Cloning And Characterization Of Stress Resistance-related Genes IbPIF1?IbPIF3 And IbAKR From Sweetpotato(Ipomoea Batatas(L.)Lam.)

Sweetpotato is an important crop for food,feed,industrial raw material and bio-energy,but the yield and planting area are restricted by drought,salt,cold and disease.Overexpression of IbPIF1,IbPIF3 and IbAKR in sweetpotato or tobacco were conducted to analyzed tolerance mechanism of these genes.The main results are as follows:1.According to the differentially expressed EST sequence,the IbPIF1 gene was cloned by homologous cloning method.The gene ORF is 1605 bp and 534 amino acids are encoded.Genomic DNA is 5165 bp,containing 7 exons and 6 introns.qRT-PCR analysis showed that IbPIFl could be upregulated by drought,cold stress and MeJA.Overexpression of IbPIF1 could significantly improve the transgenic sweetpotato plants drought resistance,salt tolerance and sweetpotato Fusarium wilt resistance.Under PEG and NaCl stress,the content of proline in L1 and L1 lwas significantly higher than those of Shangshu 19,whereas the malondialdehyde(MDA)content was significantly lower.qRT-PCR analysis showed that overexpression of IbPIF1 up-regulated the genes involved in the ROS scavenging system,ABA synthesis pathway,protective protein,pathogen-related responsive protein.Yeast two hybrid showed that IbPIF1 could interact with serpin-like protein.It is speculated that the interaction may regulate the resistance to Fusarium oxysporum disease.Under cold stress at 4?,the cold tolerance of IbPIF1-overexpressing plants was significantly lower than that of wild type.qRT-PCR analysis showed that overexpression of IbPIFl down-regulated the genes involved in the CBF pathway.Yeast one hybrid and LUC transient expression assay showed that IbPIF1 could directly bind to the promoter region of CBF2 and negatively regulate the gene expression.Therefore,it is speculated that IbPIF1 directly binds to the CBF2 promoter region and negatively regulates the expression of CBF2 gene,and then inhibits the expression of the cold stress related gene in the downstream,and reduces the cold tolerance of the transgenic lines.2.According to the differentially expressed EST sequence,the IbPIF3 gene was cloned by homologous cloning method.The gene ORF is 2001 bp and 666 amino acids are encoded.Genomic DNA is 3099 bp,containing 6 exons and 5 introns.qRT-PCR analysis showed that IbPIF3 could be upregulated by drought,cold stress and MeJA.Overexpression of IbPIF3 can significantly improve the transgenic tobacco drought resistance and sweetpotato Fusarium wilt resistance.Under PEG stress,proline content was significantly increased,whereas MDA content was significantly decreased in the transgenic plants.qRT-PCR analysis showed that overexpression of IbPIF3 up-regulated the genes involved in the ROS scavenging system and pathogen-related responsive proteins.Yeast two hybrid analysis showed that IbPIF3 could interact with G-type lectin S-receptor-like serine/threonine protein,and it is speculated that the interaction may regulate the resistance to Fusarium oxysporum disease.3.According to the differentially expressed EST sequence,the IbAKR gene was cloned by RACE method.The gene ORF is 1053 bp and 350 amino acids are encoded.Genomic DNA is 3499 bp,containing 5 exons and 4 introns.qRT-PCR analysis showed that IbAKR could be upregulated by cadmium stress and H2O2.In Escherichia coli,IbAKR was found to be able to metabolize benzaldehyde in vitro,and the expression of IbAKR can significantly improve the cadmium stress tolerance of E.coli.Overexpression of IbAKR can significantly improve the transgenic tobacco cadmium resistance.Proline content and superoxide dismutase(SOD)were significantly increased,whereas contents of MDA and H2O2 were significantly decreased in the transgenic plants under cadmium stress.qRT-PCR analysis showed that overexpression of IbAKR up-regulated the genes involved in the ROS scavenging system under cadmium stress.