Ekspresija gena za inhibitor serinskih proteinaza (BvSTI) šećerne repe (Beta vulgaris L.) i uloga u otpornosti na insekte
Expression of sugar beet (Beta vulgaris L.) serine proteinase inhibitor gene (BvSTI) and the role in insect resistance
Doctoral thesis (Published version)
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Plant proteinase inhibitor genes are among the prime candidates suitable for insect resistance improvement in plants. Expression pattern of a sugar beet serine proteinase inhibitor gene, BvSTI, was characterized in response to mechanical and fall armyworm (Spodoptera frugiperda J.E. Smith) induced wounding. BvSTI expression was analyzed in three breeding lines moderately resistant to sugar beet root maggot (Tetanops myopaeformis Roder.) and in a susceptible line, F1010. Increased mechanical wounding induced levels of BvSTI expression were observed in all resistant lines as compared to F1010. The most intensive response to wounding was observed in resistant lines, F1016 and UT-8, with a maximum up to 4- and 2,5-fold increase of BvSTI transcript levels over non-wounded roots and leaves, respectively. In contrast, slight increase of BvSTI transcript levels in leaves and even an initial decrease in roots were observed in sensitive F1010, but also in the third resistant line, F1015. BvSTI transcript accumulation in F1016 and F1010 tissues wounded by FAW showed a similar gene expression pattern, but it was delayed and less intense than the response incited by abiotic wounding. On the protein level, BvSTI-specific polyclonal antibodies confirmed increased accumulation of the 30 kDa BvSTI protein in wounded leaves but not in roots of F1016 and F1010. Using trypsin inhibition assays, the activity of BvSTI was confirmed in F1016 roots and leaves and F1010 leaves. In F1010 roots BvSTI activity was completely lacking. To confirm the potential role of the BvSTI gene in defending mechanisms to insect pests in sugar beet the same analyzed germplasm were bioassayed for resistance to fall armyworm insects. Larvae fed sugar beet leaves from all three resistant germplasms (F1016, F1015 and UT-8) had significant reductions in larval weights as compared to larvae fed on sensitive F1010 leaves. The observed daily weight increase was also the highest in larvae from sensitive vs. resistant leaves. As the larvae entered the pupal stage, pupal sizes did not reflect the overall larval weights and all developed pupae were similar. Larvae fed on roots were almost double lighter than larvae from leaves for all analyzed gemplasms. Some developmental abnormalities of the pupae fed on F1016 and F1015 leaves were noted. Since identification of gene promoters that are specifically activated in response to wounding or pest attack will facilitate regulated transgene expression in plants, we examined the induction pattern of a sugar beet promoter derived from the BvSTI proteinase inhibitor gene. BvSTI promoter was previously fused to the β-glucuronidase (GUS) reporter gene and transferred to Nicotiana benthamiana plants. GUS activity driven by BvSTI promoter was evaluated in independently derived BvSTI transgenic tobacco T2 homozygous progeny. Mechanical wounding and fall armyworm larval feeding induced GUS gene expression in tobacco leaves and roots that was localized at the wound site in mature tobacco tissues. Based on our results we can conclude that BvSTI gene expression was wound induced in the insect resistant germplasm suggesting that this gene can be used in biotechnological approaches or in breeding programs for improving insect resistance. Observation of GUS gene activity at the wound site in transgenic tobacco indicates that the BvSTI promoter is inducible in these tissues and, therefore, should prove useful for expressing resistance transgenes in leaves and roots as a first line of defense against plant pests and pathogens.
Keywords:Insect resistance; Serine proteinase inhibitor; BvSTI gene; Sugar beet; Inducible gene promoters; Transgenic tobacco; Spodoptera frugiperda
Source:University of Belgrade, Faculty of Biology, 2012, 1-157