Transient expression of a green fluorescent protein in tobacco and maize chloroplast

BACKGROUND: Maize is one of the most important crops worldwide and has been a target of nuclear-based transformation biotechnology to improve it and satisfy the food demand of the ever-growing global population. However, the maize plastid transformation has not been accomplished due to the recalcitrant condition of the crop. RESULTS: In this study, we constructed two different vectors with homologous recombination sequences from maize (Zea mays var. LPC13) and grass (Bouteloua gracilis var. ex Steud) (pZmcpGFP and pBgcpGFP, respectively). Both vectors were designed to integrate into rrn23S/rrn16S from an inverted repeat region in the chloroplast genome. Moreover, the vector had the mgfp5 gene driven by Prrn, a leader sequence of the atpB gene and a terminator sequence from the rbcL gene. Also, constructs have an hph gene as a selection marker gene driven by Prrn, a leader sequence from rbcL gene and a terminator sequence from the rbcL gene. Explants of maize, tobacco and Escherichia coli cells were transformed with both vectors to evaluate the transitory expression­an exhibition of green and red fluorescent light under epifluorescence microscopy. These results showed that both vectors were expressed; the reporter gene in all three organisms confirmed the capacity of the vectors to express genes in the cell compartments. CONCLUSIONS: This paper is the first report of transient expression of GFP in maize embryos and offers new information for genetically improving recalcitrant crops; it also opens new possibilities for the improvement in maize chloroplast transformation with these vectors.

Chloroplast Genome Analysis of Liriope spicata var. prolifera, Ophiopogon japonicus in Sichuan and Zhejiang / 中国实验方剂学杂志

Objective::To determine the chloroplast genomes of Liriope spicata var. prolifera, Ophiopogon japonicus in Sichuan and Zhejiang, analyze their sequence characteristics and complete the screening of specific DNA barcodes. Method::The chloroplast genomes of L. spicata var. prolifera, O. japonicus in Sichuan and Zhejiang were sequenced, spliced and annotated through high-throughtput sequencing technology, and the structural characteristics and phylogenetic relationships of chloroplast genomes were analyzed by bioinformatics. Result::The total length of chloroplast genome of L. spicata var. prolifera was 155 998 bp, the total content of guanine and cytosine (GC) was 37.7%, and 85 protein-coding genes, 37 transfer RNA (tRNA) genes and 8 ribosomal RNA (rRNA) genes were successfully annotated, a total of 274 simple sequence repeats (SSRs) were detected, the number of codons encoding leucine was the most, while the number of codons encoding tryptophan was the least. The total length of chloroplast genome of O. japonicus in Sichuan province was 156 078 bp, the total content of GC was 37.8%, and 85 protein-coding genes, 37 tRNA genes and 8 rRNA genes were successfully annotated, a total of 265 SSRs were detected, the number of codons encoding leucine was the most, while the number of codons encoding tryptophan was the least. The total length of chloroplast genome of O. japonicus in Zhejiang province was 156 207 bp, the total content of GC was 37.7%, and 85 protein-coding genes, 37 tRNA genes and 8 rRNA genes were successfully annotated, a total of 274 SSRs were detected with the highest number of codons encoding leucine and the lowest number of codons encoding tryptophan. Conclusion::The phylogenetic trees show that compared with O. japonicus in Sichuan province, L. spicata var. prolifera is more closely related to O. japonicus in Zhejiang province. The variation of non-coding regions of L. spicata var. prolifera, O. japonicus in Zhejiang and Sichuan is greater than that in the coding region. The entire chloroplast genome can be used as a super barcode for identifying species of Ophiopogon and Liriope.

Establishment of an Efficient Chloroplast Gene Transformation System in Sugar Beet(Beta vulgaris L.)and Obtainment of Insect and Herbicide Resistant Sugar Beet Plants / 生物化学与生物物理进展

Insects pests and weeds are the main factors that reduce the yield of sugar beet. Genetic engineering breeding is an effective method to breed insect-resisitant and herbicide-resisitant sugar beet. A transformation system for foreign genes in sugar beet chloroplast was established. The expression of the foreign genes can confers resistance in transgenic sugar beet plants to insects pests and weeds. The chloroplast transformation vector pSKARBt/bar, which carries Bt cry1Ac gene and bar gene expression cassettes, was constructed by using molecular method. The Bt gene expression cassette contained the 3.5 kb Bt cry1Ac gene under the control of psbA promoter and terminator cloned from sugar beet chloroplast genome. The bar gene expression cassette contained the bar gene, 16 S promoter and terminator cloned from sugar beet chloroplast genome, The atpB and rbcL gene cloned from sugar beet chloroplast genome were used as homologous fragment, the bar gene was the selective marker. Plasmid pSKARBt/bar were transformed into the petioles of sugar beet with particle bombardment method. The petioles were planced onto the shoot-inducing selection medium which contained spectinomycin (20 mg/L), 6-BA (1.5 mg/L) and NAA (0.2 mg/L) at first. And when the green shoots regenerated, the green shoots were transfered into the shoot-propagation medium for optimal shoot development which contained spectinomycin (20 mg/L) and 6-BA (0.5 mg/L) and NAA (1.0 mg/L) one subculture at 20-day intervals, and then the shoots were transfered into the shoot-propagation medium for optimal shoot development with herbicide (PPT 10 mg/L) several subcultures. The shoots were transfered into the root-induction medium with herbicide (PPT 10 mg/L) and the transgenic plants were obtained at last. The transgenic sugar beet plants were testsed by PCR and Southern blot. The results showed that the Bt gene and bar gene had been transferred into the chloroplast genome of sugar beet. The transgenic plants had tolerance to both PPT and bioassays testsed. The insecticidal activity (the mortality of larvaes was 33%～80%) and herbicide resistance of the transgenic plants indicated that the relevant protein had been expressed already in sugar beet. The study showed that the bar gene can also be used as an efficient selective marker gene besides antibiotic resistant markers in plant transformation. Efficient transformation system in sugar beet chloroplast had been established.

Cloning and Transformation of lba Gene of Glycine max into Chloroplast of Chlamydomonas reinhardtii / 中国生物工程杂志

To decrease the oxygen content in the cell is a key method to improve hydrogen production in Chlamydomonas reinhardtii.A new approach was developed by transforming the leghemoglobin gene lba,which has high affinity to oxygen,into the chloroplast of C.reinhardtii to get a low dissolved oxygen in the cell and result into improvement of H2 ase activity and H2 yield. The results showed that lba was successfully transformed into the chloroplast of C.reinahrdtii strain 849 and did not affect its growth significantly. The work paved the road for further regulation of lba expression in the chloroplast to improve of hydrogen production of C.reinahrdtii.