Heterologous Expression of a Recombinant Chitinase from Streptomyces olivaceoviridis ATCC 11238 in Transgenic Pea (Pisum sativum L.)

I Abstract Heterologous Expression of a Recombinant Chitinase from Streptomyces olivaceoviridis ATCC 11238 in Transgenic Pea (Pisum sativum L.) Fathi Hassan Pea is an important grain legume that gained worldwide economic importance as a source of protein (15.5-39.7 %) for human and animal nutrition. The world pea production exceeded 20 million ton in 2005. In addition, it is well suited as a rotation crop to replenish soil nitrogen levels. Improvement of the resistance to fungal diseases is a major objective in breeding, since fungal diseases can cause in pea a considerable loss to more than 30 %. The aim of the present study was to enhance the fungal resistance in pea through the heterologous expression of a chitinase gene (Chit30) from Streptomyces olivaceoviridis ATCC11238. Chit30 belongs to family 19 chitinases and can hydrolyze chitin, the backbone of the fungal cell wall. Therefore, the bacterial signal peptide was replaced by the signal sequence of an Arabidopsis thaliana basic endochitinase gene, which was Nterminally fused to the mature bacterial gene. This chimeric chitinase gene (N-Chit30) was cloned via PCR based method into the pGreenII binary basis vector 0229, which contains the selectable marker gene bar under the control of a nos-promoter and nos-terminator. The gene was regulated either by a constitutive double 35S promoter from cauliflower mosaic virus or by the plant inducible vst promoter from grape and a 35S terminator. The bar gene encodes the enzyme phosphinothricin acetyltransferase (PAT), which confers resistance to transgenic plants against phosphinotricin, the active compound of the total herbicide BASTA. The chitinase and bar genes were arranged divergently in the binary vector. Leaf disk explants from in vitro growing tobacco plants cv. Samsun and embryo axis excised from mature seeds of pea (Pisum sativum L.) cv. Sponsor were used as explants for Agrobacterium-mediated transformation. Different shoots were regenerated after transformation of tobacco and pea, which were healthy growing on media supplemented with PPT concentrations up to 15 mg/l. Shoots from tobacco were rooted and potted into greenhouse, whereas regenerated shoots from pea were grafted to recover whole plants which in turn produced T1 progeny. The total procedure from seed to seed was between 68 months until getting transgenic pea seeds with transformation efficiencies varying between 0.31 % to 1.4 % with an average of 0.9 %. Transgene detection was made by PCR using different primer combinations (chit 555, andHeterologous Expression of a Recombinant Chitinase from Streptomyces olivaceoviridis ATCC 11238 in Transgenic Pea (Pisum sativum L.) Fathi Hassan Pea is an important grain legume that gained worldwide economic importance as a source of protein (15.5-39.7 %) for human and animal nutrition. The world pea production exceeded 20 million ton in 2005. In addition, it is well suited as a rotation crop to replenish soil nitrogen levels. Improvement of the resistance to fungal diseases is a major objective in breeding, since fungal diseases can cause in pea a considerable loss to more than 30 %. The aim of the present study was to enhance the fungal resistance in pea through the heterologous expression of a chitinase gene (Chit30) from Streptomyces olivaceoviridis ATCC11238. Chit30 belongs to family 19 chitinases and can hydrolyze chitin, the backbone of the fungal cell wall. Therefore, the bacterial signal peptide was replaced by the signal sequence of an Arabidopsis thaliana basic endochitinase gene, which was Nterminally fused to the mature bacterial gene. This chimeric chitinase gene (N-Chit30) was cloned via PCR based method into the pGreenII binary basis vector 0229, which contains the selectable marker gene bar under the control of a nos-promoter and nos-terminator. The gene was regulated either by a constitutive double 35S promoter from cauliflower mosaic virus or by the plant inducible vst promoter from grape and a 35S terminator. The bar gene encodes the enzyme phosphinothricin acetyltransferase (PAT), which confers resistance to transgenic plants against phosphinotricin, the active compound of the total herbicide BASTA. The chitinase and bar genes were arranged divergently in the binary vector. Leaf disk explants from in vitro growing tobacco plants cv. Samsun and embryo axis excised from mature seeds of pea (Pisum sativum L.) cv. Sponsor were used as explants for Agrobacterium-mediated transformation. Different shoots were regenerated after transformation of tobacco and pea, which were healthy growing on media supplemented with PPT concentrations up to 15 mg/l. Shoots from tobacco were rooted and potted into greenhouse, whereas regenerated shoots from pea were grafted to recover whole plants which in turn produced T1 progeny. The total procedure from seed to seed was between 68 months until getting transgenic pea seeds with transformation efficiencies varying between 0.31 % to 1.4 % with an average of 0.9 %. Transgene detection was made by PCR using different primer combinations (chit 555, and