Establishment Of A Multi-Transgene-Stacking System By Fused Genes And Transformed Into Yeast, Tobacco And Malus Robusta RHD.

Apple(Malus×domestica Borkh) is one of the most important fruit trees in the world and in China.However,drought,soil salinity and iron deficiency,which widely exist in many apple growth areas of China,adversely affect apple productivity and quality.To deal with the problem,enhancing cultivation management or breeding new cultivars high-resistant to abiotie stress are required.On the other hand,dwarf cultivation is easy for tree management and beneficial to early fruit setting,high productivity and good fruit quality.Therefore,it is important for apple cultivation to breed rootstocks with dwarf and high resistance to stress.Conventional apple breeding is constrained by their extensive reproductive cycle with long juvenile periods,complex reproductive biology,and high degree of heterozygosity.As alternative,genetic engineering offers the possibilities introduce new characteistics into existing commercial cultivars.However,until now all genetic modifications in apple are almost single gene transformation.Multiple genes transformation is necessary for improving integration characteristics of apple.In the foot-and-mouth disease virus(FMDV) and some other picomaviruses the oligopeptide(-20 amino acids) 2A region of the polyprotein mediates cleavage at its own C terminus to release it from the 2B region.Dissociation occurs at the carboxy terminus of the 2A sequence without any requirement for cytosolic factors.2A is functional when replacing its up- and down- stream genes and is also functional in plants.In this experiment,the genes encoding glycine betaine synthase were cloned from a halophyte,Suaeda salsa and fused in a single open reading frame.The fused genes were transformed to P.pastoris GS115,Nicotiana tabacum and Malus robusta,respectively,and the expression of the fused genes in P.pastoris and plants as well as the function of 2A were analyzed.The genes DREB(encoding dehydreation responsive element binding protein),IRTl(encoding iron-regulated cation transporter) and rolC(encoding cytokinin-β-glucosidase) were cloned from Oryza sativa,Lycopersicon esculentum and Agrobacterium rhizogenes,respectively. The trivalent gene Rirol was synthesized by fusing DREB,IRTl and rolC with two copies of 2A in a single open reading frame and transformed to Malus robusta mediated by Agrobacterium tumefaciens.The main results are presented as follows:1.According to the homologous sequence of encoding two enzymes of betaine synthesis in Suaeda liaotungensis,SsCMO and SsBADH were cloned by PCR in Suaeda salsa.The nucleotide sequences of SaCMO and SsBADH show 100%and 99%homolog with SICMO and SlBADH,respectively.The deduced amino acid sequence of SsCMO and SsBADH are identical with that of SlCMO and SlBADH,respectively.To construct SsCMO and SsBADH plant co-expression vector,the SsCMO,SsBADH genes were fused into a single open reading frame(ORF) with a copy of the FMDV 2A protein gene placed between the two genes.A transitional plasmid pBKSMC2AB was constructed by introducing 2A sequence,SsCMO,SsBADH to MCS of pBKSM(modified from pBluscript KS(+)) successively.2.The fused genes 'SsCMO-2A-SsBADH' was placed under the control of the alcohol oxidase(AOXl) promoter in pPIC3B and the vector was then linearized and transformed into P.pastoris GS115.The expression of the fused genes linked by the 2A sequence in P. pastoris and the ability of recombinant yeasts to tolerate environmental stress were studied. The results showed that the activities of enzymes CMO and BADH were 26.5 and 28 U·mg^-1 protein in recombinant yeasts,which were 45-fold and 44-fold higher than those in control GS115,respectively.SDS-PAGE analysis of proteins expressed in recombinant and control yeasts showed that compared with the control recombinant yeasts,A764,A765 expressed two additional proteins of 45KDa and 54KDa,the size of which matched the predicted size of CMO and BADH.The content of glycine betaine in recombinant yeasts was 28-to 34-fold higher than that in control GS115 induced with 0.5%methanol for 96 h. Recombinant yeasts were more resistant to salt,methanol and high temperature stress than control.It was concluded that the genes fused by FMDV 2A region were expressed in Pichia pastoris successfully and the polyprotein was 'cleaved' to each functional protein. The yeasts transformed the fused genes which were more resistant to salt,methanol and high temperature stress than control as result of glycine betaine synthesis genes introduced.3.The plant expression vector harboured the fused genes 'SsCMO-2A-SsBADH' under the control of the super promoter was constructed and transformed to tobacco and Malus robusta mediated by Agrobacterium tumefaciens.Transgenic Malus robusta was not obtained by transformation via hygromycin resistant selectable marker.Tobacco transformed with the fused genes increased the content of glycine betaine and was improved the tolerance to salt stress.4.DREB,IRT1 and rolC genes were fused with two copies of FMDV 2A region and transformed to Malus robusta mediated by Agrobacterium tumefaciens.Some kanamycin-resistant plants were obtained by selection.The fused genes were proved to integrate the genome of some Km-resistant plants of Malus robusta by GUS staining,PCR and Southern bolting.The transgenic plants showed higher resistance to salt stress, shortened internodes and more branching of shoots and roots.The study primarily proved that cotransforming multiple genes fused by FMDV 2A region may be a new way introducing multiple genes into fruit trees.