PpSUT2Sucrose Transporter Gene Cloning From Peach And Primary Investigation Of Its Fuction

The formation and improvement of fruit quality has become one ofcore research areas in pomology. The synthesis, transformation, transport andaccumulation of carbohydrate in fruit is an important factor to decide fruit quality.Existing researchs are mainly concentrated in the upstream area of metabolic chain,such as photosynthetic efficiency and photosynthate loading, but the studies on sugarunloading mechanism are fewer, especially carbohydrates in sink organs unloadingfrom the phloem and metabolic pathways and molecular mechanism is still unclear. Inorganisms, sucrose transporters are critical for fruit quality and yield formation, whichare responsible for mediating sucrose transport across the cell membrane. sucrosetransporters are akey link in the sugar signaling transduction pathway, which areregulated multiplely in the processes of synthesis, transportation, consumption andstorage of carbohydrate material. As the source of peach, it is very important inpeople’s lives and gardening industry in China. It is very significant for researchingthe form mechanism of fruits to search new genes of sucrose transporters from peachand elucidate the biological function. This paper first reported a new sucrosetransporters gene from peach. The gene expression pattern was analyzed and functionand of the gene, laid the foundation for further research on the gene.In this study, one sucrose transporter gene cDNA including open reading frame(ORF) was finished by silico and homology-based cloning techniques according ESTand cDNA data, and designed two special primers, i.e. Lx3STP1f2and Lx4STP1r2. Asucrose transporter gene analogue was isolated from the leaf and fruit cDNA ofPrunus Persica (L.) Bstsch by reverse transcriptase polymerase chain reaction(RT-PCR), a length of2kb around named PpSUT2. The sequence analysis revealedthat a full length of1980bp was obtained, which contains104bp5’ untranslatedregion,39bp3’ untranslated region, and an open reading frame (ORF) of1782bp.PpSUT2encodes594amino acid residues with the predicted molecular mass of63.41kD and pH7.41.PpSUT2contains conservative domain of sucrose transporters and the deduced amino acid sequence showed high identities with other sucrose transporters. Thenucleic acid is high similar with sucrose transporter genes from Fragaria x ananassa,Vitis vinifera, Eucommia ulmoides and Citrus sinensis (>80%). Phylogenetic treeanalysis indicated that PpSUT2is close to Fragaria x ananassa (FaSUT2). Theexpression characteristic of PpSUT2was investigated at transcriptional level byReal-Time PCR. The results showed that PpSUT2transcripts were present not only inpeach leaves and fruit, but aslo in petal, sepal and cortex, whereas mRNA levels weredifferent and changed with grow period.The full length PpSUT2cDNA was subcloned into the plant expression vectorpCAMBIA3301downstream of the CaMV35S promoter to construct over-expressionvector. The over-expression vector was introduced into Agrobacterium tumefaciensEHA105by Agrobacterium-mediated transformation, and then transformed into riceand tobacco successfully. Transgenic plants were verified by PCR. It was indicatedthat the PpSUT2gene had been recombined into rice and tobacco genomerespectively and T0Transgenic plants were obtained.The research for a few transgenic tobacco to check the adaptive ability and. Thestudy showed that the growth was no difference between transgenic tobacco and CK(WT) under normal conditions. But the transgenic tobacco lines showed moreadaptive ability than CK under chilling and drought stress. It was revealed that therate of water loss of excised-leaves of transgenic tobacco was significantly lower thanCK, which indicated over-expression of PpSUT2gene maybe enhance the adversityability, and suggested that PpSUT2may have sugar transfer function. Underenviromental stress (chilling and drought stress), the content of relative electricalconductivity and MDA in leaf of transgenic tobacco and CK showed a rising trend,which indicated that the increase of membrane permeability, electrolyte leakageincreased, and membrane lipid peroxidation enhanced. But the relative conductivityand MDA content of transgenic tobacco was lower than those in CK, which showedthat the level of membrane lipid peroxidation in transgenic PpSUT2tobacco waslower than CK, cell membrane damaged lighter. While proline and soluble sugarcontent in leaf was higher than CK, which indicated that over-expression of PpSUT2gene can enhance the plant stress resistance, and thus presumed PpSUT2may directlyor indirectly drive sucrose transport function.A prokaryotic expression vector pMALc2x-PpSUT2F was constructed accordingto the fragment of central cytoplasm loop and transformed into E.coli BL21. A fusion protein was seen about48.44kD, which laid the foundation for next step antibodypreparation.