豇豆胰蛋白酶抑制剂基因转化芥菜及抗虫鉴定

用农杆菌介导将豇豆胰蛋白酶抑制剂 (CpTI)基因导入芥菜 ,获得了Kan抗性植株 .经PCR扩增、PCR Southern印迹和Northern印迹分析 ,转化再生植株大部分呈阳性 ,而非转化的再生植株均为阴性 ,证明CpTI基因已存在于芥菜基因组中 .在室内进行了喂虫试验 ,结果表明转基因芥菜抗虫性明显高于对照 ,转基因植株之间存在抗虫性差异     

豇豆胰蛋白酶抑制剂(CpTI)基因转化欧美杨的研究

本研究建立了欧美107杨的高频再生体系,用改良的根癌农杆菌介导法将豇豆胰蛋白酶抑制剂(CpTI)基因导入107杨中。经过严格的卡那霉素(Km)筛选,得到了Km抗性(Kmr)植株。对部分Kmr植株进行PCR及PCR-Southern杂交鉴定,证实CpTI基因已整合进杨树基因组中。转基因植株的饲虫实验表明,其中部分株系的叶片可在一定程度上抑制扁刺蛾幼虫的生长。     

转β-1,3-葡聚糖酶基因和几丁质酶基因棉花

为了提高棉花的抗枯、黄萎病的能力,用花粉管通道法,将烟草β-1,3-葡聚糖酶基因和菜豆几丁质酶基因导入棉花,获得了抗卡那霉素的转化植株。经PCR、PCR－Southern Blot检测,证明两种基因已插入到棉花基因组中。     

转果聚糖蔗糖转移酶基因( Sac B)美丽胡枝子的获得

采用农杆菌介导的遗传转化方法,将来自枯草杆菌的果聚糖蔗糖转移酶基因(SacB)导入美丽胡枝子,以提高胡枝子抵御干旱胁迫和盐胁迫的能力。以美丽胡枝子子叶节为外植体,通过与含有植物双元表达载体pKP的农杆菌LBA4404共培养,将SacB基因导入美丽胡枝子基因组。经卡那霉素筛选后,共获得62株卡那霉素抗性植株。经PCR特异性扩增和PCR-Southern杂交,证明有5株再生植株基因组DNA中整合了SacB基因。通过RT-PCR分析,结果表明SacB基因均获得表达。经过200mmol/LNaCl和5%PEG模拟胁迫,发现转基因植株美丽胡枝子中,可溶性糖含量在任何时候均高于未转化植株,并比对照拥有更高的抗干旱胁迫和盐胁迫能力。     

农杆菌介导抗储粮害虫转基因小麦(Triticum aestivum L.)的获得及分析

以本实验室选育的小麦优良品系的胚性愈伤组织为材料,采用农杆菌介导将抗虫基因豇豆胰蛋白酶抑制剂基因CpTI转入小麦培养细胞,经筛选获得抗卡那霉素的愈伤组织并再生植株。经PCR和实时PCR检测、PCR-Southern和Southernblot验证,确定了3株独立再生植株为含有CpTI的转基因植株。农杆菌菌浓度、侵染时间及转化处理方式对小麦转化率均有明显影响。3株转基因植株正常可育并结籽,形成转基因株系。外源基因在转基因植株T1代中的分离呈多样性,部分株系(转基因株系T-Ⅰ、T-Ⅲ)表现出孟德尔遗传规律。抗虫试验表明,3株转基因植株T2代籽粒对储粮害虫麦蛾具有一定的抗性,转基因株系T-Ⅰ、T-Ⅱ、T-Ⅲ及非转基因植株的T2代籽粒虫蛀率分别为19·8%、21·9%、32·9%和58·3%。转基因植株T1代群体农艺性状调查显示,3个株系具有良好的农艺性状,为小麦的遗传改良提供了新的种质抗虫材料。     

转果聚糖蔗糖转移酶基因(Sac B)美丽胡枝子的获得

采用农杆菌介导的遗传转化方法,将来自枯草杆菌的果聚糖蔗糖转移酶基因(Sac B) 导入美丽胡枝子,以提高胡枝子抵御干旱胁迫和盐胁迫的能力。以美丽胡枝子子叶节为外植体,通过与含有植物双元表达载体pKP的农杆菌LBA4404 共培养,将Sac B 基因导入美丽胡枝子基因组。经卡那霉素筛选后,共获得62 株卡那霉素抗性植株。经PCR特异性扩增和PCR-Southern杂交,证明有5株再生植株基因组DNA 中整合了Sac B 基因。通过RT-PCR分析,结果表明SacB 基因均获得表达。经过200 mmol/L NaCl和5% PEG模拟胁迫,发现转基因植株美丽胡枝子中,可溶性糖含量在任何时候均高于未转化植株,并比对照拥有更高的抗干旱胁迫和盐胁迫能力。     

博落回遗传转化体系的初步建立

博落回是一种重要且应用广泛的药源植物,市场前景广阔。现以博落回无菌苗叶片为材料,通过优化卡那霉素和头孢霉素两种抗生素的浓度,初步建立了博落回遗传转化体系,同时利用农杆菌介导法将MtDREB1C基因导入博落回,获得卡那霉素抗性植株。随机抽取50株转基因植株进行PCR鉴定,其中3株成功扩增出了目的条带,经初步统计,获得博落回阳性苗的概率是6%。实验结果表明植物基因工程技术可作为博落回遗传改良的新途径,为博落回分子育种奠定基础。     

高山被孢霉△6-脂肪酸脱氢酶基因转化大豆的研究

采用农杆菌介导的大豆子叶节转化系统成功地将高山被孢霉△^6-脂肪酸脱氢酶基因导入栽培大豆吉林43和黑龙37品种中。经农杆菌浸染和共培养后,在含50mg/L卡那霉素的选择培养基上连续筛选,获得一批转基因植株。经PCR检测和Southern杂交分析,证明外源基因已导入并整合到大豆的基因中组中。通过GC-MS对大豆种子进行脂肪酸色谱分析,结果表明,产生了γ-亚麻酸,其含量最高可达18.23%。     

利用根癌农杆菌和基因枪法转化获得转抗虫融合蛋白基因（Bt—CpTI）甘蓝植株

以下胚轴,带柄子叶和茎尖为外植体,利用根癌农杆菌和基因枪法将抗虫融合蛋白基因（Bt-CpTI)导入甘蓝品种“中甘8号”,得到了13株卡那霉素抗性植株,经PCR扩增反应和Southern blot分子验证表明;农杆菌介导转化下胚轴和带柄子叶来源的Ⅰ型抗性植株均为转基因植株,而农杆菌介导转化茎尖外植体得到的Ⅱ型抗性植株属“假阳性”植株,基因枪介导转化茎尖的2株Ⅲ型植株中,有1株是非转基因植株,经胰蛋白酶抑制剂活性分析和抗虫测试证明,部分转基因植株有较高的胰蛋白酶抑制剂活性和抗菜青虫能力。     

发根农杆菌介导几丁质酶基因和β-1,3-葡聚糖酶基因转化烟草的研究

用冻融法和三亲交配法将具有几丁质酶基因、β—1,3—葡聚糖酶基因和卡那霉素抗性标记基因的质牲pBLGC导入具有Ri质粒的发根农杆菌中。用叶盘法转化烟草的三个品种,经Kan抗性筛选获得126株再生植株,对119株再生植株分别以启动子CaMV35S、几丁质酶基因和β—1,3—葡聚糖酶基因的引物进行PCR检测,其中几丁质酶基因至阳性的植株有72株,β—1,3—葡聚糖酶基因至阳性的有47株,具有几丁质酶基因和β—1,3—葡聚糖酶基因的植株有36株。对转化的24株转基因经PCR-Southern杂交,结果均至阳性。实验结果表明,外源基因的转化频率与植物品种、外源基因片段的大小有关,几丁质酶基因和β—1,3—葡聚糖酶基因分别或共同整合到植物基因组中。     

转水稻NibT基因玉米植株的获得及抗病性研究

以玉米(Zea mays L.)自交系'金黄96B'为受体材料,供体为质粒pWM101并携带有水稻矮缩病病毒复制酶基因Nib的提前终止突变体基因NibT,采用超声波处理花粉介导植物基因转化方法将NibT基因导入受体,经PCR检测和Southern杂交分析证实获得转基因植株,进而对T1～T3代转基因植株(株系)进行分子分析、田间抗病鉴定和农艺性状调查.逐代分子检测分析结果证明,目的基因可稳定遗传.抗病鉴定结果证明转基因植株(株系)各代抗病水平基本一致,抗病性比对照提高3级.农艺性状调查分析表明,与对照比较,转基因植株株高增加7～18 cm、穗位高增高0～13 cm、穗长增加0.7～2.1 cm、穗粒数多8～35粒、百粒重增加1.1～2.6 g,转基因株系与阴性对照间、各代转基因株系相互间都差异显著(P＜0.05);调查还发现转基因植株的株高和穗位高随着世代的增加,与对照间的差异逐代减少.研究也说明,超声波处理花粉介导植物基因转化方法是一种简捷、快速和有效的植物转化工具.     

A wheat genomic DNA fragment reduces pollen transmission of maize transgenes by reducing pollen viability

A genomic DNA fragment from wheat carrying the Glu-1Dx5 gene has been shown to exhibit reduced pollen transmission in transgenic maize. To localize the region of the DNA fragment responsible for this reduced pollen transmission, we produced transgenic maize plants in which the wheat genomic DNA proximal to the 1Dx5 coding sequence was replaced with the maize 27 kDa gamma-zein promoter. Like the wheat promoter-driven Glu-1Dx5 transgene, this zein promoter-driven transgene functioned to produce 1Dx5 in maize endosperm. However, with the zein promoter-driven transgene, pollen transmission of the transgene loci was normal in most self- and cross-pollinations. We concluded that the wheat genomic DNA proximal to the wheat 1Dx5 coding sequence was required for reduced pollen transmission of the transgene in maize. In two of four transformation events of the wheat promoter-driven construct examined, pollen exhibited two morphological classes. In one class, pollen was normal in morphology and displayed average viability, and in the second, pollen was reduced in size and did not germinate on artificial media. DNA from the transgene was detectable in mature pollen from plants with reduced pollen transmission of transgene loci. To explain these observations, we hypothesize that elements within the transgene construct interfere with pollen development. We demonstrated that the wheat genomic DNA fragment can be used to control pollen transmission of an herbicide resistance transgene genetically linked to it. The wheat genomic DNA fragment may contain elements that are useful for controlling pollen transmission of transgene loci in commercial maize grain and seed production.     

Assessing the Effects of Bt Maize on the Predatory Mite Neoseiulus cucumeris

The investigation of Neoseiulus cucumeris in the context of the ecological risk assessment of insect resistant transgenic plants is of particular interest as this omnivorous predatory mite species is commercially available and considered important for biological control. In a multitrophic feeding experiment we assessed the impact of Bt maize on the performance of N. cucumeris when offered spider mites (Tetranychus urticae) reared on Bt (Bt11, Syngenta) or non-Bt maize (near isogenic line) and Bt or non-Bt maize pollen as a food source. Various parameters including mortality, development time, oviposition rate were measured. Spider mites were used as a prey for N. cucumeris, since these herbivores are known to contain similar levels of Cry1Ab toxin, when reared on Bt maize, as those found in the transgenic leaf material. In contrast, toxin levels in pollen of this transgenic cultivar are very low. No differences in any of the parameters were found when N. cucumeris was fed with spider mites reared on Bt and non-Bt maize. Pollen was shown to be a less suitable food source for this predator as compared to spider mites. Moreover, subtle effects on female N. cucumeris (9% longer development time and 17% reduced fecundity) were measured when fed with pollen originating from Bt maize as compared to non-Bt maize pollen. Our findings indicate that the predatory mite N. cucumeris is not sensitive to the Cry1Ab toxin as no effects could be detected when offered Bt-containing spider mites, and that the effects found when fed with Bt maize pollen can be assigned to differences in nutritional quality of Bt and non-Bt maize pollen. The significance of these findings is discussed with regard to the ecological relevance for risk assessment of transgenic plants.     

玉米Ubi－1启动子在可育转基因玉米植株中的表达活性

本工作将玉米泛素基因－1启动子（Ubi-1)与大肠杆菌β－葡萄糖苷酸酶基因（gus,uidA)的编码区融合,通过基因枪粒子轰击方法转化来自水成熟胚盾片组织的I－型愈伤组织,经PPT选择获得可育的玉米转基因植株,并采用组织化学方法分析了Ubi-1启动子驱动的gus基因在不同组织,细胞中的表达活性,发现gus基因在除花药壁以外的其它所试组织中均可以有效表达。Ubi：GUS在花粉,卵细胞中T1代转基因植株未成熟胚中的表达显示该启动子在植株发育的早期阶段即具有活性。对T0代转基因植株的花粉进行GUS组织化学染色,gus基因呈1：1分离,显示外源基因在转基因植株中以孟德尔方式遗传。同时发现,使用玉米本身的启动子Ubi-1可以降低外源基因在转基因玉米中的拷贝数,进而避免基因沉默现象的发生。目前已得到第二代转基因种子。     

Overexpression of <Emphasis Type="Italic">Zm401</Emphasis>, an mRNA-like RNA,has distinct effects on pollen development in maize

We previously identified a 0.7 Kb cDNA fragment of Zm401, a novel pollen-specific gene in maize (Zea mays). However, little information is known about the function of Zm401 in pollen development. The full-length of Zm401 cDNA was amplified by 5′ RACE and 3′ RACE and both sequence analysis and in vitro translation of Zm401 showed that it belonged to an mRNA-like non-coding gene. To analyze its possible biological roles in pollen development, the Zm401 cDNA was overexpressed in transgenic maize under the control of a pollen specific promoter Zm13 or a CaMV 35S promoter. RT-PCR and RNA gel blot analysis indicated that the expression level of Zm401 in leaves and anthers of transgenic plants was much higher than that of non-transformants. Compared with the non-transformed maize, transgenic maize showed distinct phenotypes, such as abnormal tassels and degenerate anthers. The histological observation showed that the development of pollen grains and anthers in transgenic plants were abnormal. These abnormalities include delayed degradation of tapetum, asynchronous fusion of pollen sacs, and aborted pollen grain development. Furthermore, the pollen viability in six transgenic plants ranged from 1.24% to 6.63%. The reduced pollen viability cosegregated with the transgene in a selfed progeny. These results suggest that Zm401 is involved in the regulation of pollen development. This article demonstrated Zm401, as a non-coding RNA, plays an essential role in pollen development. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Use of maize pollen by adult Chrysoperla carnea (Neuroptera: Chrysopidae) and fate of Cry proteins in Bt-transgenic varieties

We investigated the use of maize pollen as food by adult Chrysoperla carnea under laboratory and field conditions. Exposure of the insects to insecticidal Cry proteins from Bacillus thuringiensis (Bt) contained in pollen of transgenic maize was also assessed. Female C. carnea were most abundant in a maize field when the majority of plants were flowering and fresh pollen was abundant. Field-collected females contained an average of approximately 5000 maize pollen grains in their gut at the peak of pollen shedding. Comparable numbers were found in females fed ad libitum maize pollen in the laboratory. Maize pollen is readily used by C. carnea adults. When provided with a carbohydrate source, it allowed the insects to reach their full reproductive potential. Maize pollen was digested mainly in the insect's mid- and hindgut. When Bt maize pollen passed though the gut of C. carnea, 61% of Cry1Ab (event Bt176) and 79% of Cry3Bb1 (event MON 88017) was digested. The results demonstrate that maize pollen is a suitable food source for C. carnea. Even though the pollen grains are not fully digested, the insects are exposed to transgenic insecticidal proteins that are contained in the pollen.     

Can the growing of transgenic maize threaten protected Lepidoptera in Europe?

We evaluated whether protected European butterflies can potentially be at risk if transgenic maize is extensively grown in Central Europe. We explored potential consequences of both insect resistant (IR) and herbicide resistant (HR) transgenic maize. IR maize can produce pollen that is toxic to lepidopteran larvae, and this puts butterfly species at possible risk if the presence of young larvae coincides with maize flowering, during which large quantities of maize pollen can be deposited on vegetation. By considering the timing of maize flowering in Europe and the phenology of the protected Lepidoptera species, we found that 31 species had at least one generation where 50% of the larval stage overlapped with maize flowering, and 69 species for which first instar larvae were present during maize pollen shedding. HR maize allows high concentration herbicide treatments on fields without seasonal limitation, which can drastically reduce weed densities. In cases where such weed species are host plants for protected butterflies, reduced host plant/food availability can result, causing population decreases. By using published information, we first identified the important weed species in major maize-growing European countries. Subsequently, we checked whether the host plants of protected Lepidoptera included species that are common maize weeds. We identified 140 protected species having food plants that are common weeds in one or more of the major European maize-growing countries. If HR maize is grown in Europe, there is a potential hazard that their food plants will seriously decline, causing a subsequent decline of these protected species.     

转Btc-ry1 Ah基因玉米花粉对龟纹瓢虫解毒酶和中肠蛋白酶活性的影响

研究了取食转Bt-cry1Ah基因玉米花粉对龟纹瓢虫Propylaea japonica（Thunberg）体内解毒酶和中肠蛋白酶活性的影响。利用饲喂结合比色方法,比较龟纹瓢虫取食转Bt-cry1Ah基因玉米花粉和非转基因玉米花粉后体内α-乙酸萘酯酶、乙酰胆碱酯酶、谷胱甘肽-S-转移酶、中肠总蛋白酶、类胰蛋白酶和类胰凝乳蛋白酶的酶活性。结果发现：在解毒酶方面,取食Bt玉米花粉的龟纹瓢虫4龄幼虫和蛹的α-乙酸萘酯酶活性显著低于取食非Bt玉米花粉的龟纹瓢虫（对照组）,取食Bt玉米花粉的龟纹瓢虫的乙酰胆碱酯酶和谷胱甘肽-S-转移酶活性在各个发育时期与对照相比均无显著差异。在中肠蛋白酶方面,与对照组相比,取食Bt玉米花粉的龟纹瓢虫的总蛋白酶和强碱性类胰蛋白酶活性在各个发育时期均无显著差异;但取食Bt玉米花粉的龟纹瓢虫的弱碱性类胰凝蛋白酶和类胰凝乳蛋白酶活性在蛹期显著低于取食非Bt玉米花粉的龟纹瓢虫。由此可见,龟纹瓢虫取食含有Cry1Ah杀虫蛋白的玉米花粉后,体内代谢解毒酶和中肠蛋白酶与Cry1Ah杀虫蛋白相互作用,可能会引起某些酶活性的变化。因此,转cry1Ah基因玉米花粉对龟纹瓢虫的潜在影响还需要进一步的研究。     

Analysis of a maize α-tubulin gene promoter by transient expression and in transgenic tobacco plants

The pattern of expression directed by the promoter of the maize Tub α 1 gene was investigated by analysis of chloramphenicol acetyl transferase (CAT) and β-glucuronidase (GUS) activities in transient expression experiments of maize and tobacco protoplasts. The same promoter was also investigated by histochemical GUS analysis in transgenic tobacco plants containing promoter gene fusions. As determined by histochemical tests, the Tub α 1 promoter gene preferentially directs GUS expression in regenerating root tip meristems and pollen. This pattern corresponds to the distinctive features of natural expression of the gene in maize as determined by Northern analysis. However, no expression is observed in other meristematic tissues of the transgenic tobacco plants, as in shoot apex or in coleoptiles, which is weakly detected in maize. Analysis of the regulatory properties of 5' promoter deletions showed that the proximal region of the promoter, from positions −1410 or −449 to 15 bp upstream of the ATG, is sufficient to establish the qualitative pattern of expression in transgenic tobacco plants. Deletions to positions −352 or −117 abolished the expression in roots, but not in pollen, suggesting that upstream of these positions there are elements responsible for the pattern in root. Further deletions abolished all the promoter activity, suggesting that this promoter region contains the elements essential for expression in pollen. The different patterns and levels of transient and stable expression are discussed.     

Laboratory studies on the effects of pollen from Bt-maize on larvae of some butterfly species

Abstract:   Three lepidopteran species were tested to determine their susceptibility against the ingestion of pollen from genetically modified maize plants. To prove the existence of dose–response relations between the applied amount of pollen (Bt-maize) and the damages on the tested larvae, a method was developed which makes it possible to feed caterpillars with defined amounts of pollen. If their food plants were contaminated with pollen of a cultivar of the Bt-176 maize-line Pieris brassicae , Pieris rapae and Plutella xylostella -larvae fed less, grew more slowly and showed a higher mortality than caterpillars of an untreated control group. The 50% lethality (LD₅₀)-values were calculated for P.xylostella (L₄) with 19.2, for P. rapae (L₂) with 39.0 and also for P. brassicae (L₂) with 139.2 pollen of the transgenic maize Pactol CB. Studies with P. brassicae -caterpillars of different larval stages indicated, that older individuals showed a higher tolerance against pollen from Bt-maize than younger ones. It must be stated on the basis of the present studies, that ingestion of non-transgenic maize pollen has neither a positive nor a negative effect on caterpillars. It becomes clear from the information presented here that it is still difficult to make general statements about the endangering of butterflies, arising from cultivation of genetically modified maize lines. Further investigations on this issue are needed. Initially, the LD₅₀-values concerning the larvae of certain butterfly species have to be determined to anticipate the risks, and in addition the distances between habitats with caterpillar host plants and maize fields, and the abundance of these plants have to be considered.