Cloning And Functional Analysis Of PtNINV, A Neutral Invertase, In Poncirus Trifoliata

Cold strsee imposes great economic loss to the citrus industry. Therefore, it is of great importance to improving the ability of citrus chilling resistance. Transgenic technology is an alternative to traditional breeding to generate low temperature(LT) tolerant cultivars, but there must be available candidate genes. In our previous work, a LT cDNA library of Poncirus trifoliata was constructed and some LT responsive genes including a neutral invertase(PtrNINV) were screened out. In this work, the full-length of PtrNINV was cloned and characterized. The expressions under various treatments (LT, NaCl, ABA, dehydration, sucrose and glucose) were analyzed with Real Time PCR. Overexpression vector was constructed and it was introduced into tobacco via Agrobacterium-mediated transformation. The main results are as follows:1. PtrNINV gene was cloned using P. Trifoliata cDNA as template, containing an open reading frame (ORF) of2037bp and encoding678amino acids. Phylogenetic analysis revealed that it was closer to the neutral invertase in Daucus carota. Subcellular localization prediction showed that it may be localized in mitochondria.2. The expression patterns of PtrNINV under different treatments (high salt, dehydration, low temperature, ABA, sucrose and glucose) were analyzed via Real time PCR. The results revealed that PtrNINV was responsive to all the treatments. However, the expression was repressed at the beginning and then increased gradually under drought, NaCl and glucose treatments. But under4℃, ABA and sucrose treatments, PtrNINV exhibited opposite expression pattern as the previous3treatments. Comparing the expression patterns among different treatments, PtrN-INV was more responsive to low temperature and NaCl.3. Binary vector of PtrNINV-pBI121was constructed and transformed into tobacco with via Agrobacterium-mediated transformation. A total of56transgenic lines were identified by PCR. Four overexpression lines were obtained:7#,8#,22#,39#. The T1generation of three transgenic lines7#,22#,39#and the wild type (WT) were treated with500mmol/L NaCl. Under salt stress, transgenic lines showed less yellowing leaves than the wild-type, and lower malondialdehyde (MDA) accumulation, higher chlorophyll content. When the overexpression lines and wild type were exposed to low temperature, the transgenic lines exhibited better cold tolerance. Trypan blue staining revealed that less cell death was observed in the transgenic plant. The transgenic lines accumulated more reducing sugar and had higher NINV activity.