转Bt Cry1Ac基因抗虫棉花外源基因漂移研究

【目的】研究转Bt Cry1Ac基因棉花外源基因漂移规律,为转基因棉花生态安全性评价提供依据。【方法】2014年在吐鲁番市和2015年在库尔勒市,利用小区试验种植转基因棉花,翌年利用卡那霉素抗性检测结合分子检测的方法检测外源基因的漂移。【结果】在距离转基因棉花小区边缘1~120 m均能检测到含有外源基因Cry1Ac。SGK321种子漂移率最大漂移率是向南方向距离转基因棉花小区边缘1 m处,漂移率为5 %;GK19最大漂移率是向西方向达到了10.09 %。以方向因素和距离因素建立Cry1Ac基因漂移Logistic回归预测模型,方向和距离对种子漂移都有显著影响(P <0.05),但方向和距离两个因子并不能最终决定外源基因的漂移率。【结论】转基因棉花外源基因漂移率受方向和距离的影响,但最终决定外源基因的漂移率,并不是方向和距离联合作用的结果。     

转Bt基因棉花对土壤微生物的影响

在大田和盆栽条件下，以转Bt基因抗虫棉晋棉26号和其具有相似遗传背景的普通棉花品种晋棉7号为材料，研究棉花不同生育期的土壤细菌、真菌、放线菌及其生理菌种的变化。结果表明，转Bt基因棉晋棉对土壤微生物种类和数量有一定的影响，在某些生育期显著高于或低于普通非转基因棉花，或者差异不显著。因此，转基因安全评价需要对土壤微生物进行评价。     

转Bt基因棉花对棉田天敌的影响

就转Bt基因棉花对棉田害虫寄生性天敌、捕食性天敌的个体发育和种群的影响进行了综述,并且分析了影响机理。     

人工合成Bt基因对棉花的遗传转化

采用子房注射法，将构建在植物高效表达载体上的全序人工合成的Ｂｔ杀虫基因ＧＦＭＣｒｙＩＡ转入棉花品种（系）“中棉所１９”、“华农９１６”、“５４１”和“３５１７”中，获得对棉铃虫幼虫致死率在８０％以上的植株２９株。用ＰＣＲ法检测，结果表明，这些株株均呈阳性。在这２９株阳性植株中，对棉铃虫致裂纹经为１００％，且自身只有轻微伤害的植株有２６株。这说明Ｂｔ基因经改造，增加其提高转录与翻译能力的表达调控元件     

两种转Bt基因棉杀虫蛋白Cry1Ac表达量的检测

用EnvirologixCry1Ab/Cry1Ac平板试剂盒检测了两种转Cry1Ac基因的棉花品系 (NUCOTN33B、NUCOTN99B) 以及常规对照品系 (苏棉 12) 不同生育期主茎嫩叶、侧枝嫩叶、蕾及蕾的苞叶中杀虫蛋白Cry1Ac的含量。结果表明, 两种转Bt基因棉主茎嫩叶杀虫蛋白Cry1Ac的含量随生育期的推移呈明显下降趋势, 花铃期 (NUCOTN33B和NUCOTN99B分别为 4 43和 2 93μg·g-1 ) 和吐絮期 (3 87和 2 86μg·g-1 ) 的含量分别降至苗期第 6叶 (7 64和 8 38μg·g-1 )的 58%、35%和 51%、34%; 相同时期的主茎嫩叶和侧枝嫩叶中Cry1Ac的含量均显著高于蕾及蕾的苞叶, 前两者均为后两者的两倍以上。     

两种转Bt基因棉杀虫蛋白Cry1Ac表达量的检测

用Envirologix Cry1Ab/Cry1Ac平板试剂盒检测了两种转Cry1Ac基因的棉花品系(NUCOTN33B、NUCOTN99B)以及常规对照品系(苏棉12)不同生育期主茎嫩叶、侧枝嫩叶、蕾及蕾的苞叶中杀虫蛋白Cry1Ac的含量.结果表明,两种转Bt基因棉主茎嫩叶杀虫蛋白Cry1Ac的含量随生育期的推移呈明显下降趋势,花铃期(NUCOTN33B和NUCOTN99B分别为4.43和2.93 μg&#183;g-1)和吐絮期(3.87和2.86 μg&#183;g-1)的含量分别降至苗期第6叶(7.64和8.38 μg&#183;g-1)的58%、35%和51%、34%;相同时期的主茎嫩叶和侧枝嫩叶中Cry1Ac的含量均显著高于蕾及蕾的苞叶,前两者均为后两者的两倍以上.     

转Bt基因棉花对土壤微生物的影响

在大田和盆栽条件下，以转Bt基因抗虫棉晋棉26号和其具有相似遗传背景的普通棉花品种晋棉7号为材料，研究棉花不同生育期的土壤细菌、真菌、放线菌及其生理菌种的变化。结果表明，转Bt基因棉晋棉对土壤微生物种类和数量有一定的影响，在某些生育期显著高于或低于普通非转基因棉花，或者差异不显著。因此，转基因安全评价需要对土壤微生物进行评价。     

NaCl胁迫下转Bt棉花和非转Bt棉花的生理反应差异

为研究NaCl胁迫下转Bt基因棉花和非转Bt基因棉花的生理反应差异,采用盆栽条件下不同浓度NaCl溶液浇灌处理,测定叶片细胞含水量(RWC)、丙二醛(MDA)含量、游离脯氨酸(Pro)含量和Bt毒素蛋白含量。结果表明,NaCl胁迫下转Bt基因棉花和非转Bt基因棉花叶片细胞含水量比无胁迫明显降低,丙二醛含量明显升高,游离脯氨酸含量大幅增加,这些生理变化在转Bt基因棉花和非转Bt基因棉花之间没有显著差异,说明转入Bt基因对棉花的生长发育不会造成显著影响;在0.4%NaCl胁迫下,转Bt基因棉花Bt毒素蛋白含量出现了显著下降,说明在NaCl胁迫下转Bt基因棉花的抗虫性下降。     

烟粉虱的刺吸胁迫对转Bt/CpTI基因棉的生理影响

研究了转Bt/CpTI基因棉受烟粉虱(Bemisia tabaci Gennadius)危害后体内叶绿素、可溶性糖、蛋白质及主要保护酶活性的变化动态。结果表明棉叶受烟粉虱危害后,叶绿素、可溶性糖及蛋白质含量均有不同程度的变化,受害后保护酶活性有增有减,CAT酶活性增强29.21%,而POD酶活性大幅降低66.24%。     

转Bt基因棉对棉田主要害虫及其天敌种群消长的影响

１９９５～１９９６年在田间系统研究了转Ｂｔ基因棉花品系９３－４在单作和麦套２种种植方式下，对主要棉花害虫及其天敌种群消长的影响。结果表明：在单作和麦套条件下，转Ｂｔ基因棉对棉铃虫、棉造桥虫幼虫均有良好的抗性，残虫量均低于防治指标；在单作条件下，转Ｂｔ基因棉田比常规棉田的棉苗蚜、棉伏蚜和叶蝉分别增加２０．３０％，２１．３８％和６７．６２％，红蜘蛛、棉蓟马和白粉虱分别减少５８．４２％，３９．６２％和３９．０６％；在麦套条件下，转Ｂｔ基因棉田的棉苗蚜和棉蓟马分别减少５．８８％和７９．４６％，棉伏蚜、红蜘蛛、白粉虱和棉叶蝉分别增加１４．６２％，７５．４８％，４８．２２％和５８．５４％；在单作和麦套条件下，转Ｂｔ基因棉田比常规棉田捕食性天敌略增加，寄生性天敌减少，天敌总量无明显差异。     

不同药剂防治棉田烟粉虱田间药效试验

为了有效控制烟粉虱对棉花的为害、筛选出高效化学农药,用6种杀虫剂进行了防治大田棉花烟粉虱成虫的药效对比试验。结果表明,5%氯氟氰菊酯EC和4.5%高效氯氰菊酯EC防治烟粉虱成虫具有速效性强和药效持久的特点;2.8%阿维菌素EC和20%乙酰甲胺磷EC速效性较差而持久性较好;40%丙溴磷EC和2.5%联苯菊酯EC的防效较差。     

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.     

Genetic Differentiation of Different Geographical Populations of Bemisia tabaci （Gennadius） Complex

Bemisia tabaci （Gennadius） is a species complex, which includes different geographical populations with genetic differentiation. The recent progress on the genetic differentiation of various geographical populations of B. tabaci complex was introduced. The genetic differentiation was further analyzed on the basis of the sequences of mtDNA COI and rDNA ITS1 recorded in the world＇s GenBank. Five groups are defined on the basis of mtDNA COI and rDNA ITS1, including the Asia group, America group, Africa group, Australia group, and Biotype B/Mediterranean/Middle East/ Northern Africa/Biotype Ms group. There are several ungrouped geographical classifications, such as the Uganda population, Ivory Coast population, and Taiwan population. Geographical isolation may be the most important factor that contributed to the genetic differentiation of various geographical populations of B. tabaci. Many populations with biological advantages invaded new regions and caused severe economic losses within human activity. It is necessary to strengthen the research of B. tabaci biotype to prevent the spread of invaded populations and the invasion of potentially dangerous populations.     

Bt稻Cry1Ab杀虫蛋白在水体中的残留和Bt稻田三类水生昆虫数量调查

以转Btcry1Ab基因水稻克螟稻1号(KMD1)和克螟稻2号(KMD2)黄熟期的茎叶为材料,室内测定了其经浸水处理后(28℃,L16∶D8)残体和水体中Cry1Ab杀虫蛋白的含量;测定了KMD1与早籼稻嘉早935培育成的Bt稻华池B6分蘖期稻田水体中Cry1Ab蛋白的含量,同时调查了该Bt稻和对照稻嘉早935在分蘖初期、盛期和末期稻田3类水生昆虫的数量。结果表明,KMD1和KMD2茎叶经浸水100d后,水体中残留有Cry1Ab杀虫蛋白。华池B6分蘖期稻田水体中没有检测到Cry1Ab蛋白;除分蘖末期华池B6稻田中的蜉蝣目昆虫个体数量显著高于对照田嘉早935外,其他2类水生昆虫摇蚊(spp.)和鞘翅目甲虫(sp.)在两类稻田间均无显著差异。     

Enhancement of Phenylalanine Ammonia Lyase,Polyphenoloxidase, and Peroxidase in Cucumber Seedlings by Bemisia tabaci（Gennadius） （Hemiptera： Aleyrodidae） Infestation

In this study, the activities of phenylalanine ammonia lyase （PAL）, polyphenoloxidase （PPO）, and peroxidase （POD） were assayed in cucumber seedlings （Cucumis sativus L.） at 0, 6, 12, 24, 48, 72, and 96 h after they were infested by Bemisia tabaci （Gennadius） using spectrophotometric analysis. The results indicated that herbivore infestation increased the activities of PAL, PPO, and POD. The enzymes showed different activity levels at different times after the infestation. The PAL activity reached the first high peak by 23.1% at 6 h and the highest peak by 29.1% at 48 h compared to the control. The PPO activity reached the first high peak by 22.7% at 6 h and the highest peak by 52.6% at 24 h, and the POD activity reached the highest peak by 213.2% at 6 h and another higher peak value by 135.2% at 96 h. The results suggest that the enhanced activities of the enzymes may contribute to bioprotection of cucumber plants against B. tabaci infestation.     

烟粉虱持续取食对棉花叶片防御酶活性、营养物质和代谢产物含量的影响

【目的】研究不同密度与不同为害时间下,棉花植株对烟粉虱持续取食胁迫所产生防御应答响应,分析叶片中防御酶活性,营养物质和代谢产物含量的变化,分析烟粉虱与棉花的互作关系。【方法】在人工气候室内,叶片真叶生长至5叶的棉花植株(NJ-135)上,分别接入0头(对照)、50和500头烟粉虱,单株罩140目尼龙网,在烟粉虱持续取食24、48和72 h后,分别采集棉花叶片,测定总蛋白、可溶性糖、脯氨酸、丙二醛、过氧化氢酶和叶绿素的含量。【结果】50和500头/株处理组,烟粉虱持续取食24、48和72 h,叶片中总蛋白、脯氨酸、过氧化氢酶含量均与取食时间的增长呈正相关,随取食时间和密度的加大而增高(3.23, 4.87和5.55 μg/mL, 3.84, 5.38和5.92 μg/mL; 0.85, 0.92和0.99 μg/g, 0.89, 0.98和1.21 μg/g; 9.81, 10.98和18.45 U/mg prot; 12.42, 15.29和19.16 U/mg prot),叶片中可溶性糖含量随取食时间延长逐渐下降(16.77, 15.83和15.64 mg/g; 18.10, 15.75和7.84mg/g),丙二醛含量随烟粉虱持续取食时间延长先下降后升高(3.76, 3.26和4.44 nmol/mg prot; 4.45, 3.94和5.15 nmol/mg prot),叶绿素含量变化低密度与高密度处理含量动态变化不同,50头/株为先下降后上升(1.27, 0.90和1.92 mg/g),500头/株为逐渐上升(0.50, 0.86和1.98 mg/g)。【结论】 棉花叶片会对烟粉虱取食胁迫产生相应的防御应答,与棉花生理生化性质改变有关。密度与烟粉虱持续取食时间,均是诱导叶片内总蛋白、脯氨酸、丙二醛、过氧化氢酶、可溶性糖和叶绿素含量变化的因素。高密度的烟粉虱会增强这种应答的程度,随着为害时间的延长应答程度也随之增强。     

江苏棉田烟粉虱的种群动态及控制

2002～2003年通过系统调查对江苏棉田烟粉虱种群动态规律及其控制技术进行研究。结果表明：扬州市郊区棉田,7月下旬始见烟粉虱成虫,8月中、下旬形成第1个成虫高峰,10月上旬成虫消失;丰县棉田,7月中旬始见烟粉虱成虫,8月中旬出现第1个成虫高峰,10月下旬随着气温的下降田间成虫逐渐消失。棉田烟粉虱虫口密度,江苏北部棉区高于中部棉区,转Bt基因棉田高于常规棉田。黄板对烟粉虱成虫有一定的诱集作用。阿维菌素与吡虫啉混剂、灭多威和氧化乐果对棉田烟粉虱种群有一定的控制作用,其9d的校正虫口减退率分别为82．32％、84．59％和80．03％。     

Impacts of Environmental Factors on Degradation of Cry1Ab Insecticidal Protein in Leaf-Blade Powders of Transgenic Bt Rice

The determination of the environmental fate of Bt insecticidal protein released by Bt rice plants in paddy soils is a key issue in its ecological risk assessment. In this study, the impacts of soil water content, pH, and temperature on the degradation of Cry1Ab protein expressed in the leaves of Bt rice KMD2 were studied in the laboratory. Three types of paddy soils were used, i.e., blue clayey paddy soil, pale paddy soil on quaternary red soil, and marine-fluvigenic yellow loamy paddy soil. Ground powders of KMD2 leaf blades were mixed with each type of soil, and degradation dynamics of Cry1Ab were measured using enzyme-linked immunosorbent assay (ELISA). The degradation rate of Cry1Ab was high at the early experimental stage, but slowed down steadily at middle and later stages, which could be described by exponential equations, with the half-life period of degradation determined as 1.8-4.0 d. The soil water content, pH, and temperature could affect the degradation of Cry1Ab, but the effects of soil pH and temperature were relatively greater. In general,Cry 1 Ab degradations were slower under lower soil pH and temperature conditions, especially for marine-fluvigenic yellow loamy paddy soil.     

Yield, Leaf Senescence, and Cry1Ac Expression in Response to Removal of Early Fruiting Branches in Transgenic Bt Cotton

Two-year field experiments were conducted at Linqing, Yellow River valley of China, to study the plant response to the removal of early fruiting branches in transgenic Bt （Bacillus thuringiensis） cotton （Gossypium hirsutum L.） from 2003 to 2004. Plants were undamaged and treated by removing two basal fruiting branches （FB） at squaring to form the control and the removal treatment, respectively. The plant height, leaf area （LA）, dry weight of fruiting forms （DWFF）, the number of fruiting nodes （NFN）, photosynthetic （Pn） rate, and levels of leaf chlorophyll （Chl）, N, P, K, and Cry lAc protein in main- stem leaves were measured at a 10- or 20-d interval after FB removal, and the sink/source ratio as indicated by NFN/LA and DWFF/LA was determined. FB removal significantly increased the plant height, LA, and plant biomass in both years. Lint yields were increased 7.5 and 5.2% by removal compared with their controls in 2003 and 2004, respectively. Significant increases in boll size （5.7 and 5.1%） were also observed in removal than in control for both years. Either NFN/LA or DWFF/LA was significantly reduced by removal before 40 d after removal; however, both NFN/LA and DWFF/LA were significantly enhanced by FB removal at 80 d after removal compared to the untreated control. There was no significant difference in fiber quality in the first two harvests between removal and control, but fiber strength and micronarie in the third harvest were significantly improved by FB removal. In terms of leaf Chl, Pn rate, levels of total N, P, and K in late season, leaf senescence was considerably delayed by FB removal. Levels of CrylAc protein in the fully expanded young leaves were considerably higher in FB-excised plants than in control, indicating FB removal enhanced CrylAc expression. It is suggested that the yield and quality improvement with FB removal may be attributed to the increased NFN/LA or DWFF/LA in late season and delayed leaf senescence, respectively. FB removal can be a potential p