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。     

转Bt基因抗虫棉"33B"的源库特征

以转Bt基因抗虫棉"33B"及对照品种"泗棉3号"为材料,研究转Bt基因抗虫棉的源库特征。结果表明,转Bt基因抗虫棉33B源器官的生长呈前期缓慢,中后期旺盛的趋势,且叶源质量明显优于常规棉泗棉3号,表现较强的叶源生理优势;33B成铃期后延且集中,单铃库容量较泗棉3号小,但单株库容量大,结铃多,有较高的库容优势。研究结果进一步证实,单株结铃多是转Bt基因抗虫棉33B增产的主要原因。33B虽然源库基本协调,但叶片、铃壳贮存的营养物质向纤维输送速度较缓,部分贮存物质被滞留在叶片和铃壳中,铃期相对较长,铃壳率较高;营养物质流向种子偏多,导致单铃重减轻,衣分下降。针对转Bt基因抗虫棉33B的源库特征,提出适期迟播,增加密度,促进早发,适度化调,增施微肥等调控技术。     

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。     

不同氮素水平对棉花不同部位—铃叶系统生理特性及铃重空间分布的影响

 【目的】研究氮素营养对棉株主要源库关系的调节作用。【方法】以转基因抗虫杂交棉中棉所29（CCRI29）为材料，在田间进行不同氮肥水平（高氮：225.0 kg•ha-1；中氮：112.5 kg•ha-1；低氮：0 kg•ha-1）试验，较为系统地研究了CCRI29中、下部铃-叶系统生理特性及铃重的空间分布。【结果】（1）与中氮、低氮相比，高氮促进了中部果枝叶生长后期叶绿素的合成，延长了叶片功能期。（2）氮肥水平对棉花器官中碳水化合物代谢有重要影响，低氮促进了果枝叶中碳水化合物的积累，中氮水平下碳水化合物代谢较为协调。（3）施入氮肥可以增加POD、SOD酶活性。不同部位棉铃对位叶的SOD、POD酶活性变化趋势一致，下部棉铃对位叶中各处理间比较为：中氮＞高氮＞低氮，而中部棉铃对位叶各处理间顺序为高氮＞中氮＞低氮。【结论】不同氮肥水平对棉花的生长发育有一定调节效应。中氮水平可以改善棉花的早衰现象，源库关系较为协调，从而促进了铃重的增加。     

高产棉花整株铃重的提高与棉铃内源激素GA3及ZR的关系

对高产棉花棉株不同部位棉铃发育的系统研究表明 :棉株中部铃发育期赤霉素 (GA3 )含量与棉铃体积的增大密切相关 ,上部铃在内部充实期GA3 含量则对棉铃增重具有重要的诱导作用 ;不同部位棉铃中玉米素 (ZR)含量的高低则与整个棉铃发育期铃重的增长量呈显著正相关。说明整株棉铃的增重受棉铃内源激素GA3 和ZR含量水平的调节。     

钾肥运筹对高品质棉不同部位棉铃对位叶养分分配的影响

研究了钾肥运筹对高品质棉杂交种科棉3号和常规种科棉4号不同部位棉铃对位叶养分分配的影响。结果表明,通过合理的钾肥运筹能有效地促进高品质棉株中、上部棉铃对位叶中养分的合理分配,从而促进棉株上部与中部棉铃铃重的增加和纤维的发育,有利于提高整株铃重和纤维品质。高品质棉杂交种科棉3号钾肥运筹以基肥和花铃肥的比例为5∶5、常规种科棉4号基肥和花铃肥的比例为7∶3时,更有利于棉株中上部的棉铃对位叶中养分的分配,从而增加铃重,缩小上部与中部的铃重差异。     

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。     

不同铃重类型棉花不同结铃区位的源库特征研究

对5个不同铃重类型棉花品种不同区位的棉铃及对应位果枝叶主要生长与生理指标变化的规律研究,结果表明:源、库的生育性状受品种铃重类型的影响;同一品种的源、库特征受时空变化的影响。大铃型品种果枝叶中叶绿素和可溶性糖含量均高于小铃品种,且高值持续期长。受品种铃重类型和叶源质量的影响,大铃品种的籽棉干物质积累的时间较小铃品种长,且大、小铃品种单铃籽棉干物质日增量差异最大的时期在棉铃生长发育的后期。大铃品种最大单铃重集中在中、上部内围果节;小铃品种的最大单铃重集中在上部果枝     

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基因棉花品种与绿盲蝽危害相关的几个生理指标特征分析

研究了转Bt基因棉不同品种的生理代谢特征及其与棉盲蝽危害的关系。结果表明:在供试的6个棉花品种中,棉花绿盲蝽发生量和危害程度以转Bt基因杂交棉品种最高,转Bt基因常规棉品种次之,非转基因常规棉品种最低。顶芽和幼蕾的可溶性糖含量与绿盲蝽发生量和危害程度呈显著或极显著的正相关,而缩合单宁含量和PLA活性与之则呈显著或极显著的负相关。顶芽和幼蕾的可溶性糖含量以转Bt基因杂交棉品种最高,转Bt基因常规棉品种次之,非转基因常规棉品种最低;缩合单宁含量、PLA活性以非转基因常规棉品种最高,转Bt基因常规棉品种次之,转Bt基因杂交棉品种最低。棉花植株氮素积累量与顶芽和幼蕾的可溶性糖含量呈显著或极显著正相关,而与缩合单宁含量和PLA活性呈显著或极显著的负相关;蕾期和铃期的氮素积累量以转Bt基因杂交棉品种最高,转Bt基因常规棉品种次之,非转基因常规棉品种最低。上述结果表明,转Bt基因棉花品种(尤其是转Bt基因杂交棉品种)在蕾期和铃期的氮素积累量高,引起了顶芽和幼蕾的可溶性糖含量提高,缩合单宁含量和PLA活性降低。     

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.     

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.     

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.     

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。     

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.     

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"。