Study On The Expression Of H5HA And SBE-RNAi Recombinant Genes In Potato

The H5HA-encoding cDNA fragment of the avian influenza virus subtype H5N1 gene was fused with different promoters and the recombinant genes were transformed into a potato cultivar(cv.Desiree)by using Agrobacterium-mediated transformation.The transgenic plants were obtained and analyzed.In addition,in order to increase of potato amylose content by RNA interference of the starch branching enzyme ?(SBEI)and ?(SBEII),three SBE-RNA interference plasmids were constructed and transformed into potato.The level of silencing of SBE? and SBE? in transgenic plants was investigated by analysis of apparent amylose content.The results are followings:1.The H5HA fragment of H5N1 gene was,respectively,fused to the promoter fragments of potato granule-bound starch synthase I(GBSSI)and sweet potato Sporamin A(SpoA)genes.The recombinant genes were cloned into the plant expression plasmid pBIN19 to give the pBIN19/Gp-H5HA and pBIN19/Sp-H5HA plasmids.After transformation,the transgenic plants were obtained and analyzed by PCR,Western blotting and Western dot blotting.The results were compared with the expression of the CaMV35S-H5HA recombinant gene in potato.Our results showed that three recombinant proteins can be accumulated in potato tubers.The highest level of expression was the H5HA fused to GBSS ? promoter,and the lowest one was the gene fused with the Spo A pomoter.2.To develop transgenic potato plants with high-amylose starch in tubers,the endogenesis genes SBE? and SBE? were,single or simutaneously,targeted for RNA mediated gene silencing by expression of RNA interference genes in potato.The Oligo Walk module in RNA Structure 3.2 was used to find RNA oligonucleotides that could be predicted to bind with high affinity to folded target mRNA of little self-structure.In this study,three RNA interference plasmids pBIN20/SBE? F-R,pBIN20/SBE? F-R and pBN20/SBE?/? F-SBE?/? R were constructed and transformed into a potato cultivar Desiree.Three transformed series were obtained and the granule morphology,apparent amylose content,and gelatinization characteristics of the transgenic starches were analyzed.The results showed that silencing of SBEI or SBEII alone has yielded a very low frequency of high-amylose lines,and even a reduction in amylose content in most of transgenic starches.However,production of high-amylose potato lines were be achieved by simultaneous inhibition of SBE? and SBE?.The plasmid pBIN20/SBE?/? F-SBE?/?R showed to be efficient,yielding high-amylose quality in more than 50%of the transgenic lines.One line from the Ri-SBE?+? series contained higher amylose(49.1%)than the untransformed starch ones(28.5%),and enthalpy relesed of the transgenic starchgel(200.95 J/g)was much lower than the control(700.86 J/g).Furthermore,when the apparent amylose content was increase,the morphology of the transgenic granules appeared very different from being oval and regular to being irregular in shape,and with a fissure towards the surface or interior of the granules.Specially,the starch granules from the Ri-SBE?+?-53 line showed as if a large portion of the granules was built from two or more small ones,suggesting that the expression of SBE-RNA interference genes could affect granule morphology.