Kin-Ying To

Complete sequence of the binary vector pBI121 and its application in cloning T-DNA insertion from transgenic plants

The widely used expression vector pBI121 for plant transformation was reconstructed, and the complete sequence of 14758 bp is now available (accession number AF485783). The T-DNA region (6193 bp) contains the right border, expression cassettes for a neomycin phosphotransferase II (NPTII) selection marker and a β-glucuronidase (GUS) reporter gene, and the left border. The non-T-DNA region (8565 bp) was constructed according to the Bin 19 vector. We applied the vector information to clone the plant/T-DNA junction region from three independent transgenic tobacco plants. Knowledge of the complete sequence of this vector will be useful for an accurate description of vector size, determination of the integrity of T-DNA, identification of independent lines, the locus where it is inserted, the T-DNA copy number in those stable transformants, or construction of a smaller vector. In addition, the complete sequence (5667 bp) of the transient expression vector pBI221 (accession number AF502128) carrying the ampicillin resistance and gus reporter genes is also reported.

Molecular characterization of ribulose-1,5-bisphosphate carboxylase/oxygenase activase in rice leaves.

Abstract. A full-length cDNA (designated rcaII) encoding the Rubisco activase (RCA) of rice (Oryza sativa L.) has been cloned from a cDNA library constructed with mRNA from green leaves. Sequence analysis resulted in a reading frame of 432 amino acids with a calculated molecular mass of 47.9 kDa and an estimated isoelectric point of 5.97. The deduced amino acid sequence showed 74–89% identity with other Rubisco activases from higher plants. Two highly conserved motifs were identified. Southern blot analysis suggested the presence of a single rca gene in the rice genome. The accumulation of leaf rca mRNA was found to be regulated by an oscillating circadian rhythm, in rice plants grown in a light-dark photoperiod. To purify the rice RCA protein, total soluble protein from rice green leaves was fractionated by ammonium sulfate precipitation, followed by preparative gel electrophoresis. Two polypeptides, designated RCAI and RCAII, were isolated by two-dimensional gel electrophoresis and further confirmed by N-terminal sequencing. The polyclonal antibodies prepared against rice RCAI and RCAII were found to cross-react with two RCA polypeptides present in leaf extracts of spinach and tobacco. Furthermore, two different 3′ ends of rca mRNA were detected by reverse transcription-polymerase chain reaction analysis. These cDNA fragments and the related genomic DNA fragment were cloned and sequenced. The sequence of rcaI is almost identical to the corresponding sequence of rcaII, except for its having 33 additional amino acids at the C-terminal portion. It can be concluded that a novel alternative splicing mechanism for a common rca mRNA precursor near the 3′ end exists in rice plants.

Analysis of the gene cluster encoding carotenoid biosynthesis in Erwinia herbicola Eho13

Erwinia herbicola is known to synthesize carotenoids and gives an orange-coloured phenotype. These carotenoids play a role in the protection of the cells from the damage caused by near-UV irradiation in nature. The genes encoding these carotenoids in E. herbicola Eho13 are clustered in a 7 kb DNA fragment. The complete sequence of this fragment has been determined. DNA sequence analysis revealed that the entire sequence contains at least five genes, which are transcribed in the same direction. These five genes are organized in the order crtE-crtX-crtY-crtI-crtB. A gene fusion study showed that two different regions in this 7 kb gene cluster contain promoter activity. Primer-extension analysis identified two transcription start sites, located 147 bp upstream from the first gene of the cluster, crtE, and within the last gene of the cluster, crtB. An RNA-PCR study suggested that the five crt genes were organized in an operon and were transcribed from the promoter upstream from crtE.

Isolation and characterization of streptomycin-resistant mutants in Nicotiana plumbaginifolia

SummaryStreptomycin-resistant colonies were isolated from protoplast cultures of haploid Nicotiana plumbaginifolia based on their ability to green in medium containing 1 mg/ml streptomycin sulfate. The frequency of resistant colonies was 0.9×10−5 in nonmutagenized culture, and increased ten-fold following treatment of culture with 10 μg/ml N-methyl-N′-nitro-N-nitrosoguanidine. Of a total of 52 resistant clones isolated, 2 gave rise to haploid, 15 to diploid, and 3 to tetraploid plants upon transfer of calli to differentiation medium. Leaf-segment and protoplast assays showed that all diploid regenerates were resistant to streptomycin but sensitive to chloramphenicol, kanamycin, lincomycin, neomycin, and spectinomycin. Plants in most diploid clones were fertile and able to set seeds when self-fertilized and crossed reciprocally to wild-type plants. Inheritance of streptomycin resistance was studied in the diploid clones and, without exception, the resistance was transmitted maternally. Comparative studies of the ultrastructure of organelles and protein synthesis in isolated chloroplasts between wild-type and resistant clones in the presence of streptomycin suggest that streptomycin resistance is controlled by chloroplasts.

Point mutations in the chloroplast 16s rRNA gene confer streptomycin resistance in Nicotiana plumbaginifolia

In a previous paper we reported the isolation of streptomycin-resistant mutants from Nicotiana plumbaginifolia and presented evidence for chloroplast control of the resistance trait. To understand the molecular basis of the resistance in these mutants, we sequenced three regions in the chloroplast 16s rRNA gene, which correspond to the 5′ terminus, the 530 loop, and the 900 stem/loop of Escherichia coli 16s rRNA, and compared them with the sequences of the wild-type. Our results show that: (1) nine mutants have a C to T change at position 912, (2) one mutant (SR1021) has a G to A change at position 885, (3) one mutant has a C to T change at position 526, based on E. coli numbering; and (4) three mutants do not have any change in the regions analyzed. The point mutation detected in SR1021 has not been reported previously. In E. coli 16s rRNA, position 885 is protected from chemical probing by ribosomal protein S12 and is closely juxtaposed with the streptomycin-binding region (positions 912–915) in the predicted secondary structure. It is likely that the G to A transition at this position is a novel mutation for streptomycin resistance.

Transformation and characterization of transgenic Bidens pilosa L.

Bidens pilosa L. is an erect perennial herb that is used as a folk medicine for the treatment of a variety of illnesses including diabetes. Genetic engineering is an effective means of producing desired phytocompounds in certain medicinal plants; however, plant regeneration and genetic transformation have not yet been reported for B. pilosa. Here, we determined the optimal conditions for plant regeneration from cotyledon explants of in vitro-grown B. pilosa L. var. radiata, one of the three common variants of this species found in Taiwan. An Agrobacterium-mediated method was developed to transform the vector pCHS, which is carrying the Petunia chalcone synthase (chs) and neomycin phosphotransferase II (nptII) genes into B. pilosa var. radiata. From a total of 1,373 cotyledon explants, 21 putative transgenic lines were regenerated on selection medium, and 15 lines carrying both Petunia chs and nptII transgenes were obtained. Out of these, one line, BpCHS21, showed an anomalous flower phenotype. The presence of Petunia chs transgene in randomly-selected transgenic plants was confirmed by Southern blot analysis. Reverse transcription-polymerase chain reaction analysis revealed that the transgenic chs was differentially expressed in the leaf tissue of all the transformants, but not in wild type. T1 progeny assay of outcross seeds (i.e., wild-type pollens crossed with transgenic flowers) from selected transformants showed that the ratio of kanamycin-resistant and kanamycin-sensitive seedlings was close to 1 in kanamycin-containing medium, verifying again the single integration of foreign DNA into the nuclear chromosome of transformants and the inheritance of the nptII transgene. The present protocol will be useful for the introduction of genes of interest into B. pilosa paving the way for metabolic engineering in this medicinal plant.

Mendelian inheritance of streptomycin resistance in Nicotiana plumbaginifolia

In a previous study two haploid streptomycin-resistant clones of Nicotiana plumbaginifolia were isolated. The chromosome number of one of these clones has now been doubled through leaf-midvein culture and the resultant diploids were characterized genetically. Our results show that streptomycin resistance in this clone is conditioned by a recessive nuclear gene. Haploid protoplasts of this streptomycin-resistant mutant were selected for chlorate resistance. All clones obtained from the selection were deficient in nitrate reductase activity in addition to resistance to streptomycin. Genetic analysis of progeny of one of these clones revealed that the genes for streptomycin resistance and for the apoenzyme of nitrate reductase are unlinked.

In vitro regeneration, Agrobacterium-mediated transformation, and genetic assay of chalcone synthase in the medicinal plant Echinacea pallida

In vitro plant regeneration was established in Echinacea pallida, a plant that is commonly used as a folk medicine to treat the common cold, fevers, inflammation and so on. Conditions for callus induction, lateral root and shoot regeneration were determined. Subsequently, two vectors pCHS and pOSAG78, carrying different selection marker genes resistant to kanamycin and hygromycin, respectively, were independently used to transform leaf explants of E. pallida using an Agrobacterium-mediated method. Genomic PCR analysis confirmed the presence of the transgene and selection marker gene in obtained transgenic lines. Southern hybridization indicated that the T-DNA insertion in some transgenic E. pallida was single copy. Among them, transformants carrying Petunia chalcone synthase (CHS) were selected for further study. CHS is a key enzyme in the biosynthesis of diverse flavonoids including anthocyanin pigmentation. Here, we analyzed the roles and compared the gene expression of two clusters of CHSs, EpaCHS-A and EpaCHS-B (EpaCHS-B1 and EpaCHS-B2), isolated from E. pallida. Two of the genes, EpaCHS-A and EpaCHS-B1, were abundantly expressed in petals, whereas EpaCHS-B2 was expressed at high levels in leaves. The expression of EpaCHSs remained constant in leaves and roots of Petunia CHS transformants, while EpaCHS-B2 expression was changed in flowers of transgenic plants. The biosynthesis of caffeic acid derivatives, cichoric acid and caftaric acid, was increased in leaves and roots of CHS transformants, respectively, while the amount of echinacoside in roots of transgenic plants was decreased. This is the first report on genetic engineering of E. pallida. The information contained herein can be used as a tool for further study of the biological pathways and secondary metabolism of specific compounds from medicinal Echinacea species.