Although germinating seed of soybean has been successfully used as a target tissue in Agrobacterium-mediated transformation,it has not been investigated in particle bombardment-mediated transformation.In this study,factors such as bombardment times,bombardment distance,culture time,and placing orientation that affect particle bombardment-mediated transformation efficiency were studied via particle bombardment with a plant expression vector pCAMBIA1301 using 1 day-germinating half seeds of soybean as target tissue.The result showed that transformation efficiency based on GUS transient expression was significantly affected by bombardment parameters.The highest GUS transient expression(217.5 blue foci/half seed)was obtained when particle bombardment was carried out under the 2 times of bombardment,9 cm distance of bombardment,and tissue culture for 2 days after bombardment.However,the GUS transient expression was not affected by placing orientation of target tissue.The factors affecting the particle bombardment-mediated transformation of soybean germinating seeds were optimized by GUS transient expression assay.These results will provide useful information for establishment of a particle bombardment-mediated soybean transformation system.
Soybean's MADS-box genes are a large gene family involved in plant reproductive development. Among them, GmAGL1 plays an important role in the development of soybean flowers and seeds. However, the regulation mechanism of the gene expression is not completely clear. Our analysis of gene expression showed that GmAGL1 was highly expressed in flower organs, seeds and pods, weakly expressed in roots, stems and leaves. Sequence analysis of the promoter and the 3981 bp first intron of GmAGL1 isolated from soybean revealed that the promoter and the long fragment intron contains many important cis-regulatory elements. In order to investigate whether the large first intron of GmAGL1 has a regulatory function on its promoter, we constructed expression vectors that the GUS gene is driven by the GmAGL1 promoter alone and driven by the first intron in combination with the promoter, and transformed into tobacco genome. Histochemical and fluorescence analyses revealed that GUS gene driven by the GmAGL1 promoter were expressed disorderly in both vegetative and reproductive organs. However, GUS activity was significantly decreased in organs except calyx and flower stalk when the first intron was incorporated upstream or downstream of the promoter. Therefore, we believe that the large first intron of GmAGL1 contains important elements that inhibit the expression of downstream genes in tissues.
Objective:To enhance the efficiency of the multi-needle-assisted transformation of soybean(Glycine max L.Merr.).Methods:The cotyledonary node cells of half seeds germinated for 1 d were wounded by a multi-needle and inoculated with Agrobacterium tumefaciens LBA4404 harboring a vector pCAMBIA1201.The transformation efficiency was determined by the GUS activity.Results:Among wounding treatments,the highest transformation efficiency was obtained when wounding was made by puncturing 2 times with the multi-needle.The transformation efficiency was also significantly affected by genotypes,co-cultivation time and antioxidants.Conclusion:The efficiency of the multi-needle-assisted soybean transformation was increased.
We cloned a Nicotiana tomentosiformis PHLOEM PROTEIN 2-LIKE A9-like gene from the T-DNA insertion tobacco mutant, designated as NtPP2A9L1 . NtPP2A9L1 encodes a 168 amino acid and contains a conserved domain of phloem protein2 (PP2), which is the homologous gene of ArabidopsisAtPP2-A9 . NtPP2A9L1 was localized in the nucleus, cell membrane and cell wall, and the expression of NtPP2A9L1 was induced by NaCl, polyethylene glycol (PEG) 6000, cold (4℃) and abscisic acid (ABA). Overexpression of NtPP2A9L1 in transgenic tobacco significantly enhanced tolerance to salt, drought and cold. Under high salt, drought or cold treatments, NtPP2A9L1- overexpressed tobacco significantly increased the activities of antioxidant enzymes, contents of proline and chlorophyll, and decreased contents of malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ) compared with WT tobacco. Consistently, overexpression of NtPP2A9L1 significantly upregulated the transcript levels of reactive oxygen species (ROS) scavenging-related genes and stress response-related genes. However, the expression of NtPP2A9L1 was strongly inhibited in NtCBF S -overexpressing transgenic tobacco, suggesting that NtCBF S negatively regulates the expression of NtPP2A9L1 . These results suggest that the improvement of stress resistance of NtPP2A9L1 in transgenic tabacco was achieved through the expression of ROS scavenging-related genes and stress-related genes, rather than through the CBF signaling pathway.
In this study,the ERsHSP over expressing transgenic tomato plants were used to analyze the cold-resistance ability and the UPR under cold stress.Compared with non-transgenic and pROKⅡ-transformed tomato plants,transgenic tomato plants possessed higher cold-resistance ability which exhibited slighter cold-injured symptoms,less destruction of chlorophyll and electrolyte leakage,lower content of MDA and higher value of net photosynthetic rate.The results showed that the expression of BiP and Calnexin was up-regulated under cold stress.However,the mRNAs level of these two genes in transgenic tomato plants was dramatically lower than those in non-transgenic plants under the same stress conditions.In non-transgenic tomato plants,the induction of BiP and Calnexin expression peaked after cold stress treatment at 4°C for 72 h and declined thereafter.However,there was a steady and increasing expression of BiP and Calnexin in transgenic plants.On the whole,these data suggest that overexpression of ERsHSP in transgenic tomato plants could alleviate and keep the UPR under cold stress,thus reduce the ER stress induced by cold stress.
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