转Bt基因抗虫棉(Gossyposium)伤流中Bt毒蛋白的运输

本文以常规棉品种‘石远321'和抗虫棉品种‘SGK321'与‘99B'为材料,通过考马斯亮蓝和ELISA方法分析了伤流中可溶性蛋白和Bt(苏云金芽孢杆菌)毒蛋白含量.结果发现,转基因抗虫棉伤流中可溶性蛋白含量显著高于常规棉,并在抗虫棉伤流中检测到了Bt毒蛋白,而常规棉伤流中没有Bt毒蛋白的存在.通过嫁接实验研究发现,将常规棉嫁接到转Bt基因抗虫棉砧木上,叶片中也有Bt毒蛋白的积累.这些结果说明,Bt毒蛋白可以通过木质部伤流液向地上部运输,并在叶片中积累,Bt毒蛋白由根系向地上部的运输对棉花的抗虫性是有益的.     

Bt棉与常规棉根际土壤Bt毒蛋白和植物激素变化动态

通过ELISA法对常规棉（石远321）和转基因抗虫棉（99B、SGK321）根际土壤苏云金芽孢杆菌（Bt）毒蛋白和植物激素含量进行了分析,结果表明抗虫棉根际土壤Bt毒蛋白含量显著高于常规棉,特别是从苗期到盛花期抗虫棉根际土壤Bt毒蛋白含量高出常规棉200-300ng／g。抗虫棉根际土壤中脱落酸（ABA）和生长素吲哚乙酸（IAA）含量变化趋势与常规棉相比表现出较大差异。常规棉与抗虫棉根际土壤中ABA变化趋势表现为相似的单峰曲线,而抗虫棉根际土壤中ABA含量变化幅度明显小于常规棉。在棉花生长发育后期抗虫棉根际土壤中赤霉酸（GA3）含量较高,而常规棉根际土壤中GA,含量升高较抗虫棉早且变化幅度大,在高峰处常规棉高于抗虫棉,低谷时抗虫棉高于常规棉。7月17日常规棉根际土壤IAA含量显著高于抗虫棉,8月27日抗虫棉又大大高于常规棉,其他时期变化不大。上述结果表明,外源Bt基因的插入或表达能引起棉花根际土壤中Bt毒蛋白和植物激素含量的较大改变,这种改变可能会影响到抗虫棉的生长发育,并对环境产生影响。     

北疆棉区Bt棉及土壤中Bt毒蛋白含量测定

目的:转Bt基因抗虫棉植株中Bt毒蛋白含量测定.方法:采用ELISA法对转Bt基因植株中毒蛋白含量进行了定量检测.结果:在所检测的样品中,叶片中Bt毒蛋白含量最高,其次是蕾、铃、花瓣、花苞叶、蕾苞叶、花柱,而在根中的含量较低,土样中没有检测到Bt毒蛋白;不同生育期的顶叶中Bt毒蛋白含量差异显著,其在苗期叶片中最高,蕾期次之,花期、花铃期开始降低,而铃期和吐絮期又有一定的增加.结论:Bt基因在植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.     

转基因棉Bt毒蛋白含量与抗棉大卷叶螟效果间的关系分析

棉大卷叶螟Sylepta derogata Fabricius为近年来长江流域棉花中后期的一种重要害虫。在其危害盛期测定了8种转基因棉叶片Bt毒蛋白的含量与受害程度,在此基础上就转基因棉对棉大卷叶螟的抗虫性采用不同抗性指标相结合的方法进行了综合评估,同时对转基因棉对棉大卷叶螟的抗虫效果与毒蛋白含量的相关性进行分析。结果表明:各转基因棉品种的不同部位叶片的毒蛋白含量呈现顶叶最高,功能叶次之,老叶含量最低的趋势。抗蚜8017和SGK321 2个转基因棉品种的棉叶毒蛋白表达量相对较低,平均值在120μg/g以下,对棉大卷叶螟的抗性级别均为中抗。其它供试转基因棉棉叶毒蛋白表达量均值在150μg/g以上,抗性级别也均为高抗,而非转基因棉泗棉3号与石远321对棉大卷叶螟不具抗性。转基因棉对棉大卷叶螟的抗性水平与毒蛋白含量呈正相关。     

种植转Bt基因棉土壤中Bt蛋白的分布

采用盆栽试验结合酶联免疫法(ELISA)测定了转Bt基因抗虫棉及常规棉花不同生育期根际及非根际土壤中的Bt蛋白含量.结果表明:红壤、黄棕壤及黄褐土种植转Bt基因棉花后根际土中Bt蛋白含量显著高于非根际土,而种植常规棉花的根际土与非根际土中Bt蛋白含量差异不显著.在初蕾期转Bt基因棉根际土中的Bt蛋白含量为黄褐土＞黄棕壤＞红壤,分别为常规棉根际土的144%、121%和238%;而在盛花期为黄棕壤＞黄褐土＞红壤,分别为常规棉根际土的156%、116%和197%.无论种植转Bt基因棉还是常规棉,供试土壤的根际和非根际土中Bt蛋白含量都随棉花生育期的推进先增加后减少,在盛花期达最大值.整个生育期内,转Bt基因棉根际土中的Bt蛋白含量大于其非根际土;种植转Bt基因棉土壤中Bt蛋白含量高于常规棉,说明转Bt基因棉花的Bt蛋白可释放到根际土中.     

高温对Bt棉盛蕾期蕾中Bt蛋白表达及氮代谢生理的影响

以来源于中国的Bt棉品种泗抗1号(常规种)、泗抗3号(杂交种)和来源于美国的Bt棉品种99B(常规种)、岱杂1号(杂交种)为材料,研究了不同高温对Bt棉盛蕾期蕾中Bt蛋白表达量变化及氮代谢生理特征的影响.结果表明: 与对照(32 ℃)相比,在38 ℃以上高温时,4个品种棉蕾中Bt蛋白表达量均显著下降,且随温度升高,Bt蛋白含量下降幅度增大.杂交种棉蕾中Bt蛋白表达量要高于常规种,且受高温影响较小.Bt杀虫蛋白表达量下降幅度大的品种,其可溶性蛋白含量、谷氨酸丙酮酸转氨酶(GPT)活性、谷氨酸草酰乙酸转氨酶(GOT)活性下降幅度也大,游离氨基酸含量、蛋白酶活性、肽酶活性增加幅度较大.     

土壤盐分对Bt棉铃壳中Bt蛋白含量和氮代谢生理的影响

为探讨土壤盐分对转Bt基因抗虫棉棉铃抗虫性的影响,以2个Bt棉品种‘新棉33B’(盐敏感)和‘中07’(耐盐)为试验材料,设置5个土壤盐分水平(0、0.15%、0.30%、0.45%和0.60%),采用盆栽法研究了不同土壤盐分下Bt棉盛铃期铃壳Bt蛋白含量变化及氮代谢生理特征.结果表明: 铃壳中Bt蛋白含量随土壤盐分的升高而降低,与对照相比(0%),盐敏感型品种在土壤盐分0.15%、耐盐型品种在0.30%以上时,铃壳中Bt蛋白含量显著下降.相同土壤盐分水平下,花后30 d(DPA)铃壳Bt蛋白含量下降幅度明显大于10 DPA.在铃壳Bt杀虫蛋白含量下降显著时,其可溶性蛋白含量明显下降,硝酸还原酶(NR)和谷氨酸丙酮酸转氨酶(GPT)活性显著下降;游离氨基酸含量、蛋白酶和肽酶活性在土壤盐分0.30%以上时显著增加.以上结果说明,土壤盐分影响铃壳的氮代谢,从而引起Bt蛋白合成减弱,中度以上土壤盐分水平会引起Bt蛋白分解加强,从而导致杀虫蛋白表达水平进一步下降.     

盐胁迫对Bt棉棉蕾杀虫蛋白表达的影响

为探讨土壤盐分对转Bt基因抗虫棉棉蕾抗虫性的影响程度,采用盆栽试验,以2个Bt棉品种‘新棉33B’(盐敏感)和‘中07’(耐盐)为试验材料,设置5个土壤盐分水平(0、0.15%、0.30%、0.45%和0.60%), 研究盐分对蕾期Bt棉棉蕾中杀虫蛋白含量、Bt基因相对表达量及氮代谢相关酶和物质代谢的影响.结果表明: 棉蕾中Bt杀虫蛋白含量随土壤盐分的升高而降低,与对照相比(0%),2个不同类型棉花品种在土壤盐分0.30%及以上时,棉蕾中杀虫蛋白含量显著下降,且随土壤盐分水平的升高,Bt 杀虫蛋白含量下降幅度增大.土壤盐分胁迫提高了棉蕾中Bt基因的相对表达量.在相同土壤盐分水平下,盐敏感品种棉蕾中Bt杀虫蛋白含量受土壤盐分影响较大.杀虫蛋白表达量下降幅度大的品种,其可溶性蛋白含量、谷氨酸丙酮酸转氨酶(GPT)和谷氨酸草酰乙酸转氨酶(GOT)活性下降幅度较大,游离氨基酸含量、蛋白酶和肽酶活性上升幅度也较高.说明土壤盐分胁迫下,Bt棉棉蕾蛋白质合成能力下降,分解能力增强,可能是导致杀虫蛋白表达量下降的主要原因.     

高温胁迫对Bt棉铃壳中Bt蛋白含量及氮代谢的影响

以Bt基因来源于中国的棉花品种泗抗1 号(常规种)、泗抗3 号(杂交种)和来源于美国的棉花品种99B(常规棉)、岱杂1 号(杂交棉)为材料,研究了不同高温水平下Bt 棉盛铃期铃壳中Bt 蛋白含量变化及氮代谢生理特征.结果表明: 铃壳中Bt 蛋白含量随温度升高而降低,与对照相比(32 ℃),常规棉品种在38 ℃、杂交棉品种在40 ℃以上时,铃壳中Bt 蛋白含量大幅度下降.其中,常规种泗抗1号和99B在38 ℃时分别下降53.0%和69.5%;杂交种泗抗3号和岱杂1号在40 ℃时下降64.8%和54.1%.铃壳Bt 杀虫蛋白含量下降显著时,其可溶性蛋白含量明显下降,游离氨基酸含量明显提高,GPT活性显著下降,蛋白酶活性显著增加.高温影响铃壳的氮代谢引起Bt蛋白的分解加剧,合成减弱,从而造成Bt蛋白含量减少,抗虫性下降.     

The relationship between Bt toxin content and resistance to Sylepta derogata in various transgenic Bt cotton varieties

棉大卷叶螟Sylepta derogata Fabricius为近年来长江流域棉花中后期的一种重要害虫.在其危害盛期测定了8种转基因棉叶片Bt毒蛋白的含量与受害程度,在此基础上就转基因棉对棉大卷叶螟的抗虫性采用不同抗性指标相结合的方法进行了综合评估,同时对转基因棉对棉大卷叶螟的抗虫效果与毒蛋白含量的相关性进行分析.结果表明:各转基因棉品种的不同部位叶片的毒蛋白含量呈现顶叶最高,功能叶次之,老叶含量最低的趋势.抗蚜8017和SGK321 2个转基因棉品种的棉叶毒蛋白表达量相对较低,平均值在120μg/g以下,对棉大卷叶螟的抗性级别均为中抗.其它供试转基因棉棉叶毒蛋白表达量均值在150μg/g以上,抗性级别也均为高抗,而非转基因棉泗棉3号与石远321对棉大卷叶螟不具抗性.转基因棉对棉大卷叶螟的抗性水平与毒蛋白含量呈正相关.     

Changes of Bt Toxin in the Rhizosphere of Transgenic Bt Cotton and its Influence on Soil Functional Bacteria

Summary The concentrations of Bacillus thuringensis (Bt) toxin released from root exudation of Bt cotton were measured by an enzyme-linked immunosorbent assay (ELISA), and its impacts on the numbers of culturable functional bacteria in the rhizosphere were determined by cultivation. No Bt toxin was found in the rhizosphere of non-Bt cotton (SHIYUAN321), but varying levels of Bt toxin were present in the rhizosphere of two Bt cotton varieties (NuCOTN99^B and SGK321) during the entire growth period. The levels of Bt toxin in the rhizosphere of NuCOTN99^B were significantly higher (p<0.05) than those of SGK321 within all sampling dates except on June 17th in the whole growth season. Significant differences (p<0.05) were found in the numbers of the three functional bacteria between SHIYUAN321 and NuCOTN99^B within each sampling day from May 27th to October 27th. No significant differences were found in the numbers of functional bacteria among three cultivars after growth season. Fortification of pure Bt toxin into rhizospheric soil did not result in significant changes in the numbers of culturable functional bacteria, except the nitrogen-fixing bacteria when the concentration of Bt toxin was higher than 500 ng/g. The results indicated that Bt toxin was not the direct factor causing decrease of the numbers of bacteria in the rhizosphere, and other factors may be involved.     

Next‐generation transgenic cotton: pyramiding RNAi and Bt counters insect resistance

Transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are extensively cultivated worldwide. To counter rapidly increasing pest resistance to crops that produce single Bt toxins, transgenic plant ‘pyramids’ producing two or more Bt toxins that kill the same pest have been widely adopted. However, cross‐resistance and antagonism between Bt toxins limit the sustainability of this approach. Here we describe development and testing of the first pyramids of cotton combining protection from a Bt toxin and RNA interference (RNAi). We developed two types of transgenic cotton plants producing double‐stranded RNA (dsRNA) from the global lepidopteran pest Helicoverpa armigera designed to interfere with its metabolism of juvenile hormone (JH). We focused on suppression of JH acid methyltransferase (JHAMT), which is crucial for JH synthesis, and JH‐binding protein (JHBP), which transports JH to organs. In 2015 and 2016, we tested larvae from a Bt‐resistant strain and a related susceptible strain of H. armigera on seven types of cotton: two controls, Bt cotton, two types of RNAi cotton (targeting JHAMT or JHBP) and two pyramids (Bt cotton plus each type of RNAi). Both types of RNAi cotton were effective against Bt‐resistant insects. Bt cotton and RNAi acted independently against the susceptible strain. In computer simulations of conditions in northern China, where millions of farmers grow Bt cotton as well as abundant non‐transgenic host plants of H. armigera, pyramided cotton combining a Bt toxin and RNAi substantially delayed resistance relative to using Bt cotton alone.     

不同Bt棉种植年代棉铃虫种群的波动性不对称

波动性不对称 (Fluctuating asymm etry,FA)是指相对于生物两侧对称发育的一种细微的随机偏离 ,通常用来指示生物在外界环境压力下发育的稳定性程度。 2 0 0 2年在我国北方棉区 1 3个具有 1～ 5 a转 Bt基因棉种植史的市、县采集了棉铃虫上灯蛾 ,测定了各点成虫后足腿节的波动性不对称 (Fluctuating asymmetry,FA)。结果表明 ,1 2个采集地的棉铃虫后足腿节均表现出 FA;相关性分析发现 ,各采集点的 FA值与 Bt棉的种植年代间存在着显著的负相关关系 (r=- 0 .77) ,显示出棉铃虫自然种群对 Bt棉的适应性随着 Bt棉种植时间延长而渐增的趋势。讨论了棉铃虫对 Bt棉的适应以及 FA作为棉铃虫田间种群对 Bt棉早期抗性监测指标的可能性     

Impacts of elevated CO2 on expression of plant defensive compounds in Bt‐transgenic cotton in response to infestation by cotton bollworm

• 1 The allocation of defensive compounds of transgenic Bt (cv. GK‐12) and nontransgenic cotton (cv. Simian‐3) grown in elevated CO₂ in response to infestation by cotton bollworm Helicoverpa armigera (Hübner) was studied in closed‐dynamics CO₂ chambers.
• 2 A significant reduction in foliar nitrogen content and Bt toxin protein occurred when transgenic Bt cotton grew under elevated CO₂. A significantly higher carbon/nitrogen ratio as well as condensed tannin and gossypol contents was observed for transgenic Bt (cv. GK‐12) and nontransgenic cotton in elevated CO₂, in partial support of the carbon nutrient balance hypothesis as a result of limiting nitrogen and excess carbon in cotton plants in response to elevated CO₂.
• 3 The CO₂ level and infestation time significantly affected the foliar nitrogen, condensed tannin, gossypol and Bt toxin protein contents of cotton plants after feeding by H. armigera. The interaction between CO₂ levels × cotton variety had a significant effect on foliar nitrogen content after injury by H. armigera.

     

Impact of transgenic Bacillus thuringiensis cotton on a non-target pest Tetranychus spp. in northern China

Field studies were carried out to evaluate the effect of two transgenic cotton varieties (SGK321 carrying Cry1A+CpTI and DP99B carrying Cry1Ac) and the conventional variety (shiyuan321‐parental line of SGK321) on spider mites, Tetranychus spp. from 2002 to 2004. In 2002, this experiment included three treatments: Bt cotton field (SGK321) treated with acaricides against spider mites, untreated non‐Bt cotton field (Shiyuan321), and untreated Bt cotton field (SGK321). In 2003–2004, there are four types of treatments after a new transgenic Bt cotton variety, DP99B (non‐chemical control), was added into the experiments. The results showed that there were no significant difference in densities of spider mites among Bt without acaricides and non‐Bt without acaricides cotton fields, nor was there a significant difference in damage of spider mites to cotton among these treatments (P > 0.1). However, there are significant differences (P < 0.05) in densities of spider mites and damage caused by spider mites between cotton fields with and without acaricides. Acaricide significantly reduced the densities of spider mites in Bt cotton (P < 0.05). These results suggest that Bt cotton has no effect on spider mites populations. However, spider mites have the potential for severe damage in Bt cotton fields. Acaricides are essential tools in controlling cotton spider mites in northern China.     

转CpTI-Bt基因棉和转Bt基因棉对棉铃虫幼虫存活、生长及营养利用的影响

2000年7月中旬和8月中旬, 分别测定了采自田间的转CpTI-Bt基因双价抗虫棉（SGK321, 以下简称CpTI-Bt棉）和转Bt基因抗虫棉（中30,以下简称Bt棉）对棉铃虫Helicoverpa armigera幼虫存活、生长的影响。结果表明：7月中旬两种转基因抗虫棉抗虫效果均较好,尤其是CpTI-Bt棉棉叶和花瓣对4龄幼虫3天内致死率为92%以上;8月中旬两种转基因棉的抗虫活性均明显降低,且Bt棉的杀虫活性显著低于CpTI-Bt棉,其幼虫死亡率与对照受体棉中16的死亡率之间无显著差异,仅显著抑制了幼虫的生长;石远321(SGK321受体品系)的花瓣具有一定的抗虫活性,可显著降低取食幼虫的体重,甚至造成部分幼虫死亡; CpTI-Bt棉中,花瓣和棉叶的抗虫性明显高于蕾和铃心。对5龄幼虫取食棉铃1日后的营养指标测定结果显示： 两种转基因抗虫棉处理的幼虫相对生长率和相对取食量均显著低于石远321,但两者之间无显著差异; CpTI-Bt棉处理的幼虫近似消化率显著低于石远321和Bt棉,但其食物利用率显著高于石远321和Bt棉。     

转基因棉种植对土壤水解酶活性的影响

转Bt基因棉和转Bt＋cpTI基因棉种植面积不断扩大,它们种植后杀虫晶体蛋白在土壤中的残留特性及对土壤水解酶活性的影响是环境风险评价的重要组成部分,本文采用盆栽实验的方法对此进行了初步研究。结果表明,转基因棉出苗后生长到30天时可向土壤中释放Bt杀虫晶体蛋白,而双抗棉种植时CpTI杀虫晶体蛋白的释放量与品种有关;转基因棉出苗后30天时,与等价基因系非转基因棉（各对照）相比,转Bt基因棉（“中30”）和双抗棉A（转Bt＋CpTI棉“中41”）的种植并未使脲酶、蛋白酶和磷酸单脂酶活性发生显著变化,而双抗棉B（转Bt＋CpTI棉“双抗321”）的种植使土壤磷酸单脂酶活性显著下降。从杀虫晶体蛋白的释放和对酶活性的影响来看,双抗棉A的种植对土壤的生物活性扰动更小。     

Comparative incidence of cotton spider mites on transgenic Bt versus conventional cotton in relation to contents of secondary metabolites

With the wide adoption of transgenic Bacillus thuringiensis (Bt) cotton, the incidence of bollworm has reduced significantly, but secondary pests such as cotton spider mites have become serious problems in Bt cotton fields. The objective of the present study was to investigate the underlying mechanism of increased incidence of secondary pests in Bt cotton. Two transgenic cotton varieties, sGK321 and Bt-C12, and their non-transformed counterparts, SY321 and C12, were used to study differences in the incidence of spider mites in relation to secondary metabolites. Plants of each cotton cultivar were infested with five female adult spider mites and then isolated. Leaf samples with a pair of adult mites of the same age were transferred individually into Petri dishes for examination of egg laying and duration of development stages. The number of spider mites on Bt-C12 and sGK321 was more than that on C12 and SY321. The cotton spider mites feeding on Bt-C12 laid significantly more eggs than those feeding on C12; those feeding on sGK321 laid significantly more eggs than those feeding on SY321. The generation time of spider mites feeding on Bt-C12 was greatly reduced relative to those feeding on C12. Also, the generation time of mites feeding on sGK321 was shorter than those feeding on SY321. Gossypol and tannin contents in leaves of Bt-C12 were substantially lower than those in C12, and the contents in leaves of sGK321 were significantly lower than those in leaves of SY321. The occurrence of spider mites was more serious on Bt than non-Bt cotton, and the fitness of the mites on Bt cotton was higher than on non-Bt cotton. Reductions in gossypol and tannin contents in Bt cotton decreased the generation time and increased the number of eggs of cotton spider mites.