The biotechnological application and limitation of IRES to deliver multiple defence genes to plant pathogens
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文摘
Transgenic resistance often has enhanced efficacy when more than one transgene is expressed. Here, we explore the co-delivery of multiple discrete effectors via a single transgene using an internal ribosome entry site (IRES) sequence. As an example, we report the co-delivery of two distinct proteinase inhibitors in Nicotiana tabacum var. Xanthi from a bicistronic plant mRNA to examine resistance against plant parasitic nematodes. A cysteine proteinase inhibitor, Oc-IΔD86, is translated in a normal cap-dependent manner while translation of the serine proteinase inhibitor, CpTI, from the bicistronic mRNA is IRES-mediated. ELISAs using antibodies confirm the expression of the two inhibitors in aerial and root material and suggest that IRES-mediated expression in the roots is lower than normal cap-dependent expression. Analysis of Globodera tabacum recovered from transgenic Nicotiana expressing two discrete proteinase inhibitors revealed appreciable levels of resistance of up to 51±3 % . Histochemical analysis of animals recovered from transgenic Nicotiana lines expressing Oc-IΔD86, via cap-dependent translation revealed a marked reduction in cysteine proteolytic activity in comparison to those from control untransformed plants. A less dramatic reduction was observed in similar analysis of serine proteolytic activity of animals recovered from transgenic Nicotiana lines expressing CpTI via IRES-mediated translation. The utility of using an IRES element to deliver a number of discrete anti-pathogen proteins is discussed.