The rice annexin gene OsAnn5 is a positive regulator of cold stress tolerance at the seedling stage

Annexins exist widely in plants as multigene families and play critical roles in stress responses and a range of cellular processes. In this study, we report on the cloning and functional characterization of the rice annexin gene OsAnn5. We found that the expression of OsAnn5 was induced by cold stress treatment at the seedling stage of rice. GUS staining assay indicated that the expression of OsAnn5 was non tissue-specific and was detected in almost all rice tissues. Subcellular localization indicated that OsAnn5-GFP (green fluorescent protein) signals were found in the endoplasmic reticulum apparatus. Compared with wild type rice, overexpression of OsAnn5 significantly increased survival rates at the seedling stage under cold stress, while knocking out OsAnn5 using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated proteins) mediated genome editing resulted in sensitivity to cold treatments. These results indicate that OsAnn5 is a positive regulator of cold stress tolerance at the seedling stage.

Agrobacterium tumefaciens -mediated transformation of tomato (Solanum lycopersicum)

The present investigation describes the development of genetically engineered tomato plants with annexin gene. The alkaline lysis method is used to isolate the plasmid DNA of pUC 19 vector having the desired Annexin gene (3Kbp) and the plasmid DNA of the binary vector pGPTV (13 Kbp) from E.coli (DH5α strain). The purified pUC 19/Annexin and pGPTV plasmid were restriction digested using the restriction enzymes EcoRI and XbaI as a linearised band was eluted from the gel. The digested plasmid shown in the band pattern in the gel were cut by gel elution technique and purified from other reaction mixture. Then the dot spot test was done to calculate the concentration of pGPTV and Annexin gene. The recombinant PGPTV plasmid with the annexin gene in Agrobacterium tumefaciens MTCC 431 was mobilized and transferred to plant system through the mobilization helper plasmid pRK2013. The kanamycin resistance gene (NPT II) was used as a selective marker. The calli used for isolating the genomic DNA which was then amplified for confirmation of annexin gene. The nptII gene of 800 bp serves as a selectable marker system in plants and its amplification confirmed the presence of annexin gene in transgenic plants by PCR method.

Agrobacterium tumefaciens -mediated transformation of tobacco (Nicotiana tabaccum)

The present study involves the development of genetically engineered tobacco plants with annexin gene. The plasmid DNA of pUC 19 vector having the desired Annexin gene (3Kbp) and the plasmid DNA of the binary vector pGPTV (13 Kbp) were isolated from E.coli (DH5α strain) by alkaline lysis method. The purified pUC 19/Annexin and pGPTV plasmid were restriction digested using the restriction enzymes EcoRI and XbaI as a linearised band was eluted from the gel. The digested plasmid shown in the band pattern in the gel were cut by gel elution technique and purified from other reaction mixture. Then the dot spot test was done to calculate the concentration of pGPTV and Annexin gene. The recombinant PGPTV plasmid with the annexin gene in Agrobacterium tumefaciens MTCC 431 was mobilized and transferred to plant system through the mobilization helper plasmid pRK2013. The kanamycin resistance gene (NPT II) was used as a selective marker. The calli used for isolating the genomic DNA which was then amplified for confirmation of annexin gene. The nptII gene of 800 bp serves as a selectable marker system in plants and its amplification confirmed the presence of annexin gene in transgenic plants by PCR method.