*,r3= -0.706*,r4= -0.675*,子指与棉籽Bt蛋白含量极显著负关系,相关系数为r=-0.869**。人工调节棉铃发育的结果进一步表明,当通过剪叶方法使棉铃变小时,铃壳和棉籽中Bt蛋白含量增加。反之,通过去蕾使棉铃变大时,铃壳和棉子中Bt蛋白含量下降。对大小不同的棉铃的氮代谢研究进一步表明,棉铃变小时氮代谢增强,棉铃变大时氮代谢减弱。以上结果说明了棉铃的大小影响Bt棉棉铃中Bt蛋白的表达。
6. GA3及DPC对棉铃Bt蛋白的表达量和氮代谢的影响。
生长延缓剂DPC能明显提高花后10d和30d(DPA)铃壳和棉籽中Bt蛋白的含量,其中又以10 DPA铃壳和棉籽中Bt蛋白含量增加更为显著;生长促进剂GA3降低10 DPA铃壳和棉籽中Bt含量,但显著提高了30 DPA棉铃Bt蛋白含量。对棉铃氮代谢研究进一步表明,DPC提高了铃壳和棉籽中GS、GPT和GOT酶活性并增加了游离氨基酸、可溶性蛋白和全氮的含量。GA3在10 DPA降低了铃壳、棉籽上述酶活性和物质含量,但30DPA棉铃氮代谢却与之正好相反,氮代谢明显加强。相关分析表明,棉铃中Bt蛋白含量与GS、GPT和GOT酶活性呈极显著正相关关系r1=0.7786**,r2=0.7325**,r3=0.7023**。表明GA3和DPC通过影响棉铃的氮代谢,从而影响Bt蛋白的表达。
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 metabolism
In 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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