Genetic determinism of prickles in rose.

KEY MESSAGE: The genetic determinism of prickle in rose is complex, with a major locus on LG3 that controls the absence/presence of prickles on the rose stem. Rose is one of the major ornamental plants. The selection of glabrous cultivars is an important breeding target but remains a difficult task due to our limited genetic knowledge. Our objective was to understand the genetic and molecular determinism of prickles. Using a segregating diploid rose F1 population, we detected two types of prickles (glandular and non-glandular) in the progeny. We scored the number of non-glandular prickles on the floral and main stems for three years. We performed QTL analysis and detected four prickle loci on LG1, 3, 4 and 6. We determined the credible interval on the reference genome. The QTL on LG3 is a major locus that controls the presence of prickles, and three QTLs (LG3, 4 and 1) may be responsible for prickle density. We further revealed that glabrous hybrids are caused by the combination of the two recessive alleles from both parents. In order to test whether rose prickles could originate from a 'trichome-like structure,' we used a candidate approach to characterize rose gene homologues known in Arabidopsis, involved in trichome initiation. Four of these homologues were located within the overlapping credible interval of the detected QTLs. Transcript accumulation analysis weakly supports the involvement of trichome homologous genes, in the molecular control of prickle initiation. Our studies provide strong evidence for a complex genetic determinism of stem prickle and could help to establish guidelines for glabrous rose breeding. New insights into the relationship between prickles and trichomes constitute valuable information for reverse genetic research on prickles.

Cloning and characterization of DELLA genes in Artemisia annua.

Gibberellins (GA) are some of the most important phytohormones involved in plant development. DELLA proteins are negative regulators of GA signaling in many plants. In this study, the full-length cDNA sequences of three DELLA genes were cloned from Artemisia annua. Phylogenetic analysis revealed that AaDELLA1 and AaDELLA2 were located in the same cluster, but AaDELLA3 was not. Subcellular localization analysis suggested that AaDELLAs can be targeted to the nucleus and/or cytoplasm. Real-time PCR indicated that all three AaDELLA genes exhibited the highest expression in seeds. Expression of all AaDELLA genes was enhanced by exogenous MeJA treatment but inhibited by GA3 treatment. Yeast two-hybrid assay showed that AaDELLAs could interact with basic helix-loop-helix transcription factor AaMYC2, suggesting that GA and JA signaling may be involved in cross-talk via DELLA and MYC2 interaction in A. annua.

Agrobacterium tumefaciens-mediated genetic transformation of the Taxol-producing endophytic fungus Ozonium sp EFY21.

An efficient Agrobacterium tumefaciens-mediated genetic transformation method was successfully established for a newly isolated Taxol-producing fungus, Ozonium sp EFY21. A specific hygromycin B resistance expression vector, pCAMBIA1304'AN7-1, was constructed for fungal transformation. Key factors affecting transformation efficiency were thoroughly investigated and optimized. PCR amplification and Southern hybridization were used to verify the transformation events. This study should pave the way for future genetic modification studies of Ozonium sp EFY21.

Effects of artesunate and ursolic acid on hyperlipidemia and its complications in rabbit.

OBJECTIVE: To find the novel hypolipidemic agents, the effects of ursolic acid and artesunate on hyperlipidemia and its complications were determined in rabbit fed with Western-type diet. METHODS AND RESULTS: New Zealand rabbits fed a Western-type diet developed a hyperlipidemia. Rabbits received ursolic acid (25mg/kg) or artesunate (25mg/kg) alone, or in combination (12.5+12.5mg/kg), to prevent hyperlipidemia. Ursolic acid or artesunate alone significantly decreased the plasma cholesterol and triglyceride in rabbits. Furthermore, they both attenuated liver steatosis and reduced the area of aortic root lesions. The combination of ursolic acid and artesunate was more potent than either agent alone, which indicates a strong synergistic effect. CONCLUSION: The hypolipidemic effect of artesunate is firstly reported. Its combination with ursolic acid might have the potential to further develop for the treatment of atherosclerosis.

Molecular cloning and characterization of the glyceraldehyde-3-phosphate dehydrogenase gene from Penicillium expansum PE-12.

Penicillium expansum produces large amounts of lipase, which is widely used in laundry detergent and leather industry. We isolated the glyceraldehyde-3-phosphate dehydrogenase gene (PeGPD) from P. expansum PE-12 through reverse transcriptase PCR and 5'-3' rapid amplification of cDNA ends (RACE-PCR). The gene is 1266 bp long, including an ORF of 1014 bp, encoding a polypeptide chain of 337 amino acids. A phylogenetic tree based on GPD proteins showed that P. expansum is close to Aspergillus species, but comparatively distant from P. marneffei. Southern blot results revealed a single copy of PeGPD, and expression analysis gave evidence of high expression levels. PeGPD genes have potential for genetic engineering of P. expansum for industrial lipase production.

Overexpression of the cytochrome P450 monooxygenase (cyp71av1) and cytochrome P450 reductase (cpr) genes increased artemisinin content in Artemisia annua (Asteraceae).

Finding an efficient and affordable treatment against malaria is still a challenge for medicine. Artemisinin is an effective anti-malarial drug isolated from Artemisia annua. However, the artemisinin content of A. annua is very low. We used transgenic technology to increase the artemisinin content of A. annua by overexpressing cytochrome P450 monooxygenase (cyp71av1) and cytochrome P450 reductase (cpr) genes. CYP71AV1 is a key enzyme in the artemisinin biosynthesis pathway, while CPR is a redox partner for CYP71AV1. Eight independent transgenic A. annua plants were obtained through Agrobacterium tumefaciens-mediated transformation, which was confirmed by PCR and Southern blot analyses. The real-time qPCR results showed that the gene cyp71av1 was highly expressed at the transcriptional level in the transgenic A. annua plants. HPLC analysis showed that the artemisinin content was increased in a number of the transgenic plants, in which both cyp71av1 and cpr were overexpressed. In one of the transgenic A. annua plants, the artemisinin content was 38% higher than in the non-transgenic plants. We conclude that overexpressing key enzymes of the biosynthesis pathway is an effective means for increasing artemisinin content in plants.

An oleosin-fusion protein driven by the CaMV35S promoter is accumulated in Arabidopsis (Brassicaceae) seeds and correctly targeted to oil bodies.

Oleosin-fusion technology is used to express desired proteins. It was developed based on the properties of oleosin; the heterologous protein gene is fused to the oleosin gene and the fusion gene is driven by a seed-specific promoter. We replaced the seed specific promoter with the CaMV35S promoter to dive a gfp-oleosin fusion gene in transformed Arabidopsis. The heterologous oleosin-fusion protein was mainly accumulated in the transgenic Arabidopsis seeds and correctly targeted to oil bodies. This provides an alternate choice of promoter in oleosin-fusion technology.

Molecular cloning and characterization of gene coding for γ-tocopherol methyltransferase from lettuce (Lactuca sativa).

γ-tocopherol methyltransferase is an important rate-limiting enzyme involved in tocopherol biosynthesis. The full-length cDNA encoding γ-tocopherol methyltransferase (designated as LsTMT) was cloned from Lactuca sativa for the first time by rapid amplification of cDNA ends and characterized by means of quantitative RT-PCR. The full-length cDNA of LsTMT was 1131 bp, with an open reading frame of 897 bp encoding a γ-tocopherol methyltransferase protein of 298 amino acids, with a calculated molecular mass of 33.06 kDa and an isoelectric point of 5.86. Comparative analysis revealed that LsTMT has a close similarity with γ-TMTs from other plant species. Bioinformatic analysis indicated that LsTMT shares a common evolutionary origin based on sequence similarity and has the closest relationship to γ-TMT from the sunflower, Helianthus annuus. Based on quantitative RT-PCR analysis, we found that expression of LsTMT is induced and strengthened by oxidative stresses such as strong light and drought. The cloning and characterization of LsTMT will be helpful to further understanding its role in the tocopherol biosynthesis pathway. We consider it to be a candidate gene for metabolic engineering of vitamin E in vegetable crops.

Molecular cloning and characterization of a tocopherol cyclase gene from Lactuca sativa (Asteraceae).

Tocopherol cyclase is a rate-limiting enzyme involved in tocopherol biosynthesis. The full-length cDNA encoding tocopherol cyclase (designated as LsTC) was cloned from lettuce (Lactuca sativa) for the first time by rapid amplification of cDNA ends (RACE) and characterized by means of quantitative RT-PCR. The full-length cDNA of LsTC was 1675 bp, with an open reading frame of 1521 bp, encoding a tocopherol cyclase protein of 506 amino acids, with a calculated molecular mass of 56.76 kD and an isoelectric point of 6.49. Comparative analysis revealed that LsTC has a close similarity with tocopherol cyclases from other plant species. Bioinformatic analysis indicated that LsTC shares a common evolutionary origin based on sequence and has the closest relationship to tocopherol cyclase from Helianthus annuus. Quantitative RT-PCR analysis suggested that expression of LsTC is induced and strengthened by oxidative stresses, such as strong light and drought. This cloning and characterization of LsTC will be helpful for further understanding of its role in the tocopherol biosynthesis pathway and provide a candidate gene for metabolic engineering of vitamin E.

CBF-dependent signaling pathway: a key responder to low temperature stress in plants.

Plants under low temperature (LT) stress exhibit a C-repeat binding factor (CBF)-dependent responsive pathway. The transcription factors in the CBF family, existing in multiple plant species, are the key regulators of the cold-responsive (COR) genes. CBF1 and CBF3 are regulated in a different way from CBF2, and CBF4 is the only known CBF gene definitely involved in abscisic acid (ABA)-dependent signaling pathways. RAP2.1 and RAP2.6 are the downstream regulators under CBFs. The upstream regulators of the CBF named inducer of CBF expression (ICE) acts as a positive regulator of CBFs. Meanwhile, these CBF signaling pathway components could associate with many other transcription activators and repressors in regulating gene expression when plants are under LT stress. HOS1 negatively regulates ICE1, which down regulates MYB15, an upstream repressor of CBFs. ZAT12 participates in the repression of CBFs, while ZAT10 and FRY2 negatively regulate the CBF-target genes. ADF5 was recently also found to repress CBFs. LOS2 works against ZAT10, and LOS4 positively regulates CBFs. SFR6 is involved in the modification of CBFs to activate the COR genes, and SIZ1-dependent sumoylation plays a positive role in the regulation of ICE1. The utilization of CBF-dependent signaling components has a broad perspective in the field of plant breeding for enhancing crop LT tolerance.

[Single-strand conformational polymorphism markers associated with a major QTL for fusarium head blight resistance in wheat].

A major quantitative trait locus (QTL) associated with resistance to Fusarium head blight (FHB) was identified on chromosome 3BS between simple sequence repeat (SSR) markers Xgwm389 and Xgwm493 in wheat 'Ning 7840', a derivative from 'Sumai 3'. However, the marker density of SSR in the QTL region was much lower than that required for marker-assisted selection (MAS) and map-based cloning. The objective of this study was to exploit new markers to increase marker density in this QTL region by using single-strand conformational polymorphism (SSCP) markers developed from wheat expressed sequence tags (ESTs) on 3BS bin 8-0.78-1.0. Sixty-nine out of 85 SSCP primer pairs amplified PCR (polymerase chain reaction) products from the genomic DNA of 'Chinese Spring'. Thirty-four primer pairs amplified PCR products that could form clear ssDNA (single strand DNA) bands through denaturation treatment. Ten SSCP markers had polymorphisms between 'Ning 7840' and 'Clark'. Five of the ten polymorphic SSCP markers were located on chromosome 3B by nulli-tetrasomic analysis. Three SSCP markers (Xsscp6, Xsscp20, and Xsscp21) were mapped into the region between Xgwm493 and Xgwm533, and possessed higher coefficient of determination (R2) than Xgwm493 and Xgwm533. The SSCP markers, Xsscp6, Xsscp20, and Xsscp21, can be used for map-based cloning of the QTL and for marker-assisted selection in FHB resistance breeding.

[Production of herbicide-resistant rice with transforming heterogene].

Using pAHC20 (containing Bar gene), pWRG1515 (containing GUS gene and hygromycin phosphotransferase gene), and pCAMBIA3300 RG with Bar gene and snowdrop lectin (GNA) gene as donor DNA, the micro-adventitious shoots and the calli induced from mature embryos of Oryza sativa 87203, Eyi105, Shangnong aromatic glutinous rice as recipients were transformed with particle bombardment and Agrobacterium tumefaciens strain LBA4404 containing pAL4404, respectively. After chosen with phosphinothricin and antibiotic, GUS detection and PCR analysis, The results showed that the foreign genes had been transformed microprojectile-mediated to Oryza sativa Eyi105, the regeneration plants were obtained, and, 5 transgenic calli of Oryza sativa Eyi105 were obtained with Agrobacterium-mediated transformation.