棉花arf1启动子驱动Cry1A基因在烟草中特异性表达研究

在新一代Bt作物以及Bt作物安全性研究方面, Bt毒蛋白在植物特定发育阶段的表达特性渐受关注。本研究构建了植物表达载体pGBI121.A1Bt, 其携带棉花arf1启动子驱动Cry1A基因的表达盒, 启动子后面有一个Ω序列; 对照载体pGBI121.4AB携带P2E35S启动子(增强子加倍的修饰CaMV 35S启动子)驱动Cry1A基因的表达盒, 在启动子后面也有一个Ω序列。利用根癌农杆菌介导法, 将植物表达载体pGBI121.4AB和pGBI121.A1Bt转化烟草, 分别获得44株和42株转基因烟草再生植株。ELISA检测表明, 在pGBI121.A1Bt转基因烟草的蒴果、蒴果壳、花瓣和叶中Cry1A基因的平均表达水平分别为pGBI121.4AB的1.5、1.5、1.4和0.3倍。棉花arf1启动子在烟草中表达, 证明了该启动子在植物生殖器官中具有优势表达特性, 为arf1启动子应用于转基因抗虫棉, 在棉花蕾铃中优势表达Bt毒蛋白, 提高转基因棉花蕾铃抗虫性的新一代抗虫棉研究提供了依据。     

High-efficiency transformation of Gossypium hirsutum embryogenic calli mediated by Agrobacterium tumefaciens and regeneration of insect-resistant plants

A simple protocol of transformation of cotton (Gossypium hirsutum L.) at a high frequency has been developed via Agrobacterium mediation, coupled with the use of embryogenic calli as explants. Embryogenic calli derived from only one to two somatic embryogenic calli lines of two Chinese cotton cultivars, the cvs. Ekang 9 and Jihe 321 which have low embryogenic potency were first inoculated with the A. tumefaciens strain LBA4404 harbouring binary vector pBin438 carrying a synthetic Bacillus thuringiensis‐active Cry1Ac and API‐B chimeric gene. Infected embryogenic calli were co‐cultivated for 48 h and were then moved on to the selection medium with kanamycin (100 mg/l) for 7‐8 weeks. Then, the kanamycin‐resistant calli (Km¹) subcultured in proliferation medium would re‐differentiate to form somatic embryos in 30 days. Cotyledon embryos were transferred to 100‐ml Erlenmeyer flasks for germination and regeneration. Putative transformants were confirmed by polymerase chain reaction and Southern blot analysis. Forty‐five regenerated plants were successfully transferred to soil, of which 12 proved to have the active Cry1Ac and API‐B chimeric gene. Insect resistance was tested by bioassay. The transgenic plants were highly resistant to cotton bollworm (Heliothis armigera) larvae, with mortality (insect resistance) ranging from 95.8 to 100%. In comparison with the methods used in Agrobacterium‐mediated transformation of cotton hypocotyls or cotyledons, about 6 months are saved by using the method presented in this paper to obtain a large number of transgenic plants.     

Stable resistance against the leaf miner <Emphasis Type="Italic">Leucoptera coffeella</Emphasis> expressed by genetically transformed <Emphasis Type="Italic">Coffea canephora</Emphasis> in a pluriannual field experiment in French Guiana

Summary A pluriannual field trial of transgenic clones of Coffea canephora (the Robusta coffee tree) transformed for resistance to the lepidopteran coffee leaf miner Leucoptera coffeella was installed in French Guiana. Fifty-eight transformed clones produced by transformation of the C. canephora clone 126 were planted. They were harbouring the pEF1α constitutive promoter of Arabidopsis thaliana controlling either the Bacillus thuringiensis native gene for the cry1Ac insecticidal protein (eight clones) or a synthetic cry1Ac gene (53 clones). The vectors for the transformation were a strain of the bacterium Agrobacterium tumefaciens and one of Agrobacterium rhizogenes. The transformed clones were generally independent, presenting different integration patterns of the genetic construct. Four randomly distributed groups of five plants per transformed clone were planted along with 60 untransformed control trees. Over a 4-year period after plantation six releases of L. coffeella were performed. Mines on the leaves are the marks of larvae development and were counted on plants. A majority of the independent transformed clones harbouring the synthetic gene and transformed by the strain of A. tumefaciens displayed constantly much less mines than the control, therefore expressing a stable resistance. The need for complementary research is presented.     

Constitutive expression of Cry proteins in roots and border cells of transgenic cotton

Transgenic cotton plants expressing Cry1Ac and Cry2Ab, from the soil bacterium Bacillus thuringiensis (Bt), provide effective control of certain lepidopteran pests, however, little is known about the proteins below ground expression. We used ELISA to quantify in vitro expression of the Cry1Ac and Cry2Ab proteins in mucilage, root border cells and root tips in five transgenic cultivars of cotton compared to conventional cultivar Sicot 189. Expression of Cry proteins in roots and border cells of the transgenic cotton cultivars was constitutive and at detectable levels, with Cry1Ac and Cry2Ab protein expression ranging from <20 ppb to >100 pbb. To determine if genetically modified cotton demonstrated simple differences in properties of the root, when compared to an elite parental line (cv. Sicot 189), we enumerated border cells on seedling radicles. Border cell counts of 14 cultivars ranged from 0.2 to 1.1 × 10⁴ cells per root tip with an average of 5 × 10³ border cells. Border cell production in the transgenic cultivars was generally similar to that of both donor and elite parents, the exception being the cultivar Sicot 189, which had substantially more border cells than all of its transgenic derivatives. Comparison of border cell number with varietal disease resistance ranking found a limited relationship (r ² = 0.65, n = 7) between border cell numbers and the commercial resistance rank against Fusarium wilt of cotton. The implications of differences in cotton cultivar border cell number and root tip expression of Cry proteins for plant–microbe interactions in the rhizosphere and the soil ecosystem are yet to be resolved.     

<Emphasis Type="Italic">Agrobacterium</Emphasis> mediated transformation of indica rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) for insect resistance

The rice leaffolder (RLF), Cnaphalocrocis medinalis is an important pest of rice that causes severe damage in many areas of the world. The plants were transformed with fully modified (plant codon optimized) synthetic Cry1C coding sequences as well as with the hpt and gus genes, coding for hygromycin phosphotransferase and β-glucuronidase, respectively. Cry1C sequences placed under the control of doubled 35S promoter plus the AMV leader sequence, and hpt and gus genes driven by cauliflower mosaic virus 35S promoter, were used in this study. Embryogenic calli after cocultivation with Agrobacterium were selected on the medium containing hygromycin B. A total of 67 hygromycin-resistant plants were regenerated. PCR and Southern blot analyses of primary transformants revealed the stable integration of Cry1C coding sequences into the rice genome with predominant single copy integration. R₁ progeny plants disclosed a monogenic pattern (3:1) of transgene segregation as confirmed by molecular analyses. These transgenic lines were highly resistant to rice leaffolder (RLF), Cnaphalocrocis medinalis as revealed by insect bioassay.     

转Cry2A基因抗虫甘蔗植株的培育

Bt基因具有广谱杀虫性,培育转基因抗虫品种为甘蔗病虫害防治提供了新思路。为了检验Cry2A基因对甘蔗螟虫的防治效果,本研究构建了由玉米Ubi启动子驱动的Cry2A基因高效植物表达载体Cry2A-3300,利用农杆菌介导技术,对甘蔗胚性愈伤组织材料进行遗传转化,经过筛选培养及再生培养共得到46株抗性植株。对抗性植株进行PCR扩增检测,15株呈阳性;进一步利用RT-PCR,表明外源基因Cry2A在转录水平上得到有效表达。室内抗虫鉴定的结果表明Cry2A基因可有效抑制害虫的生长,这将有利于后期进行田间防治。     

Over-expression of <Emphasis Type="Italic">Arabidopsis</Emphasis><Emphasis Type="Italic">FT</Emphasis> gene reduces juvenile phase and induces early flowering in ornamental gentian plants

Ornamental gentian plants are perennial and have a juvenile period of over 1 year before flowering. We transformed gentian plants with a construct comprising the Arabidopsis FLOWERING LOCUS T (FT) gene (encoding a major component protein of the flowering hormone ‘florigen’) under the control of the rolC promoter from Agrobacterium rhizogenes, which is known to induce vascular-specific expression. The resultant rolCpro-FT transgenic gentian plants showed early flowering in vitro and the earliest line formed floral buds within 4 months after transformation. Regeneration experiments from leaf explants of these rolCpro-FT transgenic plants also confirmed the early flowering phenotype. After acclimatization, these transgenic plants showed normal floral development in a closed greenhouse. There is no effective method to induce early flowering by cultivation management in gentian, therefore these lines might be very useful as annual early season cultivars.     

Field evaluation and fiber analysis of transgenic cotton

We report the field evaluation of second generation of transgenic cotton expressing Bacillus thuringiensis (Bt) genes cry1Ac and cry2A under CaMV 35S promoter. Sixty-five transgenic lines were grown under RCBD design. Transgenic plants exhibited inherent ability to resist target insect (p < 0.05 and 0.01). Morphological studies showed significant reduction in plant height making them favorable for breeding. Yield was significantly increased for the transgenic lines. Fiber analysis showed improved gin turn out 40% for transgenic lines in comparison to 32% for non-transformed lines. Fibre quality of transgenic lines was not affected when compared with non transgenic lines. Inheritance pattern for transgenic lines suggests the need of further studies to understand the complex molecular mechanisms for resistance management and biosafety studies to develop new Bt cotton varieties.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of pigeon pea [<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) Millsp.] for resistance to legume pod borer <Emphasis Type="Italic">Helicoverpa armigera</Emphasis>

Agrobacterium-mediated genetic transformation was performed using embryonic axes explants of pigeon pea. Both legume pod borer resistant gene (cry1Ac) and plant selectable marker neomycine phosphor transferase (nptII) genes under the constitutive expression of the cauliflower mosaic virus 35S promoter (CaMV35S) assembled in pPZP211 binary vector were used for the experiments. An optimum average of 44.61% successfully hardened dot blot Southern hybridization positive plants were obtained on co-cultivation media supplemented with 200 μM acetosyringone without L-cysteine. The increased transformation efficiency from a baseline of 11.53% without acetosyringone to 44.61% with acetosyringone was further declined with the addition of different concentrations of L-cysteine to co-cultivation media. Transgenic shoots were selected on 50 and 75 mg L⁻¹ kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 20 g L⁻¹ sucrose and 0.5 mg L⁻¹ indole butyric acid in the absence of kanamycin. Furthermore, 100% seed setting was found among all the transgenic events. The plants obtained were subjected to multi- and nochoice tests to determine the behavioral responses and mortality through Helicoverpa armigera bioassays on the leaf and relate their relationship with the expression of cry1Ac protein which was found to be less in leaf as compared to the floral buds, anther, pod, and seed.     

转基因棉对棉蚜生长发育及其主要代谢物质含量的影响

近年来,转基因棉田非靶标害虫种群数量呈明显的上升趋势,棉蚜是转Bt(Bacillus thuringiensis)基因棉田发生危害较严重的非靶标刺吸性害虫之一.在实验室内,棉蚜分别用转Bt基因棉和亲本常规棉饲养40代以上,采用酶联免疫法( ELISA)检测转Bt基因棉SGK321上棉蚜体内的Bt蛋白含量,研究转Bt基因...     

GUS基因和NPTⅡ基因表达的相关性及其在转基因棉花检测研究中的应用

利用农杆菌介导转化法,将含有35S启动子驱动NPTII基因和GUS基因以及棉纤维特异表达启动子E6驱动目的基因FB的植物表达载体转入到常规棉花R15中.重点分析了GUS基因和NPTII基因在愈伤诱导阶段、T0代及T1代转基因棉花中的表达情况.综合两个基因的表达来进行转基因棉花的阳性鉴定,可以为转基因棉花后代的纯合选育提供双重保障.7个转基因株系选育到T3代共获得株行51个,卡那霉素检测多数株行阳性率在90%以上,其中21个株行阳性率达100%.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) with a fungal phytase gene improves phosphorus acquisition

A phytase gene (phyA), isolated from Aspergillus ficuum (AF537344), was introduced into cotton (Gossypium hirsutum L.) by Agrobacterium-mediated transformation to increase the phosphorus (P) acquisition efficiency of cotton. Southern and Northern blot analyses showed that the phyA was successfully incorporated into the cotton genome and expressed in transgenic lines. After growing for 45 days with phytate (Po) as the only P source, the shoot and root dry weights of the transgenic plants all increased by nearly 2.0-fold relative to those of wild-type plants, but were similar to those of transgenic plants supplied with inorganic phosphorus. The phytase activities of root extracts prepared from transgenic plants were 2.4- to 3.6-fold higher than those from wild-type plants, and the extracellular phytase activities of transgenic plants were also 4.2- to 6.3-fold higher. Furthermore, the expressed phytase was secreted into the rhizospheres as demonstrated by enzyme activity staining. The transgenic plants accumulated much higher contents of total P (up to 2.1-fold after 30 days of growth) than the wild-type plants when supplied with Po. These findings clearly showed that cotton plant transformed with a fungal phytase gene was able to secret the enzyme from the root, which markedly improved the plant’s ability to utilize P from phytate. This may serve as a promising step toward the development of new cotton cultivars with improved phosphorus acquisition.     

Transgenic cotton, expressing Amaranthus caudatus agglutinin, confers enhanced resistance to aphids

To evaluate the possible antiaphid function of Amaranthus caudatus agglutinin (ACA) in allogenetic plants, transgenic cotton plants expressing the ACA gene under the control of a phloem‐specific promoter were generated via Agrobacterium‐mediated gene transformation. Based on the results of Southern blot analyses, six plants with single or lower copy transgene and favourable agronomic traits were selected for further studies. ACA expression levels ranged from 0.02% to 0.45% of total soluble protein as determined by Western blot analysis in the six selected transgenic plants. Insect bioassays using nymphs of cotton aphid (Aphis gossypii Glover) showed that five of the six transgenic plants significantly inhibited the population growth of cotton aphid, with the highest inhibition rate of 64.5%. These results shed some new light on the antiaphid function of the ACA gene as well as the promising application of the gene for obtaining aphid‐resistant transgenic cotton plants to reduce the yield loss and honeydew contamination of fibre by aphids.     

苏云金芽孢杆菌Cry1Ab和Cry1C同源重组及转化大豆

在研究和确定Cry1Ab和Cry1C蛋白对甜菜夜蛾和斜纹夜蛾协同杀虫作用的基础上,利用同源重组技术进行Cry1Ab和Cry1C之间的结构域交换获得高效融合蛋白,利用农杆菌介导的子叶节法将融合抗虫基因表达载体导入大豆中,从而获得抗虫转基因大豆新材料。采用PCR及蛋白试纸条检测法对获得的大豆再生植株进行鉴定,测得结果初世代阳性植株为121株。对部分后代转基因植株的遗传分析表明外源基因能够稳定遗传。室内接种斜纹夜蛾鉴定表明,含有重组基因的转基因大豆对斜纹夜蛾有较强的抗性。因此,利用同源重组技术进行Cry1Aa和Cry1C之间的结构域交换,是获得高抗食叶性害虫大豆的有效途径。     

Transformation of α-EXPA1 gene leads to an improved fibre quality in Gossypium hirsutum

The Expansin protein is known for its multifaceted roles in plant growth, especially cell walls. However, very few studies have been done so far to assess the effects of expansin genes on Cotton fibre development. The present study is a successful effort to fill this gap, where the α-EXPA1 gene transformed into a local cotton variety, Gossypium hirsutum, through Agrobacterium-mediated transformation under Gossypium hirsutum Seed coat and Fibre-specific promoter (GhSCFP). The transgenic cotton plants underwent molecular characterization and fibre trait evaluation. Our results indicated that α-EXPA1 showed an up-regulated expression during the transition phase of secondary cell wall synthesis and resulted in improving the fibre parameters, especially micronaire value. Transgenic cotton fibre also showed a finer twisting under the Scanning electron microscope (SEM) as compared to non-transgenic cotton fibre samples. The fibre production is influenced by more than nine thousand genes, and the fibre improvement cannot be just achieved through a single gene transformation. However, α-EXPA1 is one of the potential candidates for cotton fibre research as it significantly improved the cotton fibre.     

抗虫棉外源Cry1A融合杀虫蛋白在土壤中的降解动态

 以转Bt基因棉GK12和转Bt+CpTI基因棉中棉所41为试验材料,以其亲本材料（泗棉3号、中棉所23）为对照,采用ELISA测定方法,研究了抗虫棉外源Bt杀虫蛋白在土壤中的降解动态。结果表明：在土壤掩埋条件下,转Bt 基因棉和转双价基因棉叶片中Bt杀虫蛋白的降解动态基本一致,降解周期达6个月;叶片掩埋后1～3个月,杀虫蛋白降解最迅速,4～6个月降解缓慢,第7个月已检测不到。不同生育期两者根系中Bt杀虫蛋白含量变化趋势基本一致,5月最高,6－9月迅速下降,10月至次年3月逐渐下降,至次年4月已检测不到。土壤中Bt杀虫蛋白的降解动态基本一致,两类抗虫棉播种前土壤中均检测不到Bt杀虫蛋白,苗期开始Bt杀虫蛋白的含量逐渐增加,至花期均达到最高峰,铃期以后逐渐下降。棉花收获后6个月内,两类抗虫棉田土壤中Bt杀虫蛋白含量迅速降低,到次年4月已检测不到。     

转Cry1A基因海岛棉的获得和鉴定

进行农杆菌介导转抗虫基因试验，以期选育抗虫性优良的海岛棉新品系(品种)；本实验在海岛棉体细胞胚再生体系的基础上，将携带Cry1A基因的表达载体pBI121通过农杆菌介导法转化到‘新海33号’的胚性愈伤组织中。经培养基筛选、PCR检测、RT-PCR检测、抗虫性鉴定等筛选抗虫后代。经培养基筛选共获得12棵抗性植株。PCR检测结果显示，这12棵抗性植株均能扩增出了大小相符的片段，检测结果呈阳性；RT-PCR结果显示，12棵转基因植株目的基因已经整合入基因组，且能反转录出mRNA；Bt-Cry1Ab/Ac转基因试纸条检测结果表明这12棵转基因植株含有目的蛋白；抗虫性鉴定结果证实了这些转基因植株对棉铃虫幼虫具有一定的抗性。本研究成功将抗虫基因导入至海岛棉的体内，获得了具有抗虫性的植株，其结果对转基因抗虫海岛棉新品种的培育具有一定的参考价值。     

Characterization of transgenic male sterility in alfalfa

Dependable male sterility would help to make hybrid cultivar development a reality in alfalfa once higher levels of heterosis are attained. Alfalfa plants obtained by genetic transformation with a construct containing the Barnase gene under the control of a tobacco anther tapetum specific promoter were studied. Vacuolization and degeneration of the tapetal cell cytoplasm at a premeiotic stage of development were observed in all five transformed plants (T₀)examined, but the severity of the abnormalities varied greatly among pollen sacs of a genotype. During the meiotic stage, some pollen sacs showed reduction in size, and the tapetum generally appeared thinner when compared to those of the non transgenic plants; tapetal cells showed abnormal vacuolization and signs of cytoplasm degeneration. Despite this, some microspores were formed and some pollen grains were shed in all the T₀ plants, but these were highly variable in size and had very low in vitro germinability. Self-fertility was negligible. The T₀ plants were crossed with one or two unrelated non transgenic male-fertile plants. Mendelian segregation was observed with two exceptions. Instability of the trait in F₁ progenies was noticed, varying for different T₀ parents. F₁ plants exhibiting higher sterility than the primary transformants were observed, indicating that it should be possible to obtain good male sterile plants by backcrossing this trait into different genetic backgrounds. The possible use of this transgenic male sterility in alfalfa breeding is briefly discussed. This revised version was published online in July 2006 with corrections to the Cover Date.