Viktor Kuvshinov

Transgenic crop plants expressing synthetic cry9Aa gene are protected against insect damage

A synthetic gene sequence of cry9Aa was made to achieve high expression levels in a plant cell. Tobacco, potato, cauliflower and turnip rape plants were transformed with this synthetic gene driven by the double 35S promoter using Agrobacterium tumefaciens LBA4404. The presence and expression of the synthetic cry9Aa gene was evaluated in Southern, Northern and Western analysis and with insect bioassays. The expression of the gene in tobacco plants reached a level of 5 pg of mRNA per 1 µg of total RNA and 0.3% of soluble protein or 1.4 µg of Cry9Aa protein per 1 g of leaf material. The expression level in the other species was three to ten times lower. Tobacco plants were also transformed with a truncated native cry9Aa gene construct and with a translational fusion construct of the truncated native cry9Aa and the uidA (GUS) gene sequence. The constructs were transformed in tobacco plants under the control of the same promoter as the synthetic cry9Aa. The expression level of the native cry9Aa gene constructs ranged from 0.03 to 1 pg of cry9Aa mRNA per 1 µg of total RNA. The protein was undetectable in Western analysis. In comparison to the native constructs the expression level of the synthetic cry9Aa gene was five to ten times higher at the mRNA level and at least 50 times higher at the translational level. Bioassays against Plutella xylostella performed with transgenic cauliflower showed high insecticidal activity of the plants expressing the synthetic cry9Aa gene.

Cloning of new rubisco promoters from Brassica rapa and determination of their activity in stably transformed Brassica napus and Nicotiana tabacum plants

The aim of our study was to identify the highest expressing rubisco small subunit (RbcS) promoters (pRbcS) from the cotyledons of germinating seedlings of Brassica rapa var. oleifera to drive high-level and preferably stage-specific transgenic protein expression in Brassicaceae plants. We cloned four new pRbcS promoters using several approaches, including the construction of a cDNA library and use of genome walking technique. Real-time PCR analysis of RbcS mRNA expression clearly showed that two of these promoters exhibited the highest activity on the germination stage of plant development. We used gusA expression as a reporter of promoter activity in Brassica napus and Nicotiana tabacum plants that were transformed with the constructs using an Agrobacterium-mediated transformation strategy. The mRNA level of RbcS and of gusA was quantified in transformed plants. The data obtained demonstrate that the promoter most active in seedlings under native conditions was also most active in transgenic constructs at the same stage of plant development. The fine structure of the promoters is discussed herein.