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.     

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。     

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蛋白表达量随种植密度增大呈上升趋势。相关分析表明,单株铃数、单铃体积、单铃干重与Bt蛋白表达量均呈显著负相关。纤维中氮代谢生理相关指标中,可溶性蛋白含量及谷氨酸丙酮酸转氨酶(GPT)和谷氨酸草酰乙酸转氨酶(GOT)活性随种植密度的增大而升高,但游离氨基酸含量、蛋白酶和肽酶活性随密度增大而下降。纤维中Bt蛋白表达量与可溶性蛋白含量、GPT和GOT活性呈显著正相关,而与游离氨基酸含量、蛋白酶和肽酶活性呈显著负相关。可见,提高种植密度降低了棉铃生长,但提高了纤维中Bt蛋白的表达量,且Bt蛋白表达量的提高与植株氮代谢生理密切相关。因此,在生产中通过协调种植密度既可以保持棉铃适度发育,又有利于提高棉纤维中Bt蛋白的表达量,为提高Bt棉产量器官抗虫性提供栽培基础。     

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.     

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.     

土壤水分亏缺对Bt棉铃壳杀虫蛋白表达的影响

【目的】研究土壤水分亏缺对Bt棉杀虫蛋白含量及Bt基因表达、氮代谢酶活性的影响,为Bt棉抗虫性安全表达提供理论参考。【方法】2014—2015年以Bt棉常规品种泗抗1号、杂交种泗抗3号为材料,采用盆栽法,2014年设置5个土壤水分处理:G1、G2、G3、G4和CK,其土壤含水量分别为最大持水量的15%、30%、45%和75%。2015年设置4个处理:G2、G3、G4和CK。观察土壤水分亏缺对盛铃期Bt棉铃壳杀虫蛋白含量影响。所有处理于盛花期前10 d控制浇水,如遇下雨,将处理盆钵移入室内。使用WET土壤三参数速测仪监测土壤水分,用称重法控制土壤水分,即当监测发现土壤水分低于设计值时,于早晨、中午、傍晚进行定量补水。2015年进一步研究水分亏缺对Bt基因表达量、氮代谢相关合成酶(硝酸还原酶和谷氨酸丙酮酸转氨酶)活性、分解酶(肽酶和蛋白酶)活性的影响。【结果】与对照(土壤含水量为最大持水量75%)相比,泗抗1号和泗抗3号铃壳中Bt蛋白含量随土壤水分亏缺程度的增加而降低,且在土壤含水量为最大持水量60%时开始显著下降,但泗抗1号下降幅度低于泗抗3号,其中2014年泗抗1号下降22.5%,泗抗3号下降41.6%。在土壤含水量为最大持水量60%时,铃壳中Bt基因表达量增加,泗抗1号、泗抗3号分别比对照提高48.6%和22.1%。氮代谢相关酶活性变化表明,水分亏缺条件下,2个类型品种的硝酸还原酶(NR)和谷氨酸丙酮酸转氨酶(GPT)活性降低,肽酶和蛋白酶活性增加。且肽酶和蛋白酶活性变化幅度高于NR和GPT。相关分析表明,NR和GPT活性与铃壳中杀虫蛋白含量呈显著或极显著正相关;肽酶和蛋白酶活性与杀虫蛋白含量呈显著负相关。【结论】水分亏缺胁迫下,供试品种铃壳中杀虫蛋白质含量下降。但在转录水平,未发现Bt基因表达量下降。但氮代谢关键合成酶(NR和GPT)活性降低,分解酶(肽酶和蛋白酶)活性增加。因此,蛋白质合成减弱、分解加强可能导致铃壳中杀虫蛋白含量下降。     

Enhancing boll protein synthesis and carbohydrate conversion by the application of exogenous amino acids at the peak flowering stage increased the boll Bt toxin concentration and lint yield in cotton

In Bacillus thuringenesis (Bt) transgenic cotton, the cotton boll has the lowest insecticidal protein content when compared to the other organs. The present study investigated the effects of amino acid spray application at the peak flowering stage on the cotton boll Bt toxin concentration and yield formation. Boll protein synthesis and carbohydrate conversion were also studied to reveal the fundamental mechanism. Three treatments (i.e., CK, the untreated control; LA1, five amino acids; LA2, 21 amino acids) were applied to two Bt cultivars of G. hirsutum (i.e., the hybrid Sikang 3 and the conventional Sikang 1) in the cotton-growing seasons during 2017 and 2018. Amino acid spray application at the peak flowering stage resulted in an increase of 5.2–16.4% in the boll Bt protein concentration and an increase of 5.5–11.3% in the seed cotton yield, but there was no difference between the two amino acid treatments. In addition, amino acid applications led to increases in the amino acid content, soluble protein content, glutamate pyruvate transaminase (GPT) activity, glutamate oxaloacetate transaminase (GOT) activity, glucose content, fructose content and soluble acid invertase (SAI) activity. This study also found that Bt protein content, enhanced boll number and the weight of opened bolls were closely related to carbon and nitrogen metabolism. The Bt protein content had significant linear positive correlations with amino acid and soluble protein contents. Enhanced boll number had significant linear positive correlations with the GPT and GOT activities from 15–25 days after flowering (DAF). The weight of opened bolls from 55–65 DAF had a significant linear positive correlation with the SAI activity. These results indicate that the enhancement of boll protein synthesis and carbohydrate conversion by amino acid application resulted in a simultaneous increase in the boll Bt protein concentration and cotton lint yield.     

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。     

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。     

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基因抗虫棉的抗虫性与主要经济性状的相关分析

分析了转Bt基因抗虫棉的抗虫性与产量性状、纤维品质性状以及抗病性状之间的相关性 ,结果表明 :转Bt基因抗虫棉的抗虫性与衣分存在极显著负相关 (R单 =-0 .870 、R偏 =-0 .972 ) ,与衣指存在显著负相关 (R单 =-0 .62 9 、R偏 =-0 .889 ) ,与籽指存在显著正向单相关 (R单 =0 .71 8 ) ,与株高、果枝数、结铃数、单铃重及籽棉产量之间相关不显著。抗虫性增强对纤维品质的不利相关效应有 :整齐度变差 (R偏 =-0 .988 )、伸长率下降 (R单 =-0 .680 )、气纱品质变差 (R偏 =-0 .992 ) ;对纤维品质的有利相关效应有 :反射率表现增强 (R偏 =0 .897 )。抗虫性与黄萎病病指之间存在极显著负相关 (R单 =-0 .868 、R偏 =-0 .90 0 ) ,但与枯萎病病指之间不存在内在相关性。     

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。     

陆地棉抗虫品种不同年份间农艺性状的比较

笔者对3个陆地棉抗虫品种在两年间的农艺性状和纤维品质性状进行了研究。结果表明,3个抗虫棉品种的产量性状年份间表现基本一致,而纤维品质性状差异较大。新棉33B在3个抗虫棉品种中产量性状表现最好。品种与年份的互作极为显著,2002年新棉33B在籽棉产量和皮棉产量上显著高于对照品种中棉所12号,而在2003年则与对照品种产量相当。JS166在单株籽棉产量和单株皮棉产量、单株铃数、单铃重等性状上两年间表现均较好,2003年还有较低的蕾铃脱落率。99035两年间在产量性状上与对照相比略有提高,但差异并不明显。所有3个抗虫棉品种2002年的纤维品质指标与对照相当,而2003年纤维长度和纤维整齐度明显高于对照。     

江苏沿海地区Bt棉主栽品种的性状评价

室内饲养结果表明,与对照品种"苏棉9号"比较,4个Bt棉品种对棉铃虫的抗性都达到极显著水平;饲喂Bt棉组织,能使棉铃虫幼虫发育延缓、蛹期缩短和蛹重减轻;1～3龄棉铃虫幼虫对Bt棉的敏感程度高于4龄以上幼虫。田间调查结果表明,Bt棉田棉铃虫的虫量和蕾铃被害量与对照棉田差异极显著,其趋势与室内试验一致。在棉花品种的生育特性方面,4个Bt棉品种的株高和果枝数与"苏棉9号"相近;蕾铃数量及成铃率高于或接近于常规棉;各品种间生长发育指标的差异均达不到显著水平。按照供试的4个Bt棉品种在当地的抗虫性及丰产性状的优劣排序,依次为"鲁棉研15">"中棉所29">"南抗3号">"科棉1号"。     

转基因抗虫玉米Bt毒蛋白的时空表达分析

对2个转Bt基因玉米抗虫系株系进行Bt毒蛋白表达量的分析测定。结果显示,转基因株系中的毒蛋白可以在其后代稳定的遗传和表达,毒蛋白的表达为组成性表达,在玉米的各个发育时期和植株的各个部位均有表达,但其表达的量有显著差异,毒蛋白表达量随着植株的生长而减少,比较植株的各个器官,叶片毒蛋白含量最高,茎次之,根、种子和花丝中的毒蛋白含量较少。     

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。     

Effects of External Chemical Regulation on Bt Transgenic Cotton Plants under Combined Stress of High Temperature and Water Deficit

[Objective] The study aimed to find a possible way to combat or alleviate the negative effects caused by high temperature and water deficit at the growth stage of peak boll-setting.[Method] With Bt transgenic cotton GK22 as the test cultivar,a potted experiment was carried out to investigate the effects of the regulation of external substances(the water solutions of pix,urea and their mixture) on the physiological parameters,insecticidal protein content,yield and yield component of cotton plants in artificial climate chambers treated with high temperature and water deficit.[Result] The application of external pix,urea or their mixture was effective in stabilizing the physiological parameters of cotton plants,insecticidal protein content,yield and yield components.Compared with the exclusive application of pix and urea,the mixture of pix and urea played the most effective role in stabilizing the content of chlorophyll,soluble sugar and insecticidal protein,alleviating the increase of the content of free amino acids and proline,and increasing boll number per plant,boll weight and seed cotton yield.[Conclusion] The water solutions of pix,urea or their mixtures can be used to combat or alleviate the stress of high temperature and water deficit if they are sprayed onto cotton plants prior to stress occurrence.     

The effects of high temperature level on square Bt protein concen-tration of Bt cotton

Higher boll worm survival rates were detected after high temperature presented during square period in Bt cotton. The objective of this study was to investigate the effects of high temperature level on the Bt efficacy of two different types of Bt cotton cultivars at squaring stage. During the 2011 to 2013 cotton growth seasons, high temperature treatments ranged from 34 to 44°C in climate chambers, and field experiments under high temperature weather with various temperature levels were conducted to investigate the effects of the high temperature level on square Bt protein concentration and nitrogen metabolism. The climate chamber experiments showed that the square insecticidal protein contents reduced after 24 h elevated temperature treatments for both cultivars, whereas significant declines of the square insecticidal protein contents were detected at temperature 38°C, and only slightly numerical reductions were observed when temperature below 38°C. Similar high temperature responses were also observed at the two field experimental sites in 2013. Correspondingly, high temperature below 38°C seems have little effect on the square amino acid concentrations, soluble protein contents, glutamic-pyruvic transaminase(GPT) and glutamic-oxalacetic transaminase(GOT) activities as well as protease and peptidase activities; however, when the temperature was above 38°C, reduced soluble protein contents, enhanced amino acid concentrations, decreased GPT and GOT activities, bolstered protease and peptidase activities in square were detected. In general, the higher the temperature is(38°C), the larger the changes for the above compound contents and key enzymes activities of the square protein cycle. The findings indicated that the unstable insect resistance of the square was related to high temperature level during square stage.     

转Bt基因棉的检测和抗虫性鉴定研究进展

综述了转Bt基因棉的检测和抗虫性鉴定的研究概况,分别从外源Bt基因的检测、Bt毒蛋白的检测、标记基因的检测和抗虫性的鉴定4个方面进行了探讨,并提出了一些有待于研究的问题.