Cloning And Functional Identification Of IbHSP90-6 And IbNHX2 Genes In Sweetpotato(Ipomoea Batatas(L.)Lam.)

Sweetpotato,Ipomoea batatas(L.)Lam.,is an important crop for food,feed,industrial materials and bio-energy resource.Its growth and productivity are seriously limited by abiotic stresses.In the present study,the differentially expressed ESTs were analyzed and IbHSP90-6 and IbNHX2 genes associated to abiotic stress tolerance were cloned from salt-tolerant line ND98 of sweetpotato.Their expression was analyzed by real-time quantative PCR(qRT-PCR)under different abiotic stresses.The functions of IbHSP90-6 and IbNHX2 genes were characterized by the Agrobacterium tumefaciens-mediated transformation system of sweetpotato.The main results are as follows:1.Based on the EST sequence selected from salt-stressed suppression subtractive hybridization(SSH)cDNA library constructed with a salt-tolerant line ND98,a HSP90 gene,named IbHSP90-6,was cloned from sweetpotato via rapid amplification of cDNA ends(RACE).The cDNA sequence of 2755 bp contained a 2385 bp open reading frame(ORF)encoding a 794 aa polypeptide with a molecular weight of 89.9 kDa and an isoelectric point(pi)of 5.31.IbHSP90-6 gene contained a conserved HATPase_c domain in N terminal,a Ribosomal protein S5 domain 2-type fold domain in C terminal and a Middle domain,a link between N terminal and terminal.The deduced amino acid sequence had 74.97%to 84.03%sequence identity to HSP90 proteins reported in Arabidopsis thaliana,Populus euphratica and Nicotiana sylvestris.The genomic DNA of IbHSP90-6 contained 20 exons and 19 introns.qRT-PCR analysis revealed that the expression of IbHSP90-6 gene was significantly induced by salt,drought,heat,cold and ABA stresses.The subcellular location result indicated that IbHSP90-6 was located in cell wall and mitochondria.ND98 plants overexpressing IbHSP90-6 gene were developed with A.tumefaciens-mediated transformation.The IbHSP90-6-overexpressing sweetpotato plants exhibited significantly higher salt,drought and heat tolerance compared with the wild-type(WT).Proline content and superoxide dismutase(SOD)and photosynthetic activities were significantly increased,whereas malonaldehyde(MDA)content was significantly decreased in the transgenic plants.H2O2 was also found to be significantly less accumulated in the transgenic plants than in WT.Overexpression of IbHSP90-6 up-regulated P5CS and P5CR genes and genes involved in photosynthesis,reactive oxygen species(ROS)-scavenging system and ABA signalling pathway under salt,drought and heat stresses.These findings suggest that overexpression of IbHSP90-6 increases proline accumulation,which enhances salt,drought and heat tolerance of the transgenic sweetpotato plants by regulating osmotic balance,protecting membrane integrity and photosynthesis and activating ROS scavenging system.2.A vacuolar Na+/H+antiporter gene,named IbNHX2,was isolated and characterized from ND98.The cDNA sequence of 2122 bp contained an 1629 bp ORF encoding a 542 aa polypeptide with a molecular weight of 59.8 kDa and an isoelectric point(pI)of 6.73.The deduced amino acid sequence had a conserved binding domain 'FFIYLLPPI' for amiloride and a cation/H+exchanger domain,and shared a high amino acid identity(73.72?96.13%)with the identified vacuolar Na+/H+antiporters in other plant species.The genomic DNA of IbNHX2 contained 14 exons and 13 introns.Expression of IbNHX2 was induced by ABA,NaCl,polyethylene glycol(PEG)and cold temperature.The subcellular location results indicated that IbNHX2 was located in tonoplast.Its overexpression significantly enhanced salt,drought and cold tolerance in the transgenic sweetpotato.The significant increase of proline content and SOD and photosynthesis activities and significant reduction of MDA and H2O2 content were found in the transgenic sweetpotato plants.Up-regulation of P5CS gene and genes involved in photosynthesis,ROS-scavenging system and ABA signalling pathway was found under salt,drought and cold stresses.The overall results demonstrate that overexpression of IbHSP90-6 increases proline accumulation,which enhances salt,drought and cold tolerance of the transgenic sweetpotato plants by regulating osmotic balance,protecting membrane integrity and photosynthesis and activating ROS scavenging system.