植物转基因座位形成的分子机制

转基因座位是指染色体上插入的转基因及相邻的特定DNA序列。大多数转基因座位是以转基因片段、基因组片段和填充DNA相间而存在,仅少数含有完整的单拷贝转基因,这是由于在转基因整合过程中,转基因及基因组DNA发生缺失、重复和染色体的重排。转基因整合主要通过双链DNA断裂修复中的异常重组所产生,而同源重组也发挥了一定的作用。异常重组主要由单链复性、合成依赖链复性和依赖Ku蛋白的非同源末端连接途径调节。     

大麦转基因座位结构的研究

利用质粒营救法获得基因枪法转化的4种转绿色荧光蛋白基因(green fluorescent protein,GFP)大麦的转基因座位序列,序列分析显示4种材料的转基因座位中均有完整栽体的串联重复现象,表明转基因整合是同源重组的结果.同时转基因座位中也存在不完整载体片段、基因组片段的混杂排列,说明转基因整合时也发生异常重组.微粒轰击的转基因整合是由异常重组和同源重组共同完成的.     

植物中转基因的整合机制

转基因在植物基因组中的整合分两步:在整合前阶段,转化的完整质粒及转基因片段经重组连接形成转基因串联子(transgenearray),其中不含任何植物基因组序列,连接反应由植物细胞本身的酶催化,仅依赖于游离的DNA末端,CaMV35S启动子和TDNA边界序列中的特定序列可能充当重组热点;在整合阶段,转基因DNA通过微同源介导的异常重组整合到植物基因组中,最初的整合位点作为整合热点,引导随后的转基因分子在该位点附近整合,不同转基因位点可被1～10kb植物DNA隔开,形成转基因簇(transgenecluster)。     

应用接头PCR克隆慢病毒介导的转基因小鼠整合位点旁侧序列

目的为鉴定慢病毒介导的转基因小鼠中外源基因的整合位点信息,应用接头PCR克隆整合位点旁侧序列。方法小鼠基因组总DNA酶解后与设计的接头片段连接,根据慢病毒的LTR序列设计巢式PCR引物,克隆转基因小鼠整合位点旁侧序列。结果成功克隆到转基因小鼠整合位点的旁侧序列,经过测序定位于小鼠染色体上。结论作为反向PCR的改进,本方法可用于转基因小鼠整合位点旁侧序列的克隆,为分析整合位点与外源基因表达之间的关系等提供了科学依据。     

精准高效外源DNA整合技术研究进展

精准高效整合技术是将外源DNA片段插入到目的细胞基因组特定位置的一项转基因技术。早期通过细胞基因组与外源DNA同源序列发生重组来完成靶向整合的目的。伴随基因编辑技术发展,特别是CRISPR/Cas9技术的出现,精准高效的外源DNA整合技术日益成熟,广泛应用到功能基因组学、转基因动物及遗传疾病治疗的研究中。围绕基因编辑研究进展、引导RNA修饰技术、单碱基整合技术、转座子技术和外源DNA整合效率等方面对精准靶向和高效整合技术进行综述。     

重组人apoE基因表达活性和转基因小鼠研究

人apoE基因组DNA,去除其自身启动子,代之以小鼠金属硫蛋白启动子,重组质粒经脂质体介导转入小鼠NIH／3T3细胞后,以人apoE基因组DNA／EcoRⅠ片段为探针检测mRNA表达,可见apoEmRNA杂交信号很强,经重金属诱导后杂交信号更强,表明MT启动子功能良好,pME表达正常．将人apoE基因组DNA用显微注射法导入小鼠受精卵雄性原核,再将胚胎移植入假孕母鼠输卵管内,仔鼠分娩四周后,自鼠尾提取DNA,鉴定人apoEDNA在小鼠染色体上的整合,最终得到有人apoE基因整合的转基因首建鼠．     

转Xa21基因水稻中T-DNA整合的遗传定位

利用转抗白叶枯病基因Xa21的水稻材料,通过TAIL－PCR方法扩增T－DNA整合的侧翼序列。从中筛选属于水稻基因组DNA的T－DNA整合的侧翼序列作为探针,将外源基因整合位点定位到窄叶青／京系17DH群体构建的水稻分子连锁图谱上。共获得属于水稻基因组DNA的T－DNA侧翼序列22个,其中的19个序列在定位群体的两个亲本之间显示RFLP多态性,分别定位在水稻基因组的第3,4,5,7,9,10,11和12染色体上。带有转基因Xa21的T－DNA整合的定位为研究外源基因在不同染色体位点的位置效应和稳定遗传打下基础。     

转基因水稻T—DNA侧翼序列的扩增与分析

利用现有的转抗白叶枯病基因Xa21的水稻材料,通过TAIL－PCR技术扩增出携带Xa21基因的T－DNA的侧翼序列,对24个有效扩增片段的序列分析结果表明,其中14个侧翼序列是水稻DNA,9个含载体主干序列,1个是外源基因Xa21片段,14个T－DNA侧翼的水稻DNA序列与直接转化法外源基因整合位点的基因组序列具有不同的特点,这些T－DNA在水稻染色体上整合后其两端序列的特点类似于在转基因双子叶植物中观察到的现象,在含主干序列的侧翼序列（37．5％,9／24）,中,载体主干序列是以不同的类型出现的。     

细菌人工染色体文库的构建及应用

细菌人工染色体（BAC）是第二代大片段DNA的克隆载体系统,具有容量大、嵌合率低、遗传特性稳定、转化效率高、插入片段易回收、操作简便等优点,因而被广泛应用于基因组较大的真核生物基因组研究中,并发挥着前所未有的重要作用。本文综述了BAC的发展,利用此载体构建基因组文库的程序和鉴定方法,及其在物理图谱构建、图位克隆、基因组测序、转基因技术等研究中的应用。     

YAC介导的天蚕丝素基因向家蚕的转移──天蚕丝素基因-YAC克隆在家蚕的表达

本文为天蚕Antheraeyamamai丝素基因在家蚕Bombyxmori成功表达的首次报道。我们构建了天蚕丝素基因的YAC克隆,然后把含有该克隆的DNA溶液导入家蚕受精卵。分子杂交实验证明天蚕丝素基因已整合到家蚕基因组中。通过丝心蛋白氨基酸组分分析以及茧丝的溶解性比较,发现有部分转基因家委表达了天蚕丝素基因。F2代的转基因家蚕蛾的染色体DNA中同时还存在YAC序列,说明YAC对丝素基因具有介导作用。天蚕丝素基因以单拷贝形式存在于转基因家蚕中。     

Complete sequence analysis of transgene loci from plants transformed via microprojectile bombardment

A substantial literature exists characterizing transgene locus structure from plants transformed via Agrobacterium and direct DNA delivery. However, there is little comprehensive sequence analysis of transgene loci available, especially from plants transformed by direct delivery methods. The goal of this study was to completely sequence transgene loci from two oat lines transformed via microprojectile bombardment that were shown to have simple transgene loci by Southern analysis. In line 3830, transformed with a single plasmid, one major and one of two minor loci were completely sequenced. Both loci exhibited rearranged delivered DNA and flanking genomic sequences. The minor locus contained only 296 bp of two non-contiguous fragments of the delivered DNA flanked by genomic (filler) DNA that did not originate from the integration target site. Predicted recognition sites for topoisomerase II and a MAR region were observed in the transgene integration target site for this non-functional minor locus. Line 11929, co-transformed with two different plasmids, had a single relatively simple transgene locus composed of truncated and rearranged sequences from both delivered DNAs. The transgene loci in both lines exhibited multiple transgene and genomic DNA rearrangements and regions of scrambling characteristic of complex transgene loci. The similar characteristics of recombined fragments and junctions in both transgenic oat lines implicate similar mechanisms of transgene integration and rearrangement regardless of the number of co-transformed plasmids and the level of transgene locus complexity.     

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.     

Transgenic trait deployment using designed nucleases

The demand for crops requiring increasingly complex combinations of transgenes poses unique challenges for transgenic trait deployment. Future value‐adding traits such as those associated with crop performance are expected to involve multiple transgenes. Random integration of transgenes not only results in unpredictable expression and potential unwanted side effects but stacking multiple, randomly integrated, independently segregating transgenes creates breeding challenges during introgression and product development. Designed nucleases enable the creation of targeted DNA double‐strand breaks at specified genomic locations whereby repair can result in targeted transgene integration leading to precise alterations in DNA sequences for plant genome editing, including the targeting of a transgene to a genomic locus that supports high‐level and stable transgene expression without interfering with resident gene function. In addition, targeted DNA integration via designed nucleases allows for the addition of transgenes into previously integrated transgenic loci to create stacked products. The currently reported frequencies of independently generated transgenic events obtained with site‐specific transgene integration without the aid of selection for targeting are very low. A modular, positive selection‐based gene targeting strategy has been developed involving cassette exchange of selectable marker genes which allows for targeted events to be preferentially selected, over multiple cycles of sequential transformation. This, combined with the demonstration of intragenomic recombination following crossing of transgenic events that contain stably integrated donor and target DNA constructs with nuclease‐expressing plants, points towards the future of trait stacking that is less dependent on high‐efficiency transformation.     

Complex transgene locus structures implicate multiple mechanisms for plant transgene rearrangement

To more fully characterize the internal structure of transgene loci and to gain further understanding of mechanisms of transgene locus formation, we sequenced more than 160 kb of complex transgene loci in two unrelated transgenic oat (Avena sativa L.) lines transformed using microprojectile bombardment. The transgene locus sequences from both lines exhibited extreme scrambling of non-contiguous transgene and genomic fragments recombined via illegitimate recombination. A perfect direct repeat of the delivered DNA, and inverted and imperfect direct repeats were detected in the same transgene locus indicating that homologous recombination and synthesis-dependent mechanism(s), respectively, were also involved in transgene locus rearrangement. The most unexpected result was the small size of the fragments of delivered and genomic DNA incorporated into the transgene loci via illegitimate recombination; 50 of the 82 delivered DNA fragments were shorter than 200 bp. Eleven transgene and genomic fragments were shorter than the DNA lengths required for Ku-mediated non-homologous end joining. Detection of these small fragments provided evidence that illegitimate recombination was most likely mediated by a synthesis-dependent strand-annealing mechanism that resulted in transgene scrambling. Taken together, these results indicate that transgene locus formation involves the concerted action of several DNA break-repair mechanisms.     

A large-scale study of rice plants transformed with different T-DNAs provides new insights into locus composition and T-DNA linkage configurations

Transgenic locus composition and T-DNA linkage configuration were assessed in a population of rice plants transformed using the dual-binary vector system pGreen (T-DNA containing the bar and gus genes)/pSoup (T-DNA containing the aphIV and gfp genes). Transgene structure, expression and inheritance were analysed in 62 independently transformed plant lines and in around 4,000 progeny plants. The plant lines exhibited a wide variety of transgenic locus number and composition. The most frequent form of integration was where both T-DNAs integrated at the same locus (56% of loci). When single-type T-DNA integration occurred (44% of loci), pGreen T-DNA was preferentially integrated. In around half of the plant lines (52%), the T-DNAs integrated at two independent loci or more. In these plants, both mixed and single-type T-DNA integration often occurred concurrently at different loci during the transformation process. Non-intact T-DNAs were present in 70–78% of the plant lines causing 14–21% of the loci to contain only the mid to right border part of a T-DNA. In 53–66% of the loci, T-DNA integrated with vector backbone sequences. Comparison of transgene presence and expression in progeny plants showed that segregation of the transgene phenotype was not a reliable indicator of either transgene inheritance or T-DNA linkage, as only 60–80% of the transgenic loci were detected by the expression study. Co-expression (28% of lines) and backbone transfer (53–66% of loci) were generally a greater limitation to the production of marker-free T₁ plants expressing the gene of interest than co-transformation (71% of lines) and unlinked integration (44% of loci).     

Label-free amperometric immunosensor for the detection of human serum chorionic gonadotropin based on nanoporous gold and graphene

Identifying a good transgenic event from the pool of putative transgenics is crucial for further characterization. In transgenic plants, the transgene can integrate in either single or multiple locations by disrupting the endogenes and/or in heterochromatin regions causing the positional effect. Apart from this, to protect the unauthorized use of transgenic plants, the signature of transgene integration for every commercial transgenic event needs to be characterized. Here we show an affinity-based genome walking method, named locus-finding (LF) PCR (polymerase chain reaction), to determine the transgene flanking sequences of rice plants transformed by Agrobacterium tumefaciens. LF PCR includes a primary PCR by a degenerated primer and transfer DNA (T-DNA)-specific primer, a nested PCR, and a method of enriching the desired amplicons by using a biotin-tagged primer that is complementary to the T-DNA. This enrichment technique separates the single strands of desired amplicons from the off-target amplicons, reducing the template complexity by several orders of magnitude. We analyzed eight transgenic rice plants and found the transgene integration loci in three different chromosomes. The characteristic illegitimate recombination of the Agrobacterium sp. was also observed from the sequenced integration loci. We believe that the LF PCR should be an indispensable technique in transgenic analysis.     

Gene stability in transgenic aspen (Populus). II. Molecular characterization of variable expression of transgene in wild and hybrid aspen

In many annual plant species, transgene inactivation occurs most often when multiple incomplete/complete copies of the transgene are present in a genome. The expression of single-copy transgene loci may also be negatively influenced by the flanking plant DNA and/or chromosomal location (position effect). To understand transgene silencing in a long-lived tree system, we analyzed several wild (Populus tremula L.) and hybrid (P. tremula L. x P. tremuloides Michx.) aspen lines transgenic to the rolC phenotypical marker system and grown under in vitro, greenhouse and field conditions. The morphological features of the 35S-rolC gene construct were used to screen lines with altered transgene expression, which was later confirmed by Northern experiments. Molecular analyses of hybrid aspen revealed that transgene inactivation was always a consequence of transgene repeats. In wild non-hybrid aspen, however, multiple-insertion-based altered or loss of rolC expression was observed only in three out of six lines showing transgene inactivation. Sequencing analysis revealed AT-rich patches at the transgene flanking genomic regions of some of the wild aspen transgenic lines. One wild aspen line showing variable rolC expression revealed characteristic integration of the transgene into genomic regions containing a high AT content (85% or more). In the remaining two wild aspen transgenic lines unstable for rolC expression, single-copy integration and non-AT-rich or repeat-free transgene flanking regions were found. A partial suppression of rolC was observed in some plants of one of the field-grown wild aspen transgenic lines. In the other wild aspen transgenic line an additional mutant phenotype along with transgene inactivation was found. This indicates that the host genome has some control over expression of a transgene, and the possible role of AT-rich regions in defense against foreign DNA.     

Transgene behaviour in populations of rice plants transformed using a new dual binary vector system: pGreen/pSoup

Transgene integration, expression level and stability have been studied, across two generations, in a population of rice plants transformed using a new dual binary vector system: pGreen/pSoup. pGreen is a small Ti binary vector unable to replicate in Agrobacterium without the presence of another binary plasmid, pSoup, in the same strain. We engineered both pGreen and pSoup to contain each a different T-DNA. Transformation experiments were conducted using a pGreen vector containing the bar and gusA expression units (no transgene in pSoup) or with a pSoup vector containing an aphIV and gfp expression units (no transgene in pGreen). High plant transformation frequencies (up to 40%) were obtained using herbicide resistance ( bar) or antibiotic resistance ( aphIV) genes. Around 80% of the independently transformed plants expressed unselected reporter genes ( gusA or gfp) present in the vectors. Backbone sequences transfer was frequent (45% of lines) and occurred often in multicopy lines. Around 15-20% of the rice plant lines contained a single T-DNA integration without backbone. Integration of additional transgene copies did not improve expression levels in either T(0) plants or T(1) progenies. Nearly all multicopy lines contained transgenes integrated at several loci in the plant genome, showing that T-DNAs from either pGreen or pSoup frequently integrated at unlinked loci. Precise determination of loci number required the analysis of transgene presence in progeny. Segregation of transgene phenotype was generally misleading and tended to underestimate the real number of transgenic loci. The contribution of this new dual-binary vector system to the development of high-throughput rice transformation systems and to the production of marker-free transgenic rice plants is discussed.     

Comparison of Biolistic and Agrobacterium -mediated Transformation Methods on Transgene Copy Number and Rearrangement Frequency in Rice

Transferring foreign DNA into plant cells by biolistic and Agrobacterium -mediated methods may result in random integration of different copy numbers of the transgene, and different proportions of intact vs. rearranged copies of the transgene. This may, in turn, affect transgene expression levels. To test the above hypothesis, we first introduced the same plasmid, pAc1PG-CAM, into rice (BX)Oryza sativa L.) calli separately by the biolistic method and by the Agrobacterium -mediated method. To show whether different plasmids may affect the results, we also introduced pTOK233 by the Agrobacterium -mediated method and pJPM44 by the biolistic method. Transgene expression of R0 plants was monitored by histochemical analysis of GUS activity. Transgene copy number was determined by Southern blot analysis after digesting genomic DNA with an enzyme that has a unique cutting site within the input plasmid. The total genomic DNA was also digested by a two-cut enzyme (the cuts are located at two sides of a given transgene expression cassette), followed by Southern blotting analysis, for determining the number of intact transgene expression cassettes. Our data showed that Agrobacterium -mediated transformation resulted in lower transgene copy number (average between 2.1 and 2.3) in transgenic rice plants, compared with those plants obtained by the biolistic method (average between 4.2 and 5.6). The frequency of DNA rearrangement in expression cassettes is lower in transgenic rice plants obtained by the Agrobacterium -mediated method than those obtained by the biolistic method. The average rearrangement frequency is 0.07 to 0.106 for the Agrobacterium -mediated method, and 0.57 to 0.66 for the biolistic method. Our results suggest that it is better to compare the number of intact expression cassettes instead of the total copy number of the transgene in demonstrating their influence on the level of transgene expression. This is the first report on the frequency of expression cassette rearrangement in transgenic plants transformed with the same plasmid by two different transformation methods.