Dynamics of Bt cotton Cry1Ac protein content under an alternating high temperature regime and effects on nitrogen metabolism

This study was conducted to investigate the effects of alternating high temperature on Cry1Ac protein content on Bt cotton cultivars Sikang 1(SK-1,a conventional cultivar)and Sikang 3(SK-3,a hybrid cultivar).In 2011 and 2012,cotton plants were subjected to high temperature treatments ranging from 32 to 40℃ in climate chambers to investigate the effects of high temperature on boll shell insecticidal protein expression.The experiments showed that significant decline of the boll shell insecticidal protein was detected at temperatures higher than 38℃ after 24 h.Based on the results,the cotton plants were treated with the threshold temperature of 38℃ from 6:00 a.m.to 6:00 p.m.followed by a normal temperature of 27℃ during the remaining night hours(DH/NN)in 2012 and 2013.These treatments were conducted at peak boll growth stage for both cultivars in study periods of 0,4,7,and 10 d.Temperature treatment of 32℃ from 6:00 a.m.to 6:00 p.m.and 27℃ in the remaining hours was set as control.The results showed that,compared with the control,after the DH/NN stress treatment applied for 7 d,the boll shell Cry1Ac protein content level was significantly decreased by 19.1 and 17.5% for SK-1 and by 15.3 and 13.7% for SK-3 in 2012 and 2013,respectively.Further analysis of nitrogen metabolic physiology under DH/NN showed that the soluble protein content and the glutamic pyruvic transaminase(GPT)activities decreased slightly after 4 d,and then decreased sharply after 7 d.The free amino acid content and the protease content increased sharply after 7 d.The changes in SK-1 were greater than those in SK-3.These results suggest that under DH/NN stress,boll shell Cry1Ac protein content decline was delayed.Reduced protein synthesis and increased protein degradation in the boll shell decreased protein content,including Bt protein,which may reduce resistance to the cotton bollworm.     

High plant density increases seed Bt endotoxin content in Bt transgenic cotton

Plant density is the cultivation practice usually employed to manipulate boll distribution, boll setting and yield in cotton production. In order to determine the effect of plant density on the insecticidal protein content of Bacillus thuringiensis(Bt) cotton plants, a study was conducted in Yangzhou University of China in 2015 and 2016. Five plant densities(PD1–PD5, representing 15 000, 30 000, 45 000, 60 000, and 75 000 plants ha–1) were imposed on two Bt cotton cultivars, Sikang 1(the conventional cultivar, SK-1) and Sikang 3(the hybrid cultivar, SK-3). The boll number per plant, boll weight and boll volume all decreased as plant density increased. As plant density increased from 15 000 to 75 000 plants ha~(–1), seed Bt protein content increased, with increases of 66.5% in SK-1 and 53.4% in SK-3 at 40 days after flowering(DAF) in 2015, and 36.8% in SK-1 and 38.6% in SK-3 in 2016. Nitrogen(N) metabolism was investigated to uncover the potential mechanism. The analysis of N metabolism showed enhanced soluble protein content, glutamic-pyruvic transaminase(GPT) and glutamate oxaloacetate transaminase(GOT) activities, but reduced free amino acid content, and protease and peptidase activities with increasing plant density. At 20 DAF, the seed Bt toxin amount was positively correlated with soluble protein level, with correlation coefficients of 0.825** in SK-1 and 0.926** in SK-3 in 2015, and 0.955** in SK-1 and 0.965** in SK-3 in 2016. In contrast, the seed Bt protein level was negatively correlated with free amino acid content, with correlation coefficients of –0.983** in SK-1 and –0.974** in SK-3 in 2015, and –0.996** in SK-1 and –0.986** in SK-3 in 2016. To further confirm the relationship of Bt protein content and N metabolism, the Bt protein content was found to be positively correlated with the activities of GPT and GOT, but negatively correlated with the activities of protease and peptidase. In conclusion, our present study indicated that high plant density elevated the amount of seed Bt protein, and this increase was associated with decreased boll number per plant, boll weight and boll volume. In addition, altered N metabolism also contributed to the increased Bt protein content under high plant density.     

Nitrogen spraying affects seed Bt toxin concentration and yield in Bt cotton

Cotton bolls exhibit the lowest insecticidal efficacy among all organs of Bt cotton, which would ultimately affect the yield formation. The objective of this study was to investigate the effects of different urea concentrations on the seed Bt protein contents, seed cotton yield and the corresponding protein metabolism mechanism. The experiments were conducted during 2017–2018 cotton growing seasons. Two cultivars, Sikang 3(hybrid, SK3) and Sikang 1(conventional, SK1), were treated with six urea concentrations and their seed Bt protein contents were compared during boll formation period. The urea spray concentration had a significant effect on the seed Bt toxin content and seed cotton yield. Spraying of either 5 or 6% urea led to higher insecticidal protein contents and higher seed cotton yield for both cultivars. Moreover, the highest amino acid and soluble protein contents, as well as GPT and GOT activities, and lower protease and peptidase activities were observed at the 5 to 6% urea levels. Significant positive correlations between the seed Bt toxin and amino acid contents, and between the seed Bt toxin content and GPT activities were detected. The lower boll worm number and hazard boll rate were also observed with the 5 to 6% urea treatments, which may be the reason why nitrogen spraying increased the seed cotton yield. Therefore, our results suggested that the seed Bt toxin content and insect resistance were impacted markedly by external nitrogen application, and 5 to 6% urea had the greatest effect on insect resistance.     

盐胁迫对转Bt基因棉苗期Bt蛋白表达量和氮代谢的影响

以棉花鲁18、鲁21为材料,以长期(播种时进行盐处理,长到4片真叶时取样)与短期(4片真叶时进行盐处理,10 d后取样)盐胁迫进行对比试验,发现长期盐胁迫后,鲁18、鲁21的Bt蛋白含量和可溶性蛋白含量在根、茎、叶中均增加,而游离氨基酸含量在根、茎、叶中均降低,盐质量分数越大,影响越显著,对叶的影响最大,其次是茎,对根的影响最小。Bt蛋白含量与游离氨基酸含量呈极显著负相关,与可溶性蛋白含量呈极显著正相关。短期盐胁迫棉苗根、茎、叶中的Bt蛋白含量、可溶性蛋白含量均降低,而游离氨基酸含量在根、茎、叶中均增加,盐质量分数越大,影响越明显,对根的影响最大,其次是叶,对茎的影响最小。短期盐胁迫的Bt蛋白含量与游离氨基酸含量呈极显著负相关,与可溶性蛋白含量呈极显著正相关。可见,与短期盐胁迫相比,长期盐胁迫棉苗抗虫性更强。     

源库调节对转Bt基因棉花中与抗虫性相关的棉铃氮代谢的影响

以转Bt基因抗虫棉小铃品种泗抗1号和大铃品种科棉6号为材料,于2009～2010年在扬州大学实验农牧场进行了源库调节对棉铃氮代谢影响的试验。结果表明,与对照相比,去除棉株每张叶片的一半后,体积膨大期棉铃铃重变小,氮代谢有关化合物含量和酶活性提高,且大铃品种科棉3号受影响较大;整株均匀去除一半蕾,则使得铃重变大,氮代谢有关化合物含量和酶活性下降,仍然是大铃品种受影响较大。相关分析结果表明,源库调节下体积增大期棉铃氮代谢强度与铃重基本上呈显著或极显著负相关。因此转Bt基因棉花群体源小库大或源大库小均不利于棉铃生理氮代谢与棉铃发育的协调,同样不利于产量和抗虫性的协同表达。     

Reduced square Bacillus thuringiensis insecticidal protein content of transgenic cotton under N deficit

To clarify the effect of the N deficit on the amount of square Bt insecticidal protein, different N application rates(0, 75, 150, 225, and 300 kg ha–1) were imposed on the conventional cultivar Sikang 1(SK-1) and hybrid cultivar Sikang 3(SK-3) during 2015–2016 cotton growth seasons. Under different N application rates, the square number per plant, square volume and square dry weight reduced when the N rates decreased from conventional rate(300 kg ha–1) to 0 kg ha–1. And the square Bt protein content decreased accordingly. The analysis of N metabolism showed that soluble protein content, GPT and GOT activities decreased, free amino acid, peptidase and protease activities increased under N deficit. Correlation analysis indicated that the reduced Bt protein content under N deficit was related to altered N metabolism. In conclusion, square development and the amount of square Bt toxin both decreased under N deficit, indicating that promoting the square development under appropriate N application rate would also promote the insect resistance during squaring stage.     

温度和土壤水分与Bt棉铃壳中杀虫蛋白表达关系及其氮代谢生理机制

【目的】明确温度和土壤水分对Bt棉杀虫蛋白含量及其氮代谢活性的影响,为生产中Bt棉抗虫性的安全稳定利用提供理论参考。【方法】2016-2017年以转Bt抗虫基因抗虫棉常规品种泗抗1号（SK1）和杂交种泗抗3号（SK3）为材料,采用盆栽法,设置29℃、32℃、35℃、38℃ 4个温度水平,土壤最大持水量的80%、70%、60%、50%、40% 5个土壤水分水平,观察温度和土壤水分对Bt棉铃壳杀虫蛋白含量的影响,各处理持续胁迫4 d。2016年主要研究各处理对Bt棉铃壳中杀虫蛋白含量的影响;在此基础上,2017年进一步探讨各处理对铃壳中可溶性蛋白含量、谷氨酸草酰乙酸转氨酶（GOT）、蛋白酶、肽酶活性的影响。【结果】SK1和SK3杀虫蛋白含量均在32℃、最大持水量为60%时最高,分别达到471.1 ng·g^-1 FW和351.7 ng·g^-1 FW。在同一土壤水分条件下,32℃最利于SK1和SK3杀虫蛋白表达;同一温度条件下,最大持水量60%利于SK1和SK3杀虫蛋白表达。对杀虫蛋白含量与温度和土壤水分关系进行二元多项式回归分析发现,Bt棉杀虫蛋白含量（Y）与温度（X₂）和土壤水分（X₁）呈二元二次方程关系,其中SK1、SK3相关方程分别为Y=-3230.2+17.2X₁+199.1X₂-0.3X₁²-3.7X₂²-0.7X₁X₂ （r＝0.829^**）、Y=-3322.0+40.7X₁+145.2X₂-0.3X₁²-2.0X₂²-0.3X₁X₂（r＝0.739^**）。SK1的杀虫蛋白表达量最大的温度和土壤水分条件为31.8℃、57.8%,SK3为33.2℃、60.8%。氮代谢相关生理特征表明,SK1和SK3均表现为在32℃和土壤含水量为60%处理下,棉铃中可溶性蛋白含量、谷氨酸草酰乙酸转氨酶（GOT）活性较高,蛋白酶、肽酶活性较低;杀虫蛋白含量与可溶性蛋白和GOT活性呈极显著正相关关系（r＝0.613**;r＝0.735**）,与蛋白酶活性和肽酶活性呈极显著负相关关系（r＝-0.724**;r＝-0.738**）。【结论】温度和土壤水分通过调控蛋白质分解和合成,共同影响Bt棉杀虫蛋白表达,且与其含量呈二元二次方程关系。     

转Bt基因抗虫棉Bt基因表达的时空动态

以转Bt基因抗虫棉 33B为材料 ,利用ELISA检测方法 ,通过对棉株不同发育时期和同一时期的不同组织或器官Bt晶体蛋白含量的测定 ,研究了转Bt基因抗虫棉Bt基因表达的时空变化。结果表明 ,随着棉株生育进程的推进和株体的老化 ,Bt晶体蛋白含量随着植株体内可溶性总蛋白含量的逐渐降低而降低 ,而Bt基因的表达强度从苗期到蕾期随着棉株营养生长的加快而呈上升趋势 ,至蕾期达到高峰 ,以后逐渐减弱。不同组织或器官Bt晶体蛋白的含量也有较大差异 ,表现在幼嫩组织或器官的含量较高 ,成熟组织或器官次之 ,衰老组织最低。这说明Bt基因表达强度的减弱和Bt晶体蛋白含量的降低是转Bt基因抗虫棉生育后期抗虫性降低的根本原因     

Bt转基因棉氮代谢生理变化的研究

在田间条件下,对Bt杂交棉科棉1号和常规棉新洋822及其亲本的氮代谢生理情况进行研究。结果表明：Bt棉的氮代谢较强,以盛铃期最为明显,表现为叶片全氮含量明显升高,科棉1号和新洋822在盛铃期分别比亲本增加36．01％和18．96％．而棉铃全氮含量显著下降。氨基酸和可溶性蛋白质含量显著提高,盛铃期科棉1号和新洋822叶片游离氨基酸分别增加10．86％和9．44％,可溶性蛋白分别增加9．82％和8．31％。叶片硝酸还原酶(NR)、丙酮酸转氨酶(GPT)活性增强,科棉1号和新洋822盛蕾期NR活性比其亲本分别高28．37％和12．83％;GPT活性在盛蕾期和盛铃期比亲本分别增加10．51％、4．38％和39．04％、29．05％,蛋白酶活性则明显下降。因此对于Bt基因导入的棉花品种在推广应用过程中应及时调控棉株氮代谢生理强度,以保持营养生长和生殖生长的平衡。     

Bt棉功能叶氮代谢对Bt蛋白表达及氮肥调节的响应

为评价外源Bt基因插入以及氮肥对Bt棉功能叶Bt蛋白表达及氮代谢的影响,对Bt棉功能叶Bt蛋白含量,硝酸还原酶、谷丙转氨酶和蛋白酶活性,可溶性蛋白质和全氮含量等指标进行了测定。结果表明:Bt棉功能叶Bt蛋白含量在蕾期最高,此后,随生育进程呈显著下降。与常规棉相比,Bt棉功能叶硝酸还原酶活性提高,在前中期谷丙转氨酶活性显著提高且有利于可溶性蛋白质的合成,前期全氮含量显著增加,对蛋白酶活性没有明显影响。Bt棉功能叶Bt蛋白含量与硝酸还原酶活性、可溶性蛋白质和全氮含量呈显著正相关,表明Bt棉前期增强的氮代谢促进了Bt蛋白的表达。施氮肥明显提高Bt棉中后期功能叶硝酸还原酶活性、可溶性蛋白质含量和Bt蛋白的表达。基施和初花期各50%的效应显著大于全部基施,而在基施氮肥基础上,初花期增施50%的氮肥效应最明显。与常规棉比较,施氮肥更有利于Bt棉中后期功能叶硝酸还原酶活性、可溶性蛋白质含量提高。     

转Bt基因抗虫棉33B的氮素代谢特征

以转B t基因抗虫棉新棉33B及其常规棉对照品种泗棉3号为材料,探讨转B t基因抗虫棉氮素代谢特征。结果显示:转基因抗虫棉33B主茎功能叶片中的全N含量、可溶性蛋白含量、硝酸还原酶(NR)活性以及主要氨基酸总量和游离氨含量均高于常规棉,其氮代谢十分旺盛;转基因抗虫棉33B的棉铃对位叶、铃壳中的全N含量亦高于常规棉,表明转基因抗虫棉33B具有较强的源、库生理优势。但在棉铃发育中后期,转基因抗虫棉33B营养物质向棉铃运输速度比常规棉低,从而导致单铃重减轻,衣分下降。     

种植密度对转Bt棉纤维杀虫蛋白表达量及氮代谢的影响

为了探讨种植密度对转基因抗虫棉纤维中杀虫(Bt)蛋白表达量的影响及其氮代谢机制,以Bt棉常规种泗抗1号和Bt棉杂交种泗抗3号为试验材料,比较1.5× 104、3.0×104、4.5×104、6.0×104、7.5×104株·hm-2五个种植密度下转Bt棉杀虫蛋白表达量及其氮代谢的差异.结果 表明,纤维中Bt蛋白表达量随...     

不同施氮量对转Bt基因棉主要害虫种群动态的影响

研究结果表明,在低、中、高3种施氮肥水平处理条件下,随着棉田施氮量的增加,转Bt基因棉棉铃虫落卵量和幼虫数量增加,棉苗蚜、伏蚜、棉叶螨种群数量减少,但差异均未达显著水平。     

转Bt基因棉对棉铃虫及其天敌发生的影响

1999年和2000年对转基因棉田棉铃虫及天敌进行了定点调查。结果表明：转基因棉对棉铃虫成虫、卵无驱避作用和抗性表现，不影响棉铃虫在植株上的落卵量，但转基因棉第二、三代棉铃虫幼虫数量与常规棉相对显著减少，存在明显抗虫性。转基因棉不影响捕食性天敌的数量。     

转Bt基因抗虫棉Bt毒蛋白表达量的时空变化

采用抗体夹心ELISA技术 ,对转Bt基因抗虫棉植株中Bt毒蛋白含量进行了测定。结果表明 :Bt基因在所检测的器官中均有表达 ,但是不同器官中的Bt毒蛋白含量明显不同。在苗期全展功能叶中Bt毒蛋白含量最高 ,根、茎和叶柄中含量较低 ;不同生育期的功能叶中Bt毒蛋白含量差异显著。Bt毒蛋白含量在苗期叶片中最高 ,蕾期次之 ,花铃期最低。随着棉花生长发育进程的推进 ,Bt基因在叶片中的表达强度逐渐减弱。Bt基因在棉株体内的表达随着器官的不同 ,生育时期的不同而表现出时空动态变化。     

高温胁迫对Bt转基因棉叶片毒蛋白含量的影响

以科棉1号为试验材料,研究连续48 h的40℃高温胁迫对棉花果枝叶片游离氨基酸、可溶性蛋白和Bt毒蛋白含量的影响.结果表明高温胁迫后叶片游离氨基酸含量一直增加,其中以胁迫后12 h内增加速度最快;可溶性蛋白含量急剧下降,胁迫后24 h内下降最快;叶片Bt毒蛋白含量的变化趋势与可溶性蛋白基本一致.高温胁迫后可溶性蛋白含量与Bt毒蛋白含量呈极显著正相关,游离氨基酸含量则与Bt毒蛋白含量呈极显著负相关.与盛花期相比,结铃盛期高温胁迫对Bt毒蛋白含量的影响更大.     

一熟转Bt基因棉田昆虫群落研究

以转Ｂｔ基因棉品系Ｒ９３－４ 为试材,常规棉中棉所１２ 为对照,研究了转Ｂｔ基因棉田昆虫群落的变化。结果表明,转基因棉田昆虫群落、害虫和天敌亚群落的种类丰富度、均匀度和优势集中性均高于常规棉,总个体数、害虫的相对丰盛度和多样性指数均低于常规棉;优势害虫种类的变化较大,棉铃虫已不是转基因棉田的优势害虫,棉蚜、红蜘蛛、叶甲类、棉蓟马等已上升为优势害虫;优势捕食性天敌种类无明显变化,仍为龟纹瓢虫和小花蝽。转基因棉田采取综合防治后昆虫群落和害虫亚群落与常规棉相比有较好的稳定性。     

转Bt基因棉与棉田绿盲蝽趋性有关的生理特征分析

以转Bt基因棉苏抗310及其轮回亲本泗棉3号为材料,研究转Bt基因棉绿盲蝽发生危害特点及棉相关生理代谢特征。结果表明:绿盲蝽在转Bt基因棉苏抗310上危害程度重于常规棉泗棉3号,且苏抗310缺氮处理轻于苏抗310适氮处理,顶芽和幼蕾的可溶性糖含量与绿盲蝽的危害程度趋势呈正相关,与缩合单宁含量和苯丙氨酸解氨酶活性呈负相关,说明转Bt基因棉顶芽和幼蕾的可溶性糖含量提高,缩合单宁含量和苯丙氨酸解氨酶活性降低,对绿盲蝽发生和发育有利,这可能是转Bt基因棉绿盲蝽危害程度加重的重要生理特征。     

转Bt基因棉花秸秆对土壤多酚氧化酶、纤维素酶活性和毒素的影响

用转Bt基因抗虫棉秸秆和非转基因棉花秸秆分别处理同种土壤,在不同时期分析土壤多酚氧化酶、纤维素酶活性及土壤毒素的变化。结果表明:两个棉花秸秆处理之间及其与对照之间的土壤多酚氧化酶没有差异;两个棉花处理均可明显提高土壤纤维素酶活性,而以非转基因棉花秸秆处理的酶活性略高一些,但两者差异不显著;与对照相比,以非转基因棉花秸秆处理的土壤毒素略强一些,而转Bt基因抗虫棉秸秆处理与对照相差不大,说明转Bt基因抗虫棉秸秆不会增加土壤的毒素。因此,转Bt基因抗虫棉残留物对土壤多酚氧化酶、纤维素酶活性及毒素没有明显影响,该研究结果可为评价转Bt基因抗虫棉的安全性提供依据。