外源基因在转基因动物中遗传和表达的稳定性

转基因技术经过近半个世纪的发展,已成为当今生物技术研究的热点。近10多年来,与核移植技术的结合,转基因效率大大提高,携带有不同外源基因的不同种类的转基因动物迅速增加。但是,成功获得转基因动物并不是转基因动物研究的最终目的,如何利用转基因技术为人类的需求服务才是科研人员始终面对的课题。在畜牧生产领域,通过转基因技术培育家畜新品种是转基因技术应用的重要体现,在我国这方面已经引起了广泛关注。但迄今为止,外源基因在转基因动物中遗传和表达的稳定性仍然是亟待解决的问题,究其原因,这主要与位置效应、外源基因的表观遗传学修饰和遗传效率相关,文章结合目前的研究进展和本实验室的研究结果,从这3方面阐述其作用机制,期望为转基因动物遗传育种向产业化的迈进提供一定的理论探讨。     

白三叶转基因及其生态适应性研究进展

牧草基因工程是近年来国内国际研究的热点之一 ,白三叶作为温带地区优良的豆科牧草 ,是人工草地建植的首选草种 ,也是各类观赏性草坪和绿地的主要组分。但白三叶的生态幅较窄 ,适宜在温暖 (适宜生长温度为 19～ 2 4℃ )湿润、年降雨量 70 0～10 0 0 mm的地区生长 ,对土壤酸碱度的要求比较严 ,适宜的土壤 p H为 6～ 7,耐酸性和耐盐碱能力都比较差 ,从而限制了其在许多地区的应用。利用转基因手段提高其抗逆性和生态适应性 ,培育抗性强、适应性广的白三叶新品种 ,对我国尤其是北方畜牧业、草坪业和生态建设都具有重要意义。在提高其对生物胁迫和非生物胁迫的抗性和生态适应性方面 ,白三叶的转基因研究已经取得了较大进展。结合国内外白三叶基因工程及生态适应性研究动态 ,系统介绍了白三叶转基因研究的主要内容和方法 ,在对已有成果进行综合分析的基础上 ,就目前白三叶转基因和生态适应性研究中亟待解决的一些问题进行了探讨。     

Genetic approaches for studying transgene inheritance and genetic recombination in three successive generations of transformed tobacco

Transgene integration into plant genomes is a complex process accompanied by molecular rearrangements. Classic methods that are normally used to study transgenic population genetics are generally inadequate for assessing such integration. Two major characteristics of transgenic populations are that a transgenic genome may harbor many copies of the transgene and that molecular rearrangements can create an unstable transgenic locus. In this work, we examined the segregation of T1, T2 and T3 transgenic tobacco progenies. Since transfer DNA (T-DNA) contains the NptII selectable marker gene that confers resistance to kanamycin, we used this characteristic in developing a method to estimate the number of functional inserts integrated into the genome. This approach was based on calculation of the theoretical segregation ratios in successive generations. Mendelian ratios of 3:1, 15:1 and 63:1 were confirmed for five transformation events whereas six transformation events yielded non-segregating progenies, a finding that raised questions about causal factors. A second approach based on a maximum likelihood method was performed to estimate recombination frequencies between linked inserts. Recombination estimates varied among transformation events and over generations. Some transgenic loci were unstable and evolved continuously to segregate independently in the T3 generation. Recombination and amplification of the transgene and filler DNA yielded additional transformed genotypes.     

Towards an optimal sampling strategy for assessing genetic variation within and among white clover (Trifolium repens L.) cultivars using AFLP

Cost reduction in plant breeding and conservation programs depends largely on correctly defining the minimal sample size required for the trustworthy assessment of intra- and inter-cultivar genetic variation. White clover, an important pasture legume, was chosen for studying this aspect. In clonal plants, such as the aforementioned, an appropriate sampling scheme eliminates the redundant analysis of identical genotypes. The aim was to define an optimal sampling strategy, i.e., the minimum sample size and appropriate sampling scheme for white clover cultivars, by using AFLP data (283 loci) from three popular types. A grid-based sampling scheme, with an interplant distance of at least 40 cm, was sufficient to avoid any excess in replicates. Simulations revealed that the number of samples substantially influenced genetic diversity parameters. When using less than 15 per cultivar, the expected heterozygosity (He) and Shannon diversity index (I) were greatly underestimated, whereas with 20, more than 95% of total intra-cultivar genetic variation was covered. Based on AMOVA, a 20-cultivar sample was apparently sufficient to accurately quantify individual genetic structuring. The recommended sampling strategy facilitates the efficient characterization of diversity in white clover, for both conservation and exploitation.     

Integration,expression and inheritance of transgenes in hexaploid oat (Avena sativa L.)

Two oat varieties, Melys (spring variety) and Bulwark (winter variety) were transformed by particle bombardment of primary embryogenic callus using either a ubi-bar-ubi-gus co-integration vector or co-transformed (Melys) with a ubi-bar plasmid together with one of three plasmids containing the beta-glucuronidase (gus) gene under the control of either a rice actin promoter, a CaMV35S promoter or a wheat high molecular weight glutenin promoter. Morphologically normal and fertile transgenic plants were regenerated following callus selection with glufosinate ammonium. Evidence for the integration and functioning of the selectable (bar) and reporter (gus) genes in T0 and T1 plants was confirmed by PCR, Southern hybridisation, fluorescence in situ hybridisation (FISH), histochemical assays, and by progeny analysis. Transformation rates varied from 0.2 to 5.0 lines/plate of callus bombarded, with co-transformation frequencies of 83 to 100%, and co-expression frequencies of 60 to 100%. Copy numbers for the bar and gus gene varied from 3 to 17 and from 2 to 20 respectively. Cell and tissue specific expression of the gus gene was evident from the different promoters, with the HMW glutenin promoter showing endosperm specific expression in T1 seed. No expression of the gus gene under the CaMV35S promoter was detected in any tissues. Progeny analysis provided evidence of Mendelian inheritance of the introduced genes suggesting either one or two unlinked integration sites. This was confirmed by fluorescence in situ hybridisation to chromosome spread preparations. No segregation of the gus gene from the bar gene was observed in any of the progeny derived from co-transformation.     

Liver-specific and seasonal expression of transgenic Atlantic salmon harboring the winter flounder antifreeze protein gene

We have analyzed the inheritance and expression of a line of transgenic salmon harboring the antifreeze protein gene from the winter flounder. The genomic clone 2A-7 coding for a major liver-type antifreeze protein gene (wflAFP-6) was integrated into the salmon genome. From a transgenic founder (# 1469), an F3 generation was produced. In this study, southern blot analysis showed that only one copy of the antifreeze protein transgene was integrated into a unique site in F3 transgenic fish. The integration site was cloned and characterized. Northern analysis indicated that the antifreeze protein mRNA was only expressed in the liver and showed seasonal variation. All of the F3 offspring contained similar levels of the antifreeze protein precursor protein in the sera and the sera of these offspring showed a characteristic hexagonal ice crystal pattern indicating the presence of antifreeze activity. In addition, the antifreeze protein precursor protein level was found to vary with the season, being highest in the month of November and lowest in May. This study had demonstrated a tissue-specific and stable expression of the antifreeze protein transgene in the F3 generation of the transgenic salmon 1469 line.     

亚热带丘陵茶园间作白三叶草的生态效应

在亚热带丘陵区1a生幼龄茶园通过连续4a的大田对比试验,研究了茶园间作白三叶草的生态效应,结果发现：茶园间作白三叶草降低了土壤容重,提高了土壤孔隙度、有机质、全N、水解N的含量、K的活性、蚯蚓的数量和生物量,消耗了部分有效P;增加了土壤关键层次（0—20cm）和关键时期（4～6月）的水分含量,延缓和缩短了夏季高温干旱与秋季持续干旱时间;获得了土壤降温时增温、保温与升温时降温的双向动态调控效果,增加了同一层次土壤温度的稳定性,延缓了高温和低温的出现时间,缩短了过度高温时间,减少了高温对茶树生长产生的影响;抑制了杂草生长,增加了天敌的种类和种群数量,减少了虫害发生率。从而形成了立地环境．茶树．伴生生物群落系统自我调节的动态平衡,促进了茶树生长,改善了茶叶品质,显著增加了茶叶产量。与清耕茶园相比,茶园间作白三叶草后,春秋茶的酚氨比分别下降了17．10％和30．90％,产量提高了32．65％。     

Transformation of oat and inheritance of bar gene expression

Fertile transgenic plants of oat (Avena sativa L. var. Melys) were produced following microprojectile bombardment of primary embryogenic calli from immature embryos with two plasmids containing the bar gene or the β-glucuronidase (uidA) gene, after selection with glufosinate ammonium. Eleven plants were regenerated from phosphinothricin resistant callus, with three of the eleven plants containing either intact or rearranged copies. No plants co-transformed with the non-selected uidA gene were detected. Stable transmission and expression of the bar gene in the T₁ inbred progenies occurred in a Mendelian manner in one line, which contained an intact bar gene, and in all six T₂ lines tested from this transformant. This revised version was published online in June 2006 with corrections to the Cover Date.     

Inheritance of phosphorus response in white clover (Trifolium repens L.)

Genotypes of white clover that exhibited divergent responses to P were identified in a glasshouse pot trial. Six high P-responding genotypes were selected from previously identified high P-responding cultivars and 5 low P-responding genotypes were selected from previously identified low P-responding cultivars. These were crossed in a full diallel design without selfing and reciprocals were kept separate. The P-response of progeny lines was compared with parents. High P-response was dominant over low P-response with progeny from crosses between high and low P-response genotypes being similar to the high P-response parent. Reciprocal effects were not significant. The general combining abilities of high P-response genotypes were generally greater than that of the low P-response genotypes, although there were significant specific combining abilities. Narrow sense heritabilities for P response were moderate, 0.46 based on the linear coefficient and 0.33 based on the quadratic coefficient of the fitted response curves.The mode of inheritance, feasibility of manipulating differences in P response by breeding and future directions of this work are discussed.Deceased.Deceased.     

No enhancing effects of plasmid-specific histone acetyltransferase recruitment system on transgene expression in vivo

Abstract

Altered levels of histone acetylation are associated with changes in chromosomal gene expression. Thus, the specific acetylation of histones bound to plasmid DNA might increase transgene expression. Previously, the expression of the histone acetyltransferase domain of CREB-binding protein fused to the sequence-dependent DNA binding domain of GAL4 (GAL4-HAT) successfully improved reporter gene expression in cultured cells [J. Biosci. Bioengng. 123, 277–280 (2017)]. In this study, the same approach was applied for transgene expression in mice. The activator and reporter plasmid DNAs bearing the genes for GAL4-HAT and Gaussia princeps luciferase, respectively, were co-administered into the mouse liver by hydrodynamics-based tail vein injection, and the Gaussia luciferase activity in serum was measured for two weeks. Unexpectedly, the co-injection of the GAL4-HAT and luciferase plasmid DNAs seemed to decrease, rather than increase, luciferase expression. Moreover, the co-injection apparently reduced the amount of luciferase DNA in the liver. These results indicated that this system is ineffective in vivo and suggested the exclusion of hepatic cells expressing GAL4-HAT.     

Temporal and spatial control of transgene expression using a heat-inducible promoter in transgenic wheat

Constitutive promoters are widely used to functionally characterise plant genes in transgenic plants, but their lack of specificity and poor control over protein expression can be a major disadvantage. On the other hand, promoters that provide precise regulation of temporal or spatial transgene expression facilitate such studies by targeting over-expression or knockdown of target genes to specific tissues and/or at particular developmental stages. Here, we used the uidA (beta-glucuronidase, GUS) reporter gene to demonstrate that the barley Hvhsp17 gene promoter can be induced by heat treatment of 38-40 °C for 1-2 h in transgenic wheat. The GUS enzyme was expressed only in those tissues directly exposed to heat and not in neighbouring leaf tissues. The induction of HSP::GUS was demonstrated in all organs and tissues tested, but expression in older tissues was lower. Generally, proximal root sections showed less GUS activity than in root tips. This heat-inducible promoter provides the ability to investigate the function of candidate genes by overexpression or by down-regulation of target gene expression (for example by RNAi) in selected tissues or developmental stages of a transgenic plant, limited only by the ability to apply a heat shock to the selected tissues. It also allows the investigation of genes that would be lethal or reduce fertility if expressed constitutively.     

基质结合区（MARs）与转基因植物的基因表达

对动植物的研究结果表明,基质结合区（MARs）能提高转基因的表达水平,并能降低其在转基因个体之间的表达差异。本文着重综述了MARs的基本特征及其在转基因植物中的研究应用,对MARs在转基因动物方面的研究成果和MARs的作用机制也作了介绍。     

Extraction buffer effects on detection of transient gene expression in white spruce

β-Glucuronidase (GUS) and luciferase (LUC) reporter genes were introduced into white spruce (Picea glauca [Moench] Voss) cultured cells via particle bombardment. Transient expression of these genes was evaluated by extracting the enzymes using 3 buffers. Different buffers resulted in significantly different sensitivities of GUS and LUC detection. In the case of cobombardment, the buffer that gave high levels of expression of one reporter gene did not necessarily result in a better detection of the second reporter gene. This study indicates that appropriate buffers should be used for maximum detection of reporter genes.     

The effect of stem nematode on establishment and early yields of white clover

The effects of stem nematode (Ditylenchus dipsaci) infestation on the establishment of white clover sown in mixed swards with perennial ryegrass, were investigated in two field trials. Clover cultivars known to have different degrees of resistance were sown on land in which stem nematode was controlled either by aldicarb (Experiment 1) or crop rotation (Experiment 2). The establishment of white clover was greatly improved and subsequent clover yields were inversely related to stem nematode infestation. At the first harvest after sowing, clover yields were 3.6 and 3.3 times greater from aldicarb and rotation treatment plots than from check plots; over the first nine months, total clover yields were increased by 3.5 and 8.9 times by aldicarb and rotation treatments. In both trials, plots of resistant cultivars had less stem nematode infestation and yielded better than the very susceptible cultivar, S184. Stem nematode infestations eventually developed on all plots, after the establishment phase. This is the first report showing that resistant cultivars improve establishment of clover in mixed swards on stem nematode infested soils. In both experiments, plots became dominated by clover and even cv. S184 eventually produced good clover swards. Aldicarb and rotation treatments also controlled clover cyst and root lesion nematodes, and numbers of these were inversely related to first harvest yields. Other soil borne pests and diseases, although not prominent, have not been ruled out as possible confounding factors. There was no correlation between grass yield and aldicarb treatment.     

Multiple effects of genetic background on variegated transgene expression in mice

BLG/7 transgenic mice express an ovine beta-lactoglobulin transgene during lactation. Unusually, transgene expression levels in milk differ between siblings. This variable expression is due to variegated transgene expression in the mammary gland and is reminiscent of position-effect variegation. The BLG/7 line was created and maintained on a mixed CBA x C57BL/6 background. We have investigated the effect on transgene expression of backcrossing for 13 generations into these backgrounds. Variable transgene expression was observed in all populations examined, confirming that it is an inherent property of the transgene array at its site of integration. There were also strain-specific effects on transgene expression that appear to be independent of the inherent variegation. The transgene, compared to endogenous milk protein genes, is specifically susceptible to inbreeding depression. Outcrossing restored transgene expression levels to that of the parental population; thus suppression was not inherited. Finally, no generation-dependent decrease in mean expression levels was observed in the parental population. Thus, although the BLG/7 transgene is expressed in a variegated manner, there was no generation-associated accumulated silencing of transgene expression.     

肠杆菌科细菌耐药基因表达的遗传和环境调控

细菌耐药性是21世纪国际关注的重要问题,也是全球面临的重大挑战.肠杆菌科细菌是医院感染的重要病原菌之一.近年来,随着抗生素的大量使用,多种肠杆菌科耐药菌,尤其是多重耐药肠杆菌开始大量出现,对人类健康形成了日益严重的威胁.细菌可以通过耐药基因突变或水平转移的方式获得耐药性,通常情况下,可以通过已知的耐药机制预测相应的耐药...     

Accumulation of a sulphur-rich seed albumin from sunflower in the leaves of transgenic subterranean clover (Trifolium subterraneum L.)

A gene encoding a sulphur-rich, sunflower seed albumin (23% cysteine plus methionine) was modified to contain the promoter for the 35S RNA of cauliflower mosaic virus, in order to obtain leaf expression in transgenic plants. In addition, a sequence encoding an endoplasmic reticulum-retention signal was added to the 3 end of the coding region so as to stabilize the protein by diverting it away from the vacuole. The modified gene was introduced into subterranean clover (T. subterraneum L.) and its expression was detected by northern and western blots and by immunogold localization. The albumin was accumulated in the lumen of the endoplasmic reticulum, and, among six independent, transformed lines, it accumulated in the leaves of T₀ transgenic plants at varying levels up to 0.3% of the total extractable protein. The level of accumulation of the sunflower albumin increased with increasing leaf age, and in the older leaves of the most highly expressing plants of the T₁ generation it reached 1.3% of total extractable protein. Expression of the SSA gene was stable in the first and second generation progeny. These results indicate that there is potential for significantly improving the nutritional value of subterranean clover for ruminant animals such as sheep by expressing genes that code for sulphur-rich, rumen-stable proteins in leaves.     

Genetic diversity in perennial ryegrass and white clover among old Dutch grasslands as compared to cultivars and nature reserves

To support conservation policies for old Dutch grasslands that are still in agricultural use, morphological variation and AFLP-based (amplified fragment length polymorphism-based) genetic diversity was studied in perennial ryegrass and white clover populations and compared with the diversity in reference varieties. In addition, AFLP variation was also studied in grasslands located in nature reserves. From principal component analysis (PCA), it appeared that date of ear emergence in perennial ryegrass and characters related to plant vigour in white clover were the main morphological characters separating the reference varieties from the old Dutch grassland populations, and some of the grassland populations from each other. In both species, intrapopulation variation was lower for the reference varieties. Lower heterogeneity within the reference varieties was also found in the AFLP analysis. All common AFLP's observed in old Dutch grasslands could also be found in the reference varieties and nature reserves. Only a small number of low-frequency alleles found in old Dutch grasslands were absent from the other two groups. However, band frequencies of markers could vary considerably between populations, which may have been caused by selection. Analysis of the AFLP data by PCA distinguished the majority of reference varieties from the old Dutch grasslands, and showed genetic differentiation only between some grasslands. Comparison of old Dutch grasslands with grasslands in nature reserves indicated that basically the same range of genetic variation is covered by the two groups. Our study indicates that the Netherlands harbour a more or less continuous population for major parts of the diversity of perennial ryegrass and white clover. It was concluded that no specific conservation measures are presently needed to maintain genetic diversity of perennial ryegrass and white clover occurring in old Dutch grasslands.     

Two human MARs effectively increase transgene expression in transfected CHO cells

Matrix attachment regions (MARs) can enhance the expression level of transgene in Chinese hamster ovaries (CHO) cell expression system. However, improvements in function and analyses of the mechanism remains unclear. In this study, we screened two new and more functional MAR elements from the human genome DNA. The human MAR‐3 and MAR‐7 element were cloned and inserted downstream of the polyA site in a eukaryotic vector. The constructs were transfected into CHO cells, and screened under G418 to produce the stably transfected cell pools. The expression levels and stability of enhanced green fluorescent protein (eGFP) were detected by flow cytometry. The transgene copy number and transgene expression at mRNA level were detected by quantitative real‐time PCR. The results showed that the expression level of eGFP of cells transfected with MAR‐containing vectors were all higher than those of the vectors without MARs under transient and stably transfection. The enhancing effect of MAR‐7 was higher than that of MAR‐3. Additionally, we found that MAR significantly increased eGFP copy numbers and eGFP gene mRNA expression level as compared with the vector without. In conclusion, MAR‐3 and MAR‐7 gene can promote the expression of transgene in transfected CHO cells, and its effect may be related to the increase of the number of copies.