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.     

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.     

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.     

施氮方式对转基因棉花Bt蛋白含量及产量的影响

为研究氮肥运筹对棉花叶片、棉蕾和棉铃不同器官中Bt蛋白含量的影响,2009—2010年,以抗虫杂交棉中棉所72为试验材料,在大田条件下进行了不同基肥：花铃肥：盖顶肥施氮比例（分别为0:0.4:0.6、0.2:0.4:0.4、0.4:0.4:0.2、0.6:0.4:0）的试验。结果表明,施氮方式对棉花不同器官中Bt蛋白含量有明显影响。总体表现为随着氮肥前移,棉花幼嫩器官中Bt蛋白含量呈明显增加的趋势,而老熟器官中Bt蛋白含量呈明显降低的趋势。施氮方式对棉花幼嫩器官中Bt蛋白含量的影响比老熟器官明显,尤其是对幼嫩叶片Bt蛋白含量的影响大于幼小的棉蕾和棉铃器官。抗虫棉采用基肥：花铃肥：盖顶肥为0.4:0.4:0.2的施氮方式,总体能提高前中期棉花器官Bt蛋白的含量,有利于提高其抗虫性能;减少后期棉花器官Bt蛋白的含量,减轻对环境的压力;而且比其余3种施氮方式的籽棉产量和皮棉产量分别增加4.15%—11.24％、3.73%—12.01%。     

咸水灌溉对沙土土壤盐分和胡杨生理生长的影响

通过咸水灌溉沙土土质生长的幼龄胡杨,分析了咸水灌溉沙土土壤盐分分布累积特点、盐分胁迫对胡杨的耗水生长关系、叶绿素、Pro、MDA的影响,结果表明:(1)在1.2—3 g/L范围内,微咸水灌溉沙土处于脱盐状态,6—12 g/L咸水灌溉使沙土积盐大增。在整个生长周期内,微咸水和咸水灌溉,0—200 cm内土体的总盐都呈累积趋势。(2)咸水灌溉胡杨,不同盐分处理的生长耗水关系可以用对数模型描述。(3)盐分胁迫下,胡杨叶片内叶绿素含量呈抛物线递减,Pro和MDA含量则呈现抛物线递增趋势。说明短期内咸水灌溉对土壤安全和胡杨的生长影响有限,可用咸水解决生态缺水现状,3种生理指标可用来衡量胡杨的盐胁迫程度,以此为指导提高人工造林的成活率。     

Bt 水稻秸秆还田对土壤养分含量的影响

将 Bt 水稻 (b2B138)和常规水稻 (安丰 A)秸秆平铺在装有 20 cm 厚土壤的塑料面包箱上,并加入一定量的腐秆剂, 研究 Bt 水稻秸秆覆盖还田对土壤养分含量的影响 , 同时探讨腐秆剂在此过程中的作用。结果表明 , 与常规水稻秸秆还田相比 , 无论有无腐秆剂的加入, Bt 水稻秸秆还田 120 d 和 180 d 后均能显著增加土壤速效钾含量, 但无腐秆剂加入时, Bt 水稻秸秆还田 120 d 后土壤全氮含量明显下降, Bt 水稻秸秆还田对土壤有机质、碱解氮、速效磷、全磷和全钾含量则没有显著影响。腐秆剂的加入使得 Bt 水稻秸秆还田 120 d 后土壤速效钾含量明显提高, 常规水稻秸秆还田 180 d后土壤速效磷含量也显著提高, 对其他养分指标则没有明显影响。     

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.     

羧甲基壳聚糖对玉米籽粒氮代谢关键酶和种子贮藏蛋白含量的影响

以羧甲基壳聚糖（ＮＣＭＣ） 处理玉米开花期果穗,发育中籽粒的谷氨酰胺合成酶（ＧＳ） 、谷氨酸脱氢酶（ＧＤＨ） 及谷丙转氨酶（ＧＰＴ） 活性均明显增强,而蛋白水解酶活性下降。羧甲基壳聚糖处理离体玉米籽粒酶提取液,酶液中ＧＤＨ 活性明显提高。羧甲基壳聚糖处理玉米果穗,其发育籽粒中可溶性蛋白和成熟种子中贮藏蛋白含量明显提高。这都表明,羧甲基壳聚糖对玉米氮代谢、蛋白质合成与积累具有明显的生理调节作用。     

羧甲基壳聚糖对玉米籽粒氮代谢关键酶和种子贮藏蛋白含量的影响

以羧甲基壳聚糖（ＮＣＭＣ）处理玉米开花期果穗,发育中籽粒的谷氨酰胺合成酶（ＧＳ）,谷氨酸脱氢酶民（ＧＤＨ）及谷丙转氨酶（ＧＰＴ）活性均明显增强,而蛋白水解酶活性下降。羧甲基壳聚糖处理离体玉米籽粒酶提取液,酶液中ＧＤＨ活性明显提高。羧甲基壳聚糖处理玉米果穗,其发育籽粒中可溶性蛋白和成熟种子中贮藏蛋白含量明显提高,这都表明,羧甲基壳聚糖对玉米氮代谢、蛋白质合成与积累具有明显的生理调节作用。     

种植Bt玉米及秸秆还田后土壤中Bt蛋白的变化及其对土壤养分的影响

在大棚水泥池内种植两个Bt玉米(5422Bt1和5422CBCL)及其同源常规玉米5422,研究了种植Bt玉米及秸秆还田过程中根际土、根围土、3层根外土(0～20、20～40和40～60 cm)中Bt蛋白含量的时空动态特征及其对土壤养分含量的影响．结果表明： 种植Bt玉米和常规玉米后,根围土(种植后90 d)和3层根外土(种植后30、60和90 d)中均检测到少量的Bt蛋白(含量<0.5 ng·g^-1),在Bt玉米5422Bt1和5422CBCL根际土中则分别检测到1.59和2.78 ng·g^-1的Bt蛋白．玉米秸秆还田后,Bt蛋白能在3 d内快速降解,在还田后第7天只检测到少量的Bt蛋白．与常规玉米5422相比,种植Bt玉米5422Bt1 90 d后根围土和3层根外土中有机质、速效养分(碱解氮、速效磷和速效钾)和全量养分(全氮、全磷和全钾)含量均没有显著差异;5422Bt1秸秆还田60 d后0～20 cm土层的有机质和全氮含量显著升高,速效钾含量显著降低,而其他养分指标则没有显著差异,20～40 cm和40～60 cm土层的所有养分指标均没有明显差异．种植Bt玉米5422CBCL后根围土中仅速效磷含量显著低于种植常规玉米5422,但0～20 cm土层中全磷含量显著提高,其他养分指标均没有差异;还田5422CBCL秸秆后仅0～20 cm土层的速效磷含量显著高于常规玉米5422．研究结果表明,通过玉米根系分泌和秸秆分解进入土壤的Bt蛋白不会在土壤中累积,对养分含量也基本没有显著影响.
     

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.     

Effects of salinity and nitrogen on cotton growth in arid environment

The influences of different N fertilization rates and soil salinity levels on the growth and nitrogen uptake of cotton was evaluated with a pot experiment under greenhouse conditions. Results showed that cotton growth measured as plant height was significantly affected by the soil salinity and N-salinity interaction, but not by N alone. Cotton was more sensitive to salinity during the emergence and early growth stages than the later developmental stages. At low to moderate soil salinity, the growth inhibition could be alleviated by fertilizer application. Soil salinity was a dominated factor affecting cotton’s above-ground dry mass and root development. Dry mass of seed was reduced by 22%, 52%, and 84% respectively, when the soil salinity level increased from control level of 2.4 dS m^?1 to 7.7 dS m^?1, 12.5 dS m^?1 and to 17.1 dS m^?1, respectively. N uptake increased with N fertilization at adequate rates at both low and medium soil salinities but was not influenced by over N fertilization. At higher salinities, N uptake was independent of N rates and mainly influenced by soil salinity. The uptake of K decreased with soil salinity. The concentration of Na, Cl and Ca in plant tissues increased with soil salinity with highest concentrations in the cotton leaf.     

Bt毒素在转基因棉花与土壤系统中的分布

研究了转Bt基因棉花与土壤系统中Bt毒素的分布.结果表明,两种转Bt基因棉花地上部(叶片、茎秆)的毒素表达量(103.5～134.1 ng·g^-1)显著高于地下部分(根系)(44.7～21.2 ng·g^-1),土壤中Bt毒素总量可通过转基因棉花地上部分秸秆的处理得到控制;Bt毒素在转Bt基因棉花根系分泌物中的含量极低,如果控制Bt毒素的其它导入来源,将显著降低转Bt基因作物释放中因Bt毒素导入而引发的对土壤生态系统的扰动.     

施氮量和种植密度对东北特早熟棉区棉铃生物量和氮素累积的影响

以辽棉19号和美棉33B为材料,研究了不同施氮量(0、240、480 kg ·hm-2)和不同种植密度(75000、97500、120000 plants·hm-2)对东北特早熟棉区棉花棉铃生物量和氮素累积特征的影响.结果表明： 棉花单铃、棉籽和纤维的生物量及其氮素累积随棉花生育进程的动态变化均符合“S”型曲线,种植密度和施氮量可以显著影响棉铃各部分生物量和氮素累积的动态特征,以及棉花产量与品质;在施氮量240 kg·hm-2和种植密度97500 plants·hm-2处理下,单铃、棉籽和纤维的生物量均达到最大,生物量和氮素累积的快速累积起始时间和终止时间较早但持续时间较短,生物量快速累积速率最大,生物量和氮素在铃壳中的分配系数最低,在棉籽和纤维中分配系数最高.     

高温和湿度对转Bt基因棉叶片Bt蛋白表达的影响

以来源于美国的转Bt基因棉DP410B(常规种)和岱杂1号(杂交种)以及来源于中国的转Bt基因棉泗抗1号(常规种)和泗抗3号(杂交种)为材料,研究高温(37℃)条件下,大气湿度(50％、70％、90％)变化对Bt蛋白表达的影响.结果表明:高温条件下,盛蕾期,温湿度对4个供试品种Bt蛋白表达均无显著影响;盛花期,与对照(温度25 ～ 30℃,湿度60％ ～70％)相比,常规种在50％湿度时叶片Bt蛋白含量显著降低2.6％ ～3.0％;盛铃期,DP410B、泗抗1号和泗抗3号的Bt蛋白含量在50％湿度处理下比对照显著降低3.3％ ～5.8％.4个转Bt基因棉品种中,DP410B和岱杂1号的Bt蛋白含量最高,而泗抗1号最低.     

盐胁迫下施肥对棉花生长及氮素利用的影响

利用海水配制不同含盐量(0、0.15%、0.3%)的土壤盆栽棉花,在可移动遮雨棚内研究了不同施肥(N、NK、NP、NPK)处理对棉花生长、氮素吸收与利用的影响.结果表明: 盐胁迫和施肥均影响棉花生物产量、棉株氮素农学利用效率、氮素生物利用效率和氮素积累量,且两者存在显著的互作效应.施肥能提高盐胁迫下棉株氮素利用效率及氮素积累量,并显著增产,不同施肥处理中以N、P、K肥料配合施用的效果最好;施肥效果受盐胁迫程度的影响,低盐胁迫(0.15%)下的施肥效果好于中度盐胁迫(0.3%).     

转双价基因抗虫棉对土壤微生物群落多样性的影响

采用Biolog技术,研究了不同生育期(30、60、90和120d)转双价(Bt+CpTI)基因抗虫棉SGK321及其亲本非转基因常规棉石远321根际土壤微生物群落多样性变化。结果表明,反映土壤微生物活性的平均颜色变化率(AWCD)呈现出以下变化规律:与亲本常规棉相比,整个培育过程中转双价基因抗虫棉AWCD值除30d显著高于亲本常规棉(P<0.05),其他3个时期均显著低于亲本常规棉(P<0.05)。两种棉花根际土壤微生物群落丰富度指数和优势度指数随棉花生育期的不同而有所不同,4个时期转双价基因抗虫棉均匀度指数与亲本常规棉无显著差异。主成分分析结果表明,转双价基因抗虫棉30d、亲本常规棉60、120d土壤微生物群落碳源利用方式相似,转双价基因抗虫棉60、90、120d与亲本常规棉30d土壤微生物群落碳源利用方式相似。土壤微生物利用的主要碳源为糖类、氨基酸类、羧酸类和聚合物。     

Effects of repeated cultivation of transgenic Bt cotton on functional bacterial populations in rhizosphere soil

The impact of multiple-year (0–5 years) cultivation of transgenic Bacillus thuringiensis (Bt) cotton on the functional bacterial populations in rhizosphere soil was investigated. The transgenic Bt + CpTI cotton line SGK321 and a non-Bt cotton line Shiyuan321 were planted in four fields in which Bt cotton had been continuously cultivated for 0, 1, 3, and 5 years. Rhizosphere soil samples were collected at the seedling, squaring, flower and boll, and boll-opening stages of cotton. Numbers of bacteria involved in nitrogen-fixing, organic phosphate-dissolving, inorganic phosphate-dissolving, and potassium-dissolving were measured with cultivation-dependent approaches. The data presented here showed no consistent statistically significant differences in the numbers of different groups of functional bacteria between rhizosphere soil of Bt and non-Bt cotton in the same field, and no obvious trends in the numbers of the various group of functional bacteria with the increasing duration of Bt cotton cultivation. These studies suggest that multiple-year cultivation of transgenic Bt cotton may not affect the functional bacterial populations in rhizosphere soil.     

Amino acid composition and level affect Bt protein concentration in Bt cotton

Low insect resistance has been widely observed in Bacillus thuringiensis (Bt) transgenic cotton. The objective of this study was to investigate the effects of amino acid compositions and their concentration on the Bt toxin content in Bt cotton to gain a better physiological understanding of the observed phenomenon. In the 2010 study, the leaf amino acid composition, contents, and Bt protein concentrations were investigated under high temperature (36?°C) and low temperature(18?°C) at both square and boll stage respectively. In 2011, the leaves were sprayed with ethionone sulfoximine (MSO), aminooxyacetic acid (AOA) or combination of MSO and AOA at the two developing stages, and the leaf amino acid composition and Bt protein concentrations were investigated. In 2012, 17 amino acid compositions were used to spray cotton plants at the same two stages, and the Bt protein concentration were measured. In comparison to the control, more amino acids changed and greater concentration changes had been observed under the extreme temperatures at boll stage compared to that at square stage, which were conformity to the changes of the leaf Bt protein concentration. More amino acids reduced and greater reduction was noted with the application of MSO, AOA or combined MSO and AOA at boll period than that at square period, which resulted in the reduction of the leaf Bt toxin concentration. The external uses of Aspartic acid, Glutamic acid, Glycine, Proline, Tyrosine, Methionine, Phenylalanine, Histidine and Arginin enhanced the leaf Bt toxin level significantly at boll stage. However, only Aspartic acid, Glutamic acid, Proline, Methionine, Arginin could increase Bt protein concentration markedly at square period and the bolstered extent were low. Therefore, it was concluded that cotton amino acid composition and concentration was more sensitive to external factors at boll stage. In addition, there are close relationship between leaf Bt toxin content and Aspartic acid, Glutamic acid, Glycine, Proline, Tyrosine, Methionine, Phenylalanine, Histidine and Arginin and their concentrations.     

不同氮素水平下CO_2倍增对转Bt棉花氮素代谢的影响

通过开顶式CO_2气室研究了盛蕾期转Bt棉花新棉33~B及其对照亲本DP5415的生长势和氮素代谢特征对土壤氮素水平(100和200 mg N·kg~(-1))和CO_2浓度倍增(750和375μl·L~(-1))的生理生态响应.结果表明:CO_2浓度升高可显著提高2种棉花的株高和茎粗,增加生物产量;氮素水平提高可显著增加转Bt棉花的株高、茎粗,以及茎和蕾的鲜质量,而对亲本棉花DP5415的影响不显著;对照棉花DP5415的谷氨酰胺合成酶(GS)活力随大气CO_2浓度的升高而显著降低,随氮素营养的提高而升高,转Bt棉花新棉33~B在低氮条件下,GS活力随大气CO_2浓度的升高而显著增加;大气CO_2浓度升高及氮素营养的增加使盛蕾期转Bt棉花的硝酸还原酶(NR)活力显著增加,DP5415的NR活力也随大气CO_2浓度的升高而显著提高;大气CO_2浓度对2种棉花的亚硝酸还原酶(NiR)活力都有明显的抑制作用,其中,高CO_2浓度条件下,DP5415的NiR活力还随氮素营养的增加而显著下降.可见,大气CO_2浓度升高下,土壤氮素水平变化对转Bt棉花的生长势影响显著,但对其氮素代谢生理的影响较对照亲本棉花小.生产中(尤其是高浓度CO_2环境下),应进一步加强转Bt棉花的氮肥优化管理.

Abstract：

By using open-top chambers, this paper studied the physiological and ecological re-sponses of transgenic Bt cotton cv. 33~B and its parent line non-transgenic cotton cv. DP5415 in their growth potential and nitrogen metabolism to doubled CO_2 concentration (750 μl·L~(-1) vs.375 μl·L~(-1)) and nitrogen fertilization level (200 mg N·kg~(-1)vs. 100 mg N·kg~(-1)). Doubled CO_2 concentration promoted the height-and stem growth and the biomass production of the two eultivars significantly, whereas doubled N fertilization level only had significant positive effects on 33~B. The leaf glutamine synthetase activity (GSA) of DP5415 decreased significantly under doubled CO_2 concentration but increased significantly under doubled N fertilization level, while the GSA of 33~B was significantly higher under doubled CO_2 concentration and low nitrogen fertili-zation level. Both the doubled CO_2 concentration and the doubled nitrogen fertilization level in-creased the leaf nitrate reductase activity (NRA) of 33~B significantly, and the NRA of DP5415 also had a significant increase under doubled CO_2 concentration. Doubled CO_2 concentration had significant inhibitory effects on the leaf nitrite reductase activity (NiRA) of both 33~B and DP5415. The NiRA of DP5415 decreased significantly under doubled CO_2 concentration and N fertilization level. All the results suggested that under doubled CO_2 concentration, N fertilization level had significant effects on the growth potential of transgenic Bt cotton but lesser effects on its nitrogen metabolism, compared with the control non-transgenic cotton. Therefore, in the planting of transgenic Bt cotton, especially,under elevated CO_2 condition, optimized N fertilization should be made.