转基因抗虫棉对根区土壤真菌影响的初步研究

以转基因抗虫棉SGK321、GK12及亲本对照石远321、泗棉三号为材料.连续2 a利用平板涂布法测定棉花根区土壤真菌数量及主要类群数量的变化,并在第2 a蕾期和花期对真菌进行DGGE检测.结果显示:第1 a全部供试品种真菌数量均随生育期增加,第2 a真菌数量变化趋势与上年不同.2 a相同生育期内转基因棉和亲本间均存在差异:①SGK321与石远321相比.第1 a蕾期、吐絮期真菌数量及吐絮期镰刀菌数量差异显著;②GK12与泗棉三号相比,第1 a花期真菌数量及蕾期、花期的毛霉数量差异显著;第2 a蕾期真菌数量及青霉数量差异显著.DGGE结果显示,在蕾期和花期,转基因棉与亲本相比,真菌数量及主要类群都有所改变;在蕾期,2个转基因棉品种与亲本间根区土壤真菌的相似度均高于在花期的相似度.说明转基因抗虫棉对根区土壤真菌数量及主要类群具有一定影响,且种植时间长短也会对其产生不同的影响.     

转基因棉花中糖类和游离氨基酸含量的变化对棉蚜泌蜜量及蜜露主要成分的影响

以3个转基因棉和2个亲本对照棉花品种为研究材料,分别测定了这5种棉花植株体内主要糖分与游离氨基酸含量;同时,分别用这5个棉花品种的叶片饲养棉蚜Aphis gossypii Glover并测定其蜜露分泌量及其主要营养成分。结果表明,转基因棉花“国抗22”叶片中葡萄糖、蔗糖、麦芽糖的平均含量及可溶性糖总量分别比亲本对照棉“泗棉3号”减少61.76%、 89.05%、77.86%和23.61%,转基因棉花“苏抗103”和“中抗310”叶片中葡萄糖、蔗糖、麦芽糖的平均含量及可溶性糖总量分别比亲本对照棉“苏棉12”下降14.15%、32.80%、92.22%、11.46% 和4 6.81%、93.19%、61.11%、43.91%,游离氨基酸总量及各种氨基酸、果糖、鼠李糖、海藻糖的含量在不同转基因棉与亲本对照棉花品种间也存在很大差异,其中一些处理间的差异达显著或极显著水平。这表明外源基因的导入已经影响到了转基因棉花品种中主要糖分与游离氨基酸的合成。棉蚜取食转基因棉花品种“国抗22”后,蜜露的日平均分泌量比取食对照品种“泗棉3号”减少40.54%,取食其他两个转基因棉花品种“苏抗103”和“中抗310”后蜜露的分泌量也比取食对照棉花品种“苏棉12”降低22.67%和30.0%,但棉蚜取食转基因棉花后蜜露中游离氨基酸的总量均高于对照棉花品种,蜜露中可溶性总糖、蔗糖和各种氨基酸含量在取食转基因棉和常规棉花品种间存在一定差异。     

棉花黄萎病菌的侵染过程

深入揭示黄萎病菌的致病机理,为培育高抗新品种提供理论依据和技术途径,是有效控制黄萎病的根本.本文系统探讨了土壤中微菌核或孢子受寄主根系分泌物的刺激,开始萌发、产生的菌丝在寄主根表面定殖、穿过表皮、在皮层中发生及在寄主体内扩展和症状形成等黄萎病菌侵染棉花的过程,阐述了寄主植物形成多级防御反应阻击病原菌的入侵.对黄萎病菌侵染寄主植物过程及其机理下一步研究的问题和内容进行了讨论.     

Effects of transgenic Cry1Ac cotton on entomopathogenic fungi in soil

利用大蜡螟Galleria mellonella L.诱菌法对转Cry1Ac抗虫棉GK-12及其亲本棉花SM-3的根系区域土壤的虫生真菌种类和数量进行监测,评价转Cry1Ac基因对棉田土壤虫生真菌的影响.实验结果显示:检测到的虫生真菌种类,在转基因棉GK-12及其亲本棉SM-3之间无显著差异,均为球孢白僵菌(Beauveria bassiana)和金龟子绿僵菌(Metarhizium anisopliae);在棉花开花初期、盛花期、结铃期或吐絮期,土壤虫生真菌数量在抗虫棉GK-12和亲本SM-3之间存在显著的差异,其他生育期无显著差异;在距棉花主根不同距离(5cm\15cm\25cm)的区域,土壤虫生真菌数量和种类在抗虫棉GK-12和亲本SM-3之间无显著差异.     

转Cry1Ac抗虫棉对土壤虫生真菌群落的影响

利用大蜡螟Galleria mellonella L.诱菌法对转Cry1Ac抗虫棉GK-12及其亲本棉花SM-3的根系区域土壤的虫生真菌种类和数量进行监测,评价转Cry1Ac基因对棉田土壤虫生真菌的影响。实验结果显示:检测到的虫生真菌种类,在转基因棉GK-12及其亲本棉SM-3之间无显著差异,均为球孢白僵菌(Beauveria bassiana)和金龟子绿僵菌(Metarhizium anisopliae);在棉花开花初期、盛花期、结铃期或吐絮期,土壤虫生真菌数量在抗虫棉GK-12和亲本SM-3之间存在显著的差异,其他生育期无显著差异;在距棉花主根不同距离(5 cm\15 cm\25 cm)的区域,土壤虫生真菌数量和种类在抗虫棉GK-12和亲本SM-3之间无显著差异。     

香蕉抗(感)病品种根系分泌物对枯萎病菌和枯草芽孢杆菌的生物效应

为明确香蕉根系分泌物对枯萎病菌及其生防枯草芽孢杆菌的生物效应,采用离位溶液培养法收集抗枯萎病香蕉品种(南天黄)和感枯萎病香蕉品种(桂蕉6号)的根系分泌物,研究根系分泌物对土壤微生物种群数量、香蕉枯萎病菌和枯草芽孢杆菌生长的影响。结果表明: 抗病品种根系分泌物能显著减少土壤真菌的数量,抑制枯萎病菌孢子的萌发;而感病品种根系分泌物能显著促进病菌菌丝生长和孢子的萌发,两个品种根系分泌物均能显著促进枯草芽孢杆菌的生长和生物膜形成。经抗(感)病香蕉品种根系分泌物处理,病菌菌丝生长速率分别为11.28和12.28 mm·d^-1,病菌孢子的萌发率分别为34.6%和79.5%;枯草芽孢杆菌培养12 h后菌体生长量的OD₆₀₀分别为1.27和1.14,生物膜形成量在静置培养72 h后OD₅₇₀分别达1.11和1.30,两个品种处理间的差异均达显著水平。枯草芽孢杆菌在香蕉感病品种根际中定殖的菌量显著高于抗病品种。通过对两个品种根系分泌物中可溶性总糖、游离氨基酸和有机酸的含量和组成分析,发现抗病品种根系分泌物中有机酸和游离氨基酸的含量明显高于感病品种,在各组成成分中,以乙酸和脯氨酸在抗(感)病香蕉品种根系分泌物中含量比值较高,分别达3.7和2.4倍。综上所述,抗病品种根系分泌物能抑制病菌生长,感病品种根系分泌物则会显著促进病菌生长,而两个品种根系分泌物均能显著促进枯草芽孢杆菌的生长和生物膜的形成。     

转Bt基因棉对棉大卷叶螟种群动态的影响

2年的研究发现,棉大卷叶螟SyleptaderogataFabricius的种群数量在不同棉花品种田均呈现上升趋势,但转基因抗虫棉“国抗2 2”的为害程度及百株虫量均小于亲本对照棉“泗棉3号”,其差异达显著或极显著水平;该虫的室内饲养结果表明,2个棉花品种均有利于棉大卷叶螟的增殖,但“国抗2 2”对棉大卷叶螟的抗性明显优于“泗棉3号”,且对棉大卷叶螟成虫存在一定的产卵排斥效应。     

Effects of the expression of foreign insect-resistant genes in cotton plants on Bemisia tabaci

以转基因棉花和对应的常规棉花亲本为材料,探讨外源基因导入对棉花烟粉虱Bemisia tabaci(Gennadius)种群的影响.结果表明,在转基因棉花GK12、33B、SGK上,烟粉虱从卵发育到成虫羽化的历期分别为15.56、15.35和15.25d,较对应的常规棉亲本SM3(19.38d)、33(20.81d)、SY321(18.76d)分别短24.55％、26.23％、18.71％;GK12和33B棉花上烟粉虱的存活率(69.16％)分别较对应的常规棉亲本SM3(54.76％)和33(64.91％)高26.29％和12.81％,而SGK(63.21％)上烟粉虱的存活率与对应的常规棉亲本SY321(62.61％)之间差异不显著;烟粉虱在GK12(84.00)和33B(77.25)上的产卵量分别较SM3(62.25)、33(70.00)高34.93％和10.35％,但SGK和SY321之间差异不显著;在转基因棉花GK12、33B、SGK上,烟粉虱的雌雄性比分别比对应的常规棉亲本高26.71％、46.23％和19.17％.结果表明,外源基因导入后,有助于烟粉虱的发育,提高了烟粉虱的产卵量和雌雄性比,从而促进了烟粉虱种群的上升.     

转Bt基因棉花对烟粉虱天敌昆虫龟纹瓢虫的影响

在实验室控制条件下,以转Bt基因棉花GK12、33B、SGK321上的烟粉虱为食料饲养龟纹瓢虫,研究转Bt基因棉花上的烟粉虱对龟纹瓢虫生长发育及捕食的影响,同时利用Y型嗅觉仪,观察龟纹瓢虫对来自不同类型棉花的棉叶及其烟粉虱的嗅觉反应和视觉反应。结果表明,转Bt基因棉花和对应的常规棉亲本上的烟粉虱对龟纹瓢虫的发育历期和存活率没有明显的影响。龟纹瓢虫对棉花叶片、烟粉虱若虫、蜜露、蜕等4种物质的视觉反应之间没有明显的差异。对4种嗅源物质的嗅觉选择性的大小依次为：烟粉虱若虫＞蜕＞棉花＞蜜露;在两类不同的棉花之间,转基因棉花和常规棉,龟纹瓢虫对GK12、33B、SGK321等三种转基因棉花上的烟粉虱若虫的嗅觉选择性明显较对应的常规棉亲本SM3、33、SY321上的烟粉虱若虫小;对烟粉虱若虫的蜕,两种转单价基因棉花GK12、33B较对应的常规棉亲本上的小,而转双价基因的棉花SGK321上与对应的常规棉之间没有明显的差异。烟粉虱密度大于200头.皿_-1时,龟纹瓢虫捕食转基因棉花上烟粉虱的数量大于对应的常规棉花亲本,但烟粉虱密度小于200头.皿_-1时,龟纹瓢虫捕食转基因棉花上烟粉虱的数量小于对应的常规棉花亲本。龟纹瓢虫对烟粉虱的捕食符合HollingⅡ型反应。龟纹瓢虫取食转基因棉花上的烟粉虱的理论极限值、瞬间攻击率均大于常规棉花。     

转基因抗虫棉苗期氮代谢

以3种不同转基因抗虫棉及其亲本对照为材料,研究了盆栽条件下不同品种棉花苗期氮代谢特征。结果表明与非转基因棉花比较,转Bt基因棉Z30叶片内的硝酸还原酶活性、肽酶活性、游离氨基酸含量均没有明显变化,但转氨酶活性显著增加(增幅为14.03%),可溶性蛋白含量显著减少(降幅为26.29%)。双价转基因棉花CCRI41叶片内除可溶性蛋白含量没有明显变化,其它所测指标均发生了显著的改变。双价转基因棉花SGK321叶片内硝酸还原酶活性、转氨酶活性、氨基酸含量没有明显变化,但肽酶活性和可溶性蛋白含量均显著增加(增幅分别为7.01%和121.32%)。随着转入基因的多样化,其可能引发转基因棉花产生的非预期效应更加不确定与复杂。     

The effect of root exudates from two transgenic insect-resistant cotton lines on the growth of Fusarium oxysporum

The attenuation of disease resistance in transgenic insect-resistant cotton has become one of the important factors restricting cotton production in China. Two transgenic insect-resistant cotton lines and their parental conventional cotton lines were used as the testing materials. The effects of root exudates of these cotton lines on the spore germination and mycelial growth of Fusarium oxysporum were studied and the components, contents of amino acids and sugars were determined. The results showed that the resistance of the two insect-resistant cotton lines to F. oxysporum was inferior to the parental lines, and that their root exudates promoted fungal spore germination and mycelial growth. Considerable differences in the components and contents of both, amino acids and sugars were found between the root exudates of transgenic cotton lines and their parental lines, where the disease indices were highly correlated with the total amount of sugars in the root exudates.     

不同品种小麦与蚕豆间作对蚕豆枯萎病的防治及其机理

通过田间小区试验和水培试验,研究了3个品种小麦（云麦42、云麦47和绵阳29）与蚕豆间作对蚕豆地上部生物量、枯萎病发生、根际微生物代谢功能多样性和枯萎病病原菌尖孢镰刀菌数量的影响,分析了3个不同小麦品种低分子量根系分泌物组分（糖、氨基酸和有机酸）的差异.结果表明： 云麦42与蚕豆间作（YM42/B）和云麦47与蚕豆间作（YM47/B）使蚕豆地上部生物量分别增加16.6%和13.4%,使蚕豆枯萎病病情指数分别降低47.6%和23.3%,绵阳29与蚕豆间作（MY29/B）对蚕豆地上部生物量和枯萎病病情指数均无显著影响.与单作蚕豆相比,YM42/B和YM47/B处理均显著提高了蚕豆根际微生物的平均颜色变化率（AWCD)及对碳源的总利用强度和Biolog ECO板中各种碳源的利用,明显改变了蚕豆根际微生物群落结构,并显著降低了蚕豆根际的尖孢镰刀菌数量;MY29/B处理对AWCD值、碳源总利用强度和Biolog ECO板中各种碳源的利用及蚕豆根际尖孢镰刀菌数量均无显著影响,也未明显改变蚕豆根际的微生物群落结构.3个小麦品种根系分泌物中可溶性糖、游离氨基酸总量和有机酸总量均表现为MY29＞YM47＞YM42.MY29根系分泌物中丝氨酸、谷氨酸、甘氨酸、缬氨酸、蛋氨酸、苯丙氨酸、赖氨酸的含量均显著高于YM42和YM47;精氨酸为YM42、YM47所特有,而亮氨酸为MY29所特有.MY29和YM47根系分泌物中检出酒石酸、苹果酸、柠檬酸、琥珀酸、延胡索酸和顺乌头酸6种有机酸,而YM42根系分泌物中检出洒石酸、苹果酸、柠檬酸和延胡索酸4种有机酸,且MY29和YM47根系分泌物中苹果酸含量显著高于YM42.小麦与蚕豆间作提高了蚕豆根际微生物的活性和碳源利用强度,改变了根际微生物的群落结构,降低了蚕豆根际尖孢镰刀菌的数量,促进了蚕豆生长,抑制了蚕豆枯萎病的发生,但小麦与蚕豆间作控病效果受小麦品种的影响,表明间作系统中非寄主作物根系分泌物的差异是影响间作对土传病害控制的重要因素.     

天麻抗真菌蛋白基因(gafp)转化彩色棉的研究

天麻抗真菌蛋白（gastrodia antifungal protein简称GAFP）是从我国传统中药天麻（Gastrodia elata Bl.）中分离到的一种具有广谱抗真菌活性的蛋白质,它对许多植物真菌病包括棉花枯萎病、黄萎病等的致病菌离体具有很强的抑制作用,因此,在植物抗真菌病基因工程上有很重要的应用价值。本研究通过花粉管通道法,将GAFP的基因gafp转入3个新疆彩色棉品种中,通过田间抗病筛选和分子检测,得到了高抗黄萎病的转基因植株,两株Southern杂交阳性植株LB-5-8和ZB-1-49对黄萎病表现整株免疫。RT-PCR的结果显示,LB-5-8和ZB-1-49中均有gafp的正确转录;离体的抑菌实验也表明,它们的蛋白粗提物对棉花黄萎病致病菌离体有明显的抑制,表明了gafp在转基因植株中的正确表达,翻译的产物具有活性。经过进一步选育和扩繁,发现转基因彩色棉后代具有稳定的、较强的抗黄萎病能力,本研究为通过植物抗病基因工程的方法防治棉花黄萎病提供了一条新的途径。     

Studies on the rhizosphere mycoflora of broad bean and cotton

Summary Seed and root exudates from three varieties of both broad bean and cotton grown aseptically were analyzed for amino acids and sugars. Broad bean varieties were found to excrete considerable amounts of amino compounds with 15 different amino acids, while cotton varieties excreted less consisting of 12 amino acids. Five sugars were identified in the seed and root exudates from broad bean varieties, while three were present in the seed and root exudates from cotton varieties. Seed and root exudates stimulated spore germination and growth of fungi isolated from the rhizosphere.     

东亚钳蝎毒素BmKT四个同源基因的克隆及基因组织分析

BmKT是从本室构建的cDNA文库中筛选到的1个α钠通道毒素,根据其全长cDNA序列设计引物,采用PCR法以蝎总基因组DNA为模板,获得4个BmKT的同源基因,分别命名为BmKT′和BmKTa、BmKTb、BmKTb′.序列分析表明：BmKT′和BmKTa基因含有大小分别为509 bp和506 bp的内含子,位于信号肽编码区内,插入信号肽-4位残基Gly密码子的第一个碱基G之后;而BmKTb和BmKTb′ 的内含子大小均为418 bp．这4个BmKT的同源基因内含子符合GT/AG拼接规律,其中BmKT′和BmKTa的内含子A+T含量分别为61.7%和61.9%,低于目前已报导的大多数蝎毒素基因A+T含量,大大低于它们第一外显子A+T含量（71.7%）,略高于第二外显子A+T含量（55.5%）;而BmKTb,BmKTb′的内含子A+T含量分别为75.8%和76.1%,与目前已报导的大多数蝎毒素基因A+T含量相似．BmKT′基因的外显子与BmKT基因cDNA所对应氨基酸序列仅在信号肽中-7位有一个氨基残基的差异（BmKT: Leu→BmKTa: Val）;而BmKTa基因外显子所推断的氨基酸序列与BmKT前体比较,则在成熟肽的+54位发生了突变（BmKT: Lys→BmKTa: Asn）,是与BmKT同源的一个新基因．BmKTb基因和BmKTb′基因所编码的前体肽与BmKT基因对应的前体肽同源性约为65%,显然BmKTb和BmKTb′是不同于BmKT的2个新基因（GenBank登录号: BmKT′, AY786186; BmKTa, AY676142; BmKTb, AY676140; BmKTb′, AY676141）     

Transgenic Bacillus thuringiensis (Bt) cotton (Gossypium hirsutum) allomone response to cotton aphid, Aphis gossypii, in a closed-dynamics CO(2) chamber (CDCC)

Allocation of allomones of transgenic Bacillus thuringiensis Gossypium hirsutum (Bt cotton) (cv. GK-12) and non-Bt-transgenic cotton (cv. Simian-3) grown in elevated CO₂ in response to infestation by cotton aphid, Aphis gossypii Glover, was studied in a closed-dynamics CO₂ chamber. Significant increases in foliar condensed tannin and carbon/nitrogen ratio for GK-12 and Simian-3 were observed in elevated CO₂ relative to ambient CO_2, as partially supported by the carbon nutrient balance hypothesis, owing to limiting nitrogen and excess carbon in cotton plants in response to elevated CO₂. The CO₂ level significantly influenced the foliar nutrients and allomones in the cotton plants. Aphid infestation significantly affected foliar nitrogen and allomone compounds in the cotton plants. Allomone allocation patterns in transgenic Bt cotton infested by A. gossypii may have broader implications across a range of plant and herbivorous insects as CO₂ continues to rise. Gang Wu and Fa Jun Chen contributed equally to this work.     

Analysis of major tomato xylem organic acids and PITC-derivatives of amino acids by RP-HPLC and UV detection

Major amino acids and organic acids in xylem exudates of tomato plants were separated by reversed phase high performance liquid chromatography (RP-HPLC) and quantified by UV detection. Before separation, amino acids were converted into their phenylisothiocyanate (PITC) derivatives. In a single run, Asp, Glu, Ser, Gln, His, Thr, Ala, Tyr, Val, Met, Cys, Ile, Leu, Phe, and Lys could be separated and detected down to the pmol level. Unresolved peaks were obtained for Asn and Gly and for Arg and Pro. For organic acid analysis, exudates were pre-treated by perfusion over a prepacked Adsorbex SCX cation exchange column, to eliminate exudate amino acids. Elution recoveries for organic acids were close to 100%. The exudate organic acids were separated by ion suppression RP-HPLC chromatography, and peaks could be resolved for L-malic acid, malonic acid, maleic acid, citric acid and fumaric acid, down to the pmol level. UV signals for exudate ascorbic acid, and succinic acid were below the limits of detection. Determination of oxalic acid and tartaric acid was impossible, due to the presence of the exudate salt peak in the chromatogram. The results indicate the potential of the methods applied, and show the applicability of RP-HPLC analysis for the determination of both amino acids and organic acids in xylem exudates.     

甘肃鼢鼠骨骼19种氨基酸含量的测定

用121型氨基酸分析仪对甘肃鼢鼠(Myospalax cansus)躯干、四肢、头骨骨骼中19种氨基酸含量进行测定,并与高原鼢鼠及其他哺乳动物骨骼中氨基酸含量进行差异显著性分析。结果表明,甘肃鼢鼠各部位骨骼中19种氨基酸含量均为甘氨酸最高,谷氨酸次之,胱氨酸最低。同一氨基酸在甘肃鼢鼠不同骨骼部位含量顺序为躯干>四肢(头骨。甘肃鼢鼠骨骼总氨基酸含量低于塞隆骨,与虎骨相近,高于其他哺乳动物。其中羟脯氨酸含量明显高于虎骨、塞隆骨等。     

Amino acids in root exudates of cotton

Summary Cotton-field soils from a wilt-free zone (KPT) and from a wilt-sick zone (PLD), and root exudates of diploid and amphidiploid cotton strains grown in KPT and PLD soils were analyzed for 13 free amino acids employing microbiological assay techniques. Test organisms used wereLactobacillus arabinosus 17/5 andLeuconostoc mesenteroides P. 60.Arginine, proline, lysine, serine, glycine, cystine, and tyrosine occurred in both the soils; amounts of these were higher in PLD soil than in KPT soil.Seven to eleven amino acids were found in the root exudates of cotton. Aspartic acid, threonine (in one), phenylalanine and leucine were found in root exudates but not in the control soils. Diploid strains were found to exude greater amounts of amino acids than the amphidiploid strains. Comparing the effect of the two soils tested, it was found that amino acids in exudates of plants grown in PLD soil were higher except in the case of one diploid strain.In the inoculated series, amino acids in the exudates were markedly lower than the respective healthy controls in the diploid strains. In the exudates of amphidiploid strains also, amino acid contents were reduced but to a lesser extent.Significance of occurrence of cystine in the exudates of some strains is indicated.Part of Doctoral thesis, University of Madras.     

甜瓜根系分泌物中酚酸物质对尖孢镰孢菌的化感效应

采用HPLC法对甜瓜根系分泌物进行分离鉴定,检测到甜瓜根系分泌物中含有没食子酸、邻苯二甲酸、丁香酸、水杨酸、阿魏酸、苯甲酸和肉桂酸7种酚酸物质,通过外源添加法研究该类物质对尖孢镰孢菌的化感效应.室内试验结果表明: 阿魏酸、苯甲酸、肉桂酸在0.1、0.25 mmol·L^-1处理浓度下能够显著促进尖孢镰孢菌的孢子萌发,水杨酸则对孢子萌发具有一定的抑制作用;丁香酸、阿魏酸在菌丝培养后期表现出较强的促进作用.盆栽结果显示,在0.05、0.1和0.5 mmol·L^-1处理浓度下肉桂酸、阿魏酸、苯甲酸可显著促进甜瓜枯萎病病情.