انتقال سازه بیانی pCaPR10 حامل پروموتر القائی ژن PR-10 نخود به سویه LBA4404 اگروباکتریوم و تایید مولکولی آن

Chickpea (Cicer aritinum L.) is one of the most important legumes in the world which has been produced and consumed in many countries since long. It is also the third widely cultivated legume in the world after bean and pea. Unfortunately, the yield of this crop is very low compared to the surface of the planted area. Ascochyta blight caused by the fungus Ascochyta rabiei (telomorph: Mycosphaerella rabiei) is the most important disease that reduces the yield of this crop. The current strategy to control this disease is the cultivation of resistant varieties containing PR proteins. The PR-10 protein plays a crucial role on the growth and adaptation of chickpea in environment. The PR-10 gene can be traced in this plant so that the promoter sequence of this gene can quickly be induced in response to the pathogens similar to the other PR proteins. Isolation and analysis of the promoter sequence belong to the pathogenesis-related genes can be very beneficial in the meaningful expression of resistance gene. In the present study, in order to prepare the Agrobacterium strains used in promoter analysis of chickpea PR-10 gene, the expression vector pCaPR10 carrying this promoter (which had already been constructed in the preliminary studies) along with the following vectors pCAMBIA3301 and pBI121 as control, were transferred to the LBA4404 strain using heat shock method. The transformed colonies were confirmed by colony PCR method using PSh3-F/R and PKa-F/4R primers. As expected, three bands related to the PCR products of 1212, 1021 and 600 with the size of 1000, 1000 and 500 were observed on agarose gel. The prokaryotic expression pattern of reporter gene under control of this promoter in confirmed strains was studied in different colonies. The results showed only the expression of GUS reporter gene in the colonies harboring pBI121 vector. No prokaryotic expression was observed in the colonies containing pCAMBIA3301 vector or pCaPR10 expression construct either. These results indicated the absence of prokaryotic expression in the intron-GUS reporter gene.