Expression of foreign genes, GUS and hygromycin resistance, in the halophyte Kosteletzkya virginica in response to bombardment with the Particle Inflow Gun

A ‘Particle Inflow Gun’ was constructed to introducethe glucuronidase (GUS) and hygromycin resistance genes intohalophytic suspension cells of Kosteletzkya virginica. The transientexpression of the GUS gene was associated with the cell cultureconditions, physical parameters during the use of the ParticleInflow Gun, and different promoters coupled to GUS. When theCaMV35S promoter was used, the cells adapted at 85 mM NaCl hada similar gene transfer efficiency to those of the non-salt-adaptedcontrol, while expression was less in the 170 mM and 255 mMNaCl-adapted cells. Both elevating bombardment pressure to 1.65mPa and shortening the distance between the cells and the particleholder from 21 cm to 9 cm enhanced GUS expression in the cellsgrown in four salinity treatments. An ABA-responsive promoterinduced the expression of the GUS gene either with 10^–4M ABA or with salts in the post-bombardment medium in both controland NaCl-adapted cell tines. Stable transgenic callus lineswere isolated by using hygromycin containing medium after bombardingthe suspension cells with the Particle Inflow Gun. The presenceof the GUS gene in stable transformants was confirmed not onlyby histochemical and fluorimetric assays for the GUS activity,but also by Southern hybridization of RT-PCR amplified mANA. Key words: Transgenic hatophyte, Particle Inflow Gun, bombardment, salt tolerance, transformation     

Test pigments in media for tissue culture and transformation

Accurate uses of key components and right media during tissue cultures are vital for a particular process and repeatable outcome in plant transformation. If additional components, as indicators, can be added into media, it could avoid some of the issues, such as adding a wrong component or misrecognition of a right medium. We tested to add a specific pigment into a set of media sharing a key common element, such as a selection agent, so that all media containing the same selection agent appeared in a certain coloration to provide a convenient marker for all people involved in the process of transformation. The effect of four color pigments on tissue cultures of corn, rice, and soybean was evaluated in this study. The results show pigment brilliant blue, Pyla-Cert green, and mixture purple neither impacted soybean cotyledon growth nor callus formation and shoot regenerations of corn and rice. In soybean hair root transformation, addition of pigment blue, green, erythrosine red and mixture purple into the selection media did not reduce its transformation frequency and copy number. However, erythrosine red resulted in lower callus formation rates and low regeneration rates among rice and corn. Further, fresh weight and dry weight of soybean cultures were increased on the media containing erythrosine red. Therefore, we recommend using pigment brilliant blue, Pyla-Cert green and mixture purple as indicators for selection agents in the transformation process of maize, soybean, and rice to prevent misuse of wrong components or wrong media.     

Activity of the 5′ regulatory regions of the rice polyubiquitin <Emphasis Type="Italic">rubi3</Emphasis> gene in transgenic rice plants as analyzed by both <Emphasis Type="Italic">GUS</Emphasis> and <Emphasis Type="Italic">GFP</Emphasis> reporter genes

Ubiquitin is an abundant protein involved in protein degradation and cell cycle control in plants and rubi3 is a polyubiquitin gene isolated from rice (Oryza sativa L.). Using both GFP and GUS as reporter genes, we analyzed the expression pattern of the rubi3 promoter as well as the effects of the rubi3 5'-UTR (5' untranslated region) intron and the 5' terminal 27 bp of the rubi3 coding sequence on the activity of the promoter in transgenic rice plants. The rubi3 promoter with the 5'-UTR intron was active in all the tissue and cell types examined and supported more constitutive expression of reporter genes than the maize Ubi-1 promoter. The rubi3 5'-UTR intron mediated enhancement on the activity of its promoter in a tissue-specific manner but did not alter its overall expression pattern. The enhancement was particularly intense in roots, pollen grains, inner tissue of ovaries, and embryos and aleurone layers in maturing seeds. The translational fusion of the first 27 bp of the rubi3 coding sequence to GUS gene further enhanced GUS expression directed by the rubi3 promoter in all the tissues examined. The rubi3 promoter should be an important addition to the arsenal of strong and constitutive promoters for monocot transformation and biotechnology.     

Functional Analysis of Two Matrix Attachment Region (MAR) Elements in Transgenic Maize Plants

Matrix attachment regions (MARs) are binding sites for nuclear scaffold proteins in vitro, and are proposed to mediate the attachment of chromatin to the nuclear scaffold in vivo. Previous reports suggest that MAR elements may stabilize transgene expression. Here, we tested the effects of two maize MAR elements (P-MAR from the P1-rr gene, and Adh1-MAR from the adh1 gene) on the expression of a gusA reporter gene driven by three different promoters: the maize p1 gene promoter, a wheat peroxidase (WP) gene promoter, or a synthetic promoter (Rsyn7). The inclusion of P-MAR or Adh1-MAR on P::GUS transgene constructs did not reduce variation in the levels of GUS activity among independent transformation events, nor among the progeny derived from each event. The Adh1-MAR element did not affect GUS expression driven by the WP promoter, but did modify the spatial pattern of expression of the Rsyn7::GUS transgene. These results indicate that, in transgenic maize plants, the effects of MAR elements can vary significantly depending upon the promoter used to drive the transgene.     

Gene expression enhancement mediated by the 5′ UTR intron of the rice <Emphasis Type="Italic">rubi3</Emphasis> gene varied remarkably among tissues in transgenic rice plants

Introns are important sequence elements that modulate the expression of genes. Using the GUS reporter gene driven by the promoter of the rice (Oryza sativa L.) polyubiquitin rubi3 gene, we investigated the effects of the 5' UTR intron of the rubi3 gene and the 5' terminal 27 bp of the rubi3 coding sequence on gene expression in stably transformed rice plants. While the intron enhanced GUS gene expression, the 27-bp fused to the GUS coding sequence further augmented GUS expression level, with both varying among different tissues. The intron elevated GUS gene expression mainly at mRNA accumulation level, but also stimulated enhancement at translational level. The enhancement on mRNA accumulation, as determined by realtime quantitative RT-PCR, varied remarkably with tissue type. The augmentation by the intron at translational level also differed by tissue type, but to a lesser extent. On the other hand, the 27-bp fusion further boosted GUS protein yield without affecting mRNA accumulation level, indicating stimulation at translation level, which was also affected by tissue type. The research revealed substantial variation in the magnitudes of intron-mediated enhancement of gene expression (IME) among tissues in rice plants and the importance of using transgenic plants for IME studies.     

CRISPR/Cas9基因组定点编辑中脱靶现象的研究进展

近年来, CRISPR定点编辑技术发展迅猛, 在动物、植物和微生物中均得到广泛应用。其中, 备受关注的脱靶现象也是研究的热点, 迄今已取得了重要进展。该文介绍了脱靶现象的产生原理及体内和体外检测脱靶现象的方法, 评价了通过改进sgRNA设计和优化CRISPR系统等来降低脱靶率的方法。在植物基因组定点编辑过程中, 应适时检测脱靶现象, 提高脱靶检测的精确度和准确度。     

Development of protoporphyrinogen oxidase as an efficient selection marker for Agrobacterium tumefaciens-mediated transformation of maize

In this article, we report the isolation of plant protoporphyrinogen oxidase (PPO) genes and the isolation of herbicide-tolerant mutants. Subsequently, an Arabidopsis double mutant (Y426M + S305L) was used to develop a selectable marker system for Agrobacterium tumefaciens-mediated transformation of maize (Zea mays) and to obtain multiple events tolerant to the PPO family of herbicides. Maize transformants were produced via butafenacil selection using a flexible light regime to increase selection pressure. Butafenacil selection per se did not change transgene copy number distribution relative to other selectable marker systems, but the most tolerant events identified in the greenhouse were more likely to contain multiple copies of the introduced mutant PPO gene. To date, more than 2,500 independent transgenic maize events have been produced using butafenacil selection. The high frequency of A. tumefaciens-mediated transformation via PPO selection enabled us to obtain single-copy transgenic maize lines tolerant to field levels of butafenacil.