Genetic Engineering and Gene Transformation
Mohamad Amin Neycee; Motahhareh Mohsenpour; Hassan Rahnama
Abstract
Safflower with low oleic acid content is one of the native plants of Iran. Generally,, high oleic acid oils have more oxidative stability than the oils with high linoleic acids . Genome editing technology enable us to obtain oilseeds with high oleic acid. In this research, two guide RNA sequences ...
Read More
Safflower with low oleic acid content is one of the native plants of Iran. Generally,, high oleic acid oils have more oxidative stability than the oils with high linoleic acids . Genome editing technology enable us to obtain oilseeds with high oleic acid. In this research, two guide RNA sequences were designed to target of Fatty Acid Desaturase 2 (FAD2-1) gene, which were located within the coding region and at a distance of 640 base pairs from each other. The guide sequences along with the codon optimized Cas9 gene were cloned in the T-DNA region of the Agrobacterium construct and transferred to the safflower by the In-planta method. The resulting seeds were cultivated and the plants were screened to track changes in the fatty acid profile of the seeds. The results showed that the amount of oleic acid in the seeds of one of the lines reached 53.14% on average. This line had four amino acid changes (L66F, N204D, S236A and I238V) at the same time. This is while the amount of oleic acid in the control plant was measured as 11.62% on average. The results showed that in the segregating generation, the change in fatty acid profile occurred in the line with homozygous amino acid change, and the heterozygous plants have the same oil profile as the control plants. Also, the results of this research can indicate the possibility of increasing the amount of oleic acid in oilseeds by changing the FAD2 enzyme sequence and without gene knockout.
Genetic Engineering and Gene Transformation
Nahid Ahmadi; Hasan Rahnama
Volume 3, Issue 4 , September 2013, , Pages 77-85
Abstract
Transient expression of foreign genes in plant tissues is a valuable tool for plant biotechnology and to shorten the time for gene functional analysis. Transient expression is a fast, flexible, simple and easy method that could be used in fully differentiated plant tissues. 2A11 promoter is a tomato ...
Read More
Transient expression of foreign genes in plant tissues is a valuable tool for plant biotechnology and to shorten the time for gene functional analysis. Transient expression is a fast, flexible, simple and easy method that could be used in fully differentiated plant tissues. 2A11 promoter is a tomato fruit specific promoter whose expression occurs in fruit significantly. Cloning of fruit-specific promoter (2A11) is the main purpose of this study. 2A11 promoters were amplified from genomic DNA of tomato by PCR using specific primers. The promoter fragments were cloned into cloning vector PTZ57R/T. Recombinant plasmids were transferred into E. coli XLI-blue strain. Fragments of the interest were digested using restriction enzymes BamH1 and HindIII and were then purified and substituted in CaMV35S promoter in binary pBI121. Binary pBI121 was selected for cloning of 2A11 promoters as it is an ideal vector for agroinfiltration due to the presence of the CaMV35S promoter and the GUS reporter gene within its T-DNA region. Recombinant plasmids were transformed into Agrobacterium tumefaciens LBA4404 strain with freeze and thawing method. The expression of GUS gene was analyzed in tomato with agroinfiltration method. The results showed that, 2A11 promoter was found as efficient as CaMV35S promoter in expression of GUS gene specifically in tomato fruit. Then, we can use this promoter for tissue specific expression of recombinant proteins in tomato fruits.
