油菜BnCr4基因RNAi载体构建及植株转化

该实验构建了含甘蓝型油菜黄化相关基因BnCr4特异片段反向重复结构的RNA干扰(RNAi)载体pFGC5941-Cr4,通过根癌农杆菌介导转化油菜,获得47株抗Basta的抗性再生油菜植株,其中10株经PCR鉴定为阳性转基因植株.随机选取3株经鉴定的转基因阳性油菜植株进行半定量RT-PCR分析,结果显示,相对于非转基因的野生型油菜,3株转基因植株中BnCr4基因的表达量分别降低了78.5%、8.5%、11.8%,表明该干扰载体转入油菜能特异引起植株BnCr4基因表达量下降.     

Expression of the S receptor kinase in self-compatible Brassica napus cv. Westar leads to the allele-specific rejection of self-incompatible Brassica napus pollen

Expression of an S receptor kinase (SRK910) transgene in the self-compatible Brassica napus cv. Westar conferred on the transgenic pistil the ability to reject pollen from the self-incompatible Brassica napus W1 line, which carries the S910 allele. In one of the SRK transgenic lines, 1C, virtually no seeds were produced when the transgenic pistils were pollinated with W1 pollen (Mean number of seeds per pod = 1.22). This response was specific to the W1 pollen since pollen from a different self-incompatible Brassica napus line (T2) and self-pollinations were fully compatible. Westar plants expressing an S locus glycoprotein transgene (SLG910) did not show any self-incompatibility response towards W1 pollen. Transgenic Westar plants resulting from crosses between the 1C SRK transgenic line and three SLG910 transgenic lines were also tested for rejection of W1 pollen. The additional expression of the SLG910 transgene in the SRK910 transgenic plants did not cause any significant further reduction in seed production (Mean seeds/pod = 1.04) or have any detectable effects on the number of pollen grains that adhered to the pistil. Thus, while the allele-specific SLG gene was previously reported to have an enhancing effect on the self-incompatibility response, no evidence for such a role was found in this study.     

Characterization and immunolocalization of a cytosolic calcium-binding protein from Brassica napus and Arabidopsis pollen.

Two low-molecular-weight proteins have been purified from Brassica napus pollen and a gene corresponding to one of them has been isolated. The gene encodes an 8.6-kD protein with two EF-hand calcium-binding motifs and is a member of a small gene family in B. napus. The protein is part of a family of pollen allergens recently identified in several evolutionarily distant dicot and monocot plants. Homologs have been detected in Arabidopsis, from which one gene has been cloned in this study, and in snapdragon (Antirrhinum majus), but not in tobacco (Nicotiana tabacum). Expression of the gene in B. napus was limited to male tissues and occurred during the pollen-maturation phase of anther development. Both the B. napus and Arabidopsis proteins interact with calcium, and the potential for a calcium-dependent conformational change was demonstrated. Given this affinity for calcium, the cloned genes were termed BPC1 and APC1 (B. napus and Arabidopsis pollen calcium-binding protein 1, respectively). Immunolocalization studies demonstrated that BPC1 is found in the cytosol of mature pollen. However, upon pollen hydration and germination, there is some apparent leakage of the protein to the pollen wall. BPC1 is also concentrated on or near the surface of the elongating pollen tube. The essential nature of calcium in pollen physiology, combined with the properties of BPC1 and its high evolutionary conservation suggests that this protein plays an important role in pollination by functioning as a calcium-sensitive signal molecule.     

Glyphosate-drift but not herbivory alters the rate of transgene flow from single and stacked trait transgenic canola (Brassica napus) to nontransgenic B. napus and B. rapa

? Transgenic plants can offer agricultural benefits, but the escape of transgenes is an environmental concern. In this study we tested the hypothesis that glyphosate drift and herbivory selective pressures can change the rate of transgene flow between the crop Brassica napus (canola), and weedy species and contribute to the potential for increased transgene escape risk and persistence outside of cultivation. ? We constructed plant communities containing single transgenic B. napus genotypes expressing glyphosate herbicide resistance (CP4 EPSPS), lepidopteran insect resistance (Cry1Ac), or both traits ('stacked'), plus nontransgenic B. napus, Brassica rapa and Brassica nigra. Two different selective pressures, a sublethal glyphosate dose and lepidopteran herbivores (Plutella xylostella), were applied and rates of transgene flow and transgenic seed production were measured. ? Selective treatments differed in the degree in which they affected gene flow and production of transgenic hybrid seed. Most notably, glyphosate-drift increased the incidence of transgenic seeds on nontransgenic B. napus by altering flowering phenology and reproductive function. ? The findings of this study indicate that transgenic traits may be transmitted to wild populations and may increase in frequency in weedy populations through the direct and indirect effects of selection pressures on gene flow.     

甘蓝型油菜PEP基因片段的克隆和种子特异性反义表达载体的构建及转基因油菜的获得

丙酮酸羧化酶(PEP)是控制油菜蛋白质/油脂含量比例的一个种子的含油量.本研究利用PCR法从甘蓝型油菜花油5号(H045)克隆了PEP基因片段,并与载体pBI121-B构建了反义PEP基因的种子特异性植物表达载体,通过激光微束穿刺法将其转化到甘蓝型油菜中,目前已获得了转基因植株.     

雄性不育相关基因msLTP的反义RNA验证

根据普通白菜雄性不育相关的脂质转移蛋白基因(msLTP)的cDNA序列设计引物,从普通白菜花蕾的cDNA中扩增出312bp的片段,然后将该片段连接至双元载体pBI12l中,得到反义RNA植物表达载体并导入农杆菌LBA4404菌株中,通过农杆菌介导法转化菜心;利用PCR和Southern blot分析检测得到了25株转基因植株,转基因植株的花粉部分畸形或空瘪,花粉离体萌发率为38.56%,较未转化植株的萌发率(76.32%)降低了37.76个百分点.研究表明,反义RNA技术使msLTP基因沉默而导致了菜心转基因植株的部分花粉发育不良,说明msLTP基因在普通白菜和菜心等花粉发育中具有重要作用.     

BnC15 and BnATA20, the different putative components, control anther development in Brassica napus L

In Brassica napus, male fertility depends on proper cell differentiation in the anther. However, relatively little is known about the genes regulating anther cell differentiation and function. Here, we report two floral organ specific genes, BnC15 and BnATA20, derived from a B. napus two-line Rs1046A/B floral subtractive library. Although BnC15 and BnATA20 genes have a different expression pattern in anthers demonstrated by in situ hybridization and real-time PCR analysis, silencing of both genes in B. napus by antisense suppression resulted in pollen abortion after microspore release. Light and electron microscopy observation revealed the lack of plastoglobuli, lipid bodies and sporopollenin secreted from the tapetum leading to aberrations in exine sculpturing and the formation of a pollen coat. In addition, the microspores were squeezed to the irregular shape in the locule in the end. As shown by gene expression analysis in transgenic plants and the comparison of anther development between bnc15 or bnata20 mutants and Rs1046A, BnC15 and BnATA20 were positively regulated downstream of Rf gene controlling the fertility of Rs1046B in the same pathway. The results support the hypothesis that BnC15 and BnATA20 are crucial components of a genetic network that controls tapetum development and exine sculpturing.     

以基因枪介导获转ps1—barnase基因的工程雄性不育水稻植株

以ps1-barnase(brn)为目的基因,pHcintG(PG)为选择/标记基因进行共转化,以PDS-1000-氦气基因枪介导,将brn及PG基因转化到水稻台北309及秋光的核DNA中,得到了转ps1-barnase基因的工程雄性不育植株。以悬浮细胞作为基因枪轰击的靶材料,转化植株再生频率较初级愈伤组织的为高。转brn基因植株的其他主要性状与供体亲本无显著差异,但却表现不育。其不育的程度在不同的植株之间表现不同。在转brn基因植株中观察到全不育(占全部brn阳性植株的40.6％)、高不育(占15.6％)及半不育的个体(占43.7％)。全不育的转基因植株自交完全不能结实(结实率为零),除个别植株外,花粉完全不被I-KI染色;而人工授以正常的花粉则可以获得杂交种子。而brn基因的阴性植株及未进行转化的对照植株则完全可育,表明转基因植株之雄性不育乃brn基因所致。结果表明,brn基因在水稻中是完全可以正常表达的,其表达的时期推测在花粉母细胞减数分裂前至花粉形成之间的整个时期。     

Silencing gene expression of the ethylene-forming enzyme results in a reversible inhibition of ovule development in transgenic tobacco plants

To study the role of ethylene in plant reproduction, we constructed transgenic tobacco plants in which the expression of a pistil-specific gene coding for the ethylene-forming enzyme 1-aminocyclopropane-1-carboxylate oxidase was inhibited. Flowers from transgenic plants showed female sterility due to an arrest in ovule development. Megasporogenesis did not occur, and ovules did not reach maturity. When pollinated, pollen tubes were able to reach the ovary but did not penetrate into the immature ovule in transgenic plants. Flower treatment with an ethylene source resulted in a functional recovery of ovule development and restored guidance of the pollen tube tip into the ovule micropyle that resulted in seed set. The recovery was abolished if inhibitors of ethylene action were present. These results demonstrate that the plant hormone ethylene is required during the very early stages of female sporogenesis and ultimately to enable fertilization.     

Antisense-mediated suppression of transgene expression targeted specifically to pollen

A potential problem in the field release of transgenic plants is the spread of foreign gene products via pollen. Therefore, the use of the tomato pollen-specific lat52 gene promoter was investigated as a means of targeting antisense RNA to pollen without affecting transgene expression elsewhere in the plant. A transgenic tobacco line T115, which showed GUS expression in pollen, leaves and roots were retransformed with a construct containing the pollen-specific lat52 promoter driving the GUS encoding uid A gene in antisense orientation. From 24 independent transformants obtained, 19 showed a significant reduction in pollen GUS activity. Of these lines, four showed a reproducible antisense effect in pollen in the next generation, while it was shown in one line that GUS activity in leaves and roots was also unaffected. To ascertain the effectiveness of the antisense strategy to downregulate very high levels of pollen expression, a lat52-gus antisense construct was introduced into tobacco lines containing lat52-gus, which had pollen GUS activity of up to 250 times greater than in line T115. Results showed that 30 out of 34 independent lines exhibited a significant antisense effect in pollen, confirming the effectiveness of pollen-targeted antisense strategy to reduce undesirable expression in pollen independent of expression level in pollen.     

The Arabidopsis copper transporter COPT1 functions in root elongation and pollen development

Copper plays a dual role in aerobic organisms, as both an essential and a potentially toxic element. To ensure copper availability while avoiding its toxic effects, organisms have developed complex homeostatic networks to control copper uptake, distribution, and utilization. In eukaryotes, including yeasts and mammals, high affinity copper uptake is mediated by the Ctr family of copper transporters. This work is the first report on the physiological function of copper transport in Arabidopsis thaliana. We have studied the expression pattern of COPT1 in transgenic plants expressing a reporter gene under the control of the COPT1 promoter. The reporter gene is highly expressed in embryos, trichomes, stomata, pollen, and root tips. The involvement of COPT1 in copper acquisition was investigated in CaMV35S::COPT1 antisense transgenic plants. Consistent with a decrease in COPT1 expression and the associated copper deprivation, these plants exhibit increased mRNA levels of genes that are down-regulated by copper, decreased rates of (64)Cu uptake by seedlings and reduced steady state levels of copper as measured by atomic absorption spectroscopy in mature leaves. Interestingly, COPT1 antisense plants also display dramatically increased root length, which is completely and specifically reversed by copper addition, and an increased sensitivity to growth inhibition by the copper-specific chelator bathocuproine disulfonic acid. Furthermore, COPT1 antisense plants exhibit pollen development defects that are specifically reversed by copper. Taken together, these studies reveal striking plant growth and development roles for copper acquisition by high affinity copper transporters.     

Gene Flow,Risk Assessment and the Environmental Release of Transgenic Plants

The release of genetically modified plants is governed by regulations that aim to provide an assessment of potential impact on the environment. One of the most important components of this risk assessment is an evaluation of the probability of gene flow. In this review, we provide an overview of the current literature on gene flow from transgenic plants, providing a framework of issues for those considering the release of a transgenic plant into the environment. For some plants gene flow from transgenic crops is well documented, and this information is discussed in detail in this review. Mechanisms of gene flow vary from plant species to plant species and range from the possibility of asexual propagation, short- or long-distance pollen dispersal mediated by insects or wind and seed dispersal. Volunteer populations of transgenic plants may occur where seed is inadvertently spread during harvest or commercial distribution. If there are wild populations related to the transgenic crop then hybridization and eventually introgression in the wild may occur, as it has for herbicide resistant transgenic oilseed rape (Brassica napus). Tools to measure the amount of gene flow, experimental data measuring the distance of pollen dispersal, and experiments measuring hybridization and seed survivability are discussed in this review. The various methods that have been proposed to prevent gene flow from genetically modified plants are also described. The current “transgenic traits” in the major crops confer resistance to herbicides and certain insects. Such traits could confer a selective advantage (an increase in fitness) in wild plant populations in some circumstances, were gene flow to occur. However, there is ample evidence that gene flow from crops to related wild species occurred before the development of transgenic crops and this should be taken into account in the risk assessment process.