GMOs： The new hotspot in ecological research

Gene flow is an important ecological and genetic process, and the fitness conferred by the insertion of a foreiga gene could be important in ecology after it becomes introgressed into the wild genetic background of organisms,which is the determinative factor for the fate of GMOs or foreign genes in natural ecosystem and for the competition with native species. Trophic interrelation fulfilled by food chains is the most important correlation between species in ecosystem and could be affected by the modified genotypes after the introduction of GMOs. The technology enabling the precise manipulation of genotype is a great opportunity for the stndy of ecological genetics in an unknown way prior to the biotechnology era. Especially interesting is the impact of genes on ecosystem function when GMOs are released into environment. A variety of ecological terminology has been frequently used in assessment of ecological risks related to the release of GMOs in environment, e.g. gene flow, fitness,interspecific competition and food chain. The value of GMOs in ecological research is illustrated through several examples in biasafety assessment related to those terms here. It is proposed that ecolegists should pay attention to the study of gene function in ecosystem and take the opportunity to enrich ecological theory with the release of GMOs.     

Genome editing and animal models

Transgenic technology allows a gene of interest to be introduced into the genome of a laboratory animal, and provides an extremely powerful tool to dissect the molecular mechanisms of disease. Transgenic mouse models made by microinjection of DNA into zygotic pro- nuclei in particular have been widely used by the genetics community for 30 years. However, it remains a rather crude approach： injected sequences randomly insert in multiple copies as concatamers, they can be mutagenic, and they have variable or silenced expression depending on the site of integration, a phenomenon called position effects. As a result, multiple lines are required in order to confirm appropriate transgene expression. This can be partially overcome by flanking transgenes with insulator sequences to protect the transgene from the influence of the sur- rounding regulatory elements. Large （〈300 kb） BAC- based transgenic vectors have also been shown to be more resistant to position effects. However, animals carrying extra copies of fairly large regions of the genome could have unpredictable phenotypes. The most effective method used to control for position effects is to target transgene insertion to specific genomic loci, the so-called targeted transgenesis; for instance, the fast, site-specific transgenic technology TargattTM. The purpose of this review is to provide an overview on the current existing methods for making targeted transgenic mouse models.     

Pollen-mediated gene flow in Chinese commercial fields of glufosinate-resistant canola （Brassica napus）

One of the most pressing issues surrounding transgenic oilseed rape cultivation is the potential impact of GM genes entering neighboring non-GM oilseed rape plants, related weeds or closely related wild relatives. Following the principle of a case-by-case for transgenic crop safety assessment, we designed experiments to study pollen-mediated gene flow from herbicide-resistant transgenic oilseed rape to Chinese commercial varieties. The pollen dispersal was detected as the progeny of recipient plants that were identified as glufosinate resistant. The results indicated that pollen dispersal occurred up to 2000 m and its rate sharply decreased as the distance from the pollen source increased. However, the rate of long-distance pollen dispersal from 33 to 2000 m was less than 0.015% and did not present a gradual decrease, indicating the randomization of dispersal and pollination. Most pollen dispersed within a 4.5 m area around the transgenic pollen source plot, with a maximum of 1.19% at the sampling site of 1.4 m. Wind direction significantly affected the direction and the distance of pollen dispersal, resulting in the more and farther dispersal in southwest direction. However, the number of potentially pollinating bees was not correlated with direction and distance from the pollen source plot, suggesting that bee density and distribution would not contribute to the differences in pollen dispersal among different directions. This paper is the first report on research in a large-scale experiment of pollen-mediated gene flow under the China＇s environmental conditions and provided scientific evidence for the effective commercialization of transgenic oilseed rape and its safe management. Our results also provided important data on pollen spatial distribution pattern.     

Pollen-mediated gene flow in Chinese commercial fields of glufosinate-resistant canola (Brassica napus)

One of the most pressing issues surrounding transgenic oilseed rape cultivation is the potential impact of GM genes entering neighboring non-GM oilseed rape plants, related weeds or closely related wild relatives. Following the principle of a case-by-case for transgenic crop safety assessment, we designed experiments to study pollen-mediated gene flow from herbicide-resistant transgenic oilseed rape to Chinese commercial varieties. The pollen dispersal was detected as the progeny of recipient plants that were identified as glufosinate resistant. The results indicated that pollen dispersal occurred up to 2000 m and its rate sharply decreased as the distance from the pollen source increased. However, the rate of long-distance pollen dispersal from 33 to 2000 m was less than 0.015% and did not present a gradual decrease, indicating the randomization of dispersal and pollination. Most pollen dispersed within a 4.5 m area around the transgenic pollen source plot, with a maximum of 1.19% at the sampling site of 1.4 m. Wind direction significantly affected the direction and the distance of pollen dispersal, resulting in the more and farther dispersal in southwest direction. However, the number of potentially pollinating bees was not correlated with direction and distance from the pollen source plot, suggesting that bee density and distribution would not contribute to the differences in pollen dispersal among different directions. This paper is the first report on research in a large-scale experiment of pollen-mediated gene flow un-der the China’s environmental conditions and provided scientific evidence for the effective commer-cialization of transgenic oilseed rape and its safe management. Our results also provided important data on pollen spatial distribution pattern.     

Over-expression of an Arabi-dopsisd δ-OAT gene enhances salt and drought tolerance in transgenic rice

δ-OAT, ornithine-δ-aminotransferase, is the key enzyme involved in proline biosynthesis. In this study the Arabidopsis δ-OAT gene was transferred into rice (Oryza sativa L. ssp japonica cv. Zhongzuo 321), whose successful integration was demonstrated by PCR and Southern blot analysis. The over-expression of the gene in transgenic rice was also confirmed. Biochemical analysis showed that, under salt or drought stress conditions, proline contents in the leaves and roots in transgenic rice plants were 5- to 15-fold of those in non-transgenic controls. Under stress conditions, germinating rate of transgenic lines is higher than that of controls. Although the growth of rice plants tested were more and more retarded with the increasing of NaCI concentration, the transgenic plants grow faster compared to the controls under the same stress condition. Meanwhile, the resistance to KCl and MgSO4 stresses was also found enhanced in transgenie rice. Furthermore, the over-expression of δ-OAT also improved the yield of transgenic plants under stress conditions. The average yield per plant of transgenic lines increases about 12%--41% more than that of control line sunder 0.1 mol/L NaCI stress. These data indicated that the over-expression of δ-OAT, with the accumulation of proline, resulted in the enhancement of salt and drought tolerance and an increase of rice yield, which is of significance in agriculture.     

Identification and mapping of quantitative trait loci controlling cold-tolerance of Chinese common with rice （O.rufipogon Griff.） at booting to flowering stages

An advanced backcross population of rice was used to identify the quantitative trait locus(QTL) controlling the cold-tolerance at booting to flowering stages.The recipient,Guichao 2(GC2),was a commercial Indica rice;the donor Dongxiang common wild rice,was an accession of common wild rice(DXCWR,Oryza rufipogon Griff.).Three QTLs for cold-tolerance were detected on chromosomes 1,6 and 11.Two of them coming from DXCWR could enhance the cold-tolerance of the backcross progenies.Moreover,one sterility QTL that could reduce the seed set rate of the backcross progenies by 78% was mapped on chromosome5.     

Review and prospect of transgenic rice research

Rice is one of the most important crops as the staple food for more than half of the world＇s population. Rice improvement has achieved remarkable success in the past half-century, with the yield doubled in most parts of the world and even tripled in certain regions, which has contributed greatly to food security globally. Rapid population growth and economic development pose a constantly increased food requirement. However, rice yield has been hovering in the past decade, which is mainly caused by the absence of novel breeding technologies, reduction of genetic diversity of rice cultivars, and serious yield loss due to increasingly severe occurrences of insects, diseases, and abiotic stresses. To address these challenges, Chinese scientists proposed a novel rice breeding goal of developing Green Super Rice to improve rice varieties and realize the sustainable development of agriculture, by focusing on the following 5 classes of traits： insect and disease resistance, drought-tolerance, nutrient-use effi- ciency, quality and yield potential. As a modern breeding approach, transgenic strategy will play an important role in realizing the goal of Green Super Rice. Presently, many transgenic studies of rice have been conducted, and most of target traits are consistent with the goal of Green Super Rice. In this paper, we firstly review technical advances of rice transformation, and then outline the main progress in transgenic rice research with respect to the most important traits： insect and disease-resistance, drought-tolerance, nutrient-use efficiency, quality, yield potential and herbicide-tolerance. The prospects of developing transgenic rice are also discussed.     

Genetic structure and eco-geographical differentiation of cultivated Hsien rice (Oryza sativa L. subsp. indica) in China revealed by microsatellites

Indica is not only an important rice subspecies widely planted in Asia and the rest of the world,but it is also the genetic background of the majority of hybrid varieties in China.Studies on genetic structure and genetic diversity in indica germplasm resources are important for the classification and utilization of cultivated rice in China.Using a genetically representative core collection comprising 1482 Chinese indica landraces,we analysed the genetic structure,geographic differentiation and diversity.Model-based structure analysis of varieties within three ecotypes revealed nine eco-geographical types partially accordant with certain ecological zones in China.Differentiation of eco-geographical types was attributed to local ecological adaption and physical isolation.These groups may be useful for developing heterotic groups of indica.To facilitate the identification of different ecotypes and eco-geographical types,we identified characteristic SSR alleles of each ecotype and eco-geographical type and a rapid index of discrimination based on characteristic alleles.The characteristic alleles and rapid discrimination index may guide development of heterotic groups,and selection of hybrid parents.     

Obtaining transgenic rice resistant to rice fungal blast disease by controlled cell death strategy

The strategy of the two-component system,composed of Barnase and Barstar which encode RNase and a specific inhibitor to the RNase respectively, is adopted to obtain transgenic rice resistant to rice fungal blast disease. In this study, two chimeric promoters, induced by rice blast fungus pathogen (Magnaporthe grisea), are fused with Barnase respectively to construct two plant expression vectors, pWBNBS and pPBNBS together with the Barstar driven by CaMV 35S promoter. The resistance of the transgenic rice lines to rice blast fungus disease and rice blight disease are evaluated. The results show that (1) the expression of Barnase is induced in rice leaves when inoculated with the spores of Magnaporthe grisea; (2) the induced expression level of Barnase surpasses the level of Barstar, which elicits a similar hypersensitive response (HR) in the leaves, and the transgenic plant shows high resistance to the rice fungal blast disease; and (3) transgenic rice plants also show obvious resistance to rice bacterial blight disease. Taken together, these results suggest that the transgenic rice plants harboring this two-component system acquire relatively broad spectrum resistance against pathogens, especially high resistance to rice fungal pathogen.     

The Recent Advances in Plant Protection Researches of China

There are eight examples briefly given in this paper, namely, (1) Polymyxa graminis and the cereal viruses it transmits; (2) the geographical types and facultative migration of cotton bollworm as well as the safety of Bt transgenic cotton; (3) development of crop near-isogenic lines with resistance to diseases; (4) molecular-biological researches induced resistance of rice by infection of blast fungus;(5) to use cytological and molecular-biological techniques for breeding wheat varieties resistant to barley yellow dwarf virus; (6) mass rearing and field releasing of Microplitis mediator for cotton bollworm control; (7) identification and recombination of insecticidal crystal genes of Bacillus thuringiensis; and (8) interplanting of diverse resistance rice varieties for sustainable control of blast disease; which reflect the general situation of recent advances in plant protection researches of China.     

The characteristics of CO2 assimilation of photosynthesis and chlorophyll fluorescence in transgenic PEPC rice

With PEPC, PPDK, NADP-ME and PEPC+ PPDK transgenic and untransformed rice (Orysa sativa L.), the activities of related C₄ photosynthesis enzymes, the chlorophyll fluorescence parameters, CO₂ exchange and other physiological indexes were compared, in which the physiological characteristics of PEPC transgenic rice were mainly studied. The results were as follows: (ⅰ) The activities of PEPC in PEPC transgenic rice were 20-fold higher than those in untransformed rice; the light-saturation photosynthetic rates and the carboxylation efficiency of PEPC transgenic rice were increased by 55% and 50% more than those of untransformed rice, respectively, while the CO₂ compensation point decreased by 27%. (ⅱ) The PSⅡ photochemical efficiency (F_v/F_m) and photochemical quenching (q_P) of transgenic PEPC rice decreased less in comparison with those of untransformed rice after the treatment with high light intensity (3 h) or methyl viologen (MV), a photooxidative reagent, which demonstrated that the tolerance of PEPC transgenic rice to photoinhibition and photooxidation was enhanced. (ⅲ) Under the condition of high light intensity, the activity of RuBPCase in PEPC transgenic rice did not obviously vary while the activity induced of carbonic anhydrase (CA) in PEPC transgenic rice increased by 1.8 fold. These results would provide some beneficial enlightment for revealing the mechanism of high photosynthetic efficiency and breeding with high photosynthetic efficiency in rice.     

Overexpression of OsRAA1 promotes flowering and hypocotyls elongation in Arabidopsis

Previously, OsRAA1, an AtFPF1 homologue gene, was found to play an important role in modulating rice root development. In the current study, OsRAA1 was overexpressed in Arabidopsis, and the transgenic plants showed early flowering and elongated hypocotyl phenotypes as compared with the wild-type under white-light conditions. The hypocotyls of transgenic lines were twice as long as those of wild-type plants under red-light conditions but were indistinguishable from those of the wild-type under blue and far-red light and darkness. In addition, the phenotypes of AtFPF1 transgenic lines were similar to those of OsRAA1 transgenic lines. These results suggested that OsRAAI/AtFPF1 protein is involved in regulating flowering time and plays an important role in the inhibition of hypocotyl elongation under continuous red light. The functions were preserved during the evolution.     

Digestive stability and acute toxicity studies of exogenous protein in transgenic rice expressing lysine-rich fusion proteins

Evaluating exogenous protein expressed in transgenic crops is one of the most effective methods of assessing the safety of transgenic plants. The objective of this study was to assess the food safety of genetically modified (GM) rice containing a lysine-rich fusion protein gene (transgenic GL gene rice) by in vitro digestion and acute toxicity testing of exogenous protein, according to the national standard of the People’s Republic of China. The exogenous protein was rapidly degraded in the simulated gastric and intestinal fluids. In the acute experiment, the exogenous protein was injected into Institute of Cancer Research (ICR) mice via the tail vein at a dose of 438 mg kg-1 body weight. No adverse effects on animal behavior or mortality were observed during the following 15-day period and there were no significant biological changes in body weight, serum biochemistry parameters, relative organ weights or histopathological examinations, compared with the control group. Therefore, exogenous protein in transgenic GL gene rice has a low potential allergenicity or toxicity risk.     

Transgene inheritance and quality improvement by expressing novel HMW glutenin subunit （HMW-GS）genes in winter wheat

The expression vector pBPC30, which carries the high molecular weight glutenin subunit (HMW-GS) 1Dx5 and 1Dy10 genes, was transferred into hexaploid winter wheat cv. Jinghua No. 1, Jing411 and Jingdong No. 6 explants of immature embryos and immature inflorescence by particle bombardment. A large number of resistant transgenic plants were obtained under the selection of herbicide bialaphos or phosphinothricin (PPT). Confirmed transgenic plants of To generation showed successful integration of HMW-GS genes and bar gene into the wheat genome. T1 generation of transgenic plants can resist 20--150 mg/L PPT.Protein analysis of T2 seed by SDS-PAGE showed that HMW-GS 1Dx5 and 1DylO genes were well expressed in offspring seed of transgenic lines by co-expression with or substitution of endogenous 1Dx2 or 1DylO. In one transgenic line, TG3-74, a new protein band between endogenous protein subunits 7 and 8 (marked as 8*) of glutenin appeared,but endogenous subunit 8 (encoded by 1By8 gene) was absent. Analysis of gluten rheological quality on seed proteins of 102 T3 plants showed that the sedimentation value of 5 transgenic lines (44.2149.0 mL) was remarkably improved,59.6%---64.3% higher than that of wild type Jinghua No. 1 and Jingdong No. 6, similar to bread wheat Cheyenne (48.0 mL). Analysis of dough rheological properties of transgenic lines showed that the dough stable time of 5 transgenic lines range from 16 to 30 min, whereas the dough stable time of wild type was only between 3--7 min. Our research suggests that introducing novel HMW-GS genes into wheat is an efficient way to improve its bread-making quality.     

Ancient DNA sequences of rice from the low Yangtze reveal significant genotypic divergence

Rice (Oryza sativa) was first domesticated in the lower and middle Yangtze regions of China, and rice remains have been found in many Chinese archaeological sites. Until now, only phenotypic archeobotanical evidence, such as the spikelet bases of ancient grains, has been used to speculate on the domestication process and domestication rate of rice. In this study, we sequenced 4 genomic segments from rice remains in Tianluoshan, a site of the local Hemudu Neolithic culture in the low Yangtze and two other archaeological sites (~2400 and 1200 BC, respectively). We compared our sequences with those of the current domesticated and wild rice (O. rufipogon) populations. At least two genotypes were found in the remains from each site, suggesting a heterozygotic state of the rice seeds. One ancient genotype was not found in the current domesticated population and might have been lost. The rice remains belonged to the japonica group, and most if not all were japonica-type, suggesting that the remains might be at an early stage of indica-japonica divergence or an indica-japonica mixture. We also identified sequences with significant similarity to those from species of Sapindales, Zygophyllales, and Brassicales, which is consistent with the identification of other plant remains in the Tianluoshan site and the common rice field weeds such as mustards in southern China.     

Fine mapping of the awn gene on chromosome 4 in rice by association and linkage analyses

Awnness is a key trait in rice domestication, yet no studies have been conducted on fine mapping or association mapping of the rice awn gene. In this study, we investigated the awnness and genotype of a core collection of 303 cultivated rice varieties and a BC5F2 segregating population of 200 individuals. Combining association and linkage analyses, we mapped the awnness related genes to chromosome 4. Primary association analysis using 24 SSR markers revealed five loci significantly associated with awnness on chromosome 4. The associated markers cover previously identified regions. Fine association mapping was conducted using another 29 markers within a 4-Mb region, covering the associated marker in34, which is close to the awn gene Awn4.1. Seven associated markers were revealed, distributed over an 870-kb region. Combining the fine association mapping and linkage analysis of awnness in the 200 BC5F2 segregating population, we finally identified a 330-kb region as the candidate region for Awn4.1. The results indicate that combining association mapping and linkage mapping provides an efficient and precise approach to both genome-wide mapping and fine mapping of rice genes.