Cloning And Functional Characterization Of Stress-Responsive Genes From Trifoliate Orange(Poncirus Trifoliata (L.) Raf.)

Citrus is one of the most important fruit trees in the southern part of China and possesses a pivotal position in the regional economy promotion. Though tremendous progress for the citrus industry has been achieved in recent decades, the developments of quality and yield are impeded by various stresses, such as drought, salt and low temperature, making stress tolerance one of the important objectives for citrus breeding. Due to polyembryony and sterility of reproductive organs, it is difficult for traditional breeding to obtain stress-tolerant germplasm, which can be complemented by gene engineering as an alternative. But so far, few stress-related genes were reported. ABA responsive element binding factor (ABF), Mitogen-activated protein kinase (MAPK), bHLH and ICE1are four well-known stress-related genes. In this study, all these three genes were cloned from Poncirus trifoliate and transferred into either tobacco or Arabidopsis for functional analysis to dissect the corresponding mechanism of stress tolerance and expand the pool of stress-related genes in Citrus. Main results were as follows:1. PtrABF was obtained via in silico cloning, containing an open reading frame (ORF) of1347bp and encoding448amino acids with protein molecular weight (Mw)48kD and isoelectric point (pI)9.66. Positioning in the nucleus, strong transcriptional activation and binding with ABRE, these data proved that PtrABF is a transcription factor (TF), which can be induced by dehydration, low temperature and ABA. PtrABF transgenic tobacco showed improved dehydration and drought tolerance. It may be due to lower accumulation of reactive oxygen species (ROS) since the expression of anti-oxidant enzymes in transgenic plants were higher, so were the activities. In additional,9stress-related genes were observed higher expression in transgenic tobacco before and after drought treatment.2. In silico cloning and5’-RACE were applied to obtain the full length of PtrMAPK with an ORF of1128bp, Mw43kD, pI5.51. Bioinformatics analysis revealed that PtrMAPK possesses11conserved kinase domains and a phosphorylation site. And subcellular localization analysis showed that it is situated in nucleus. The expression of PtrMAPK was induced by drought and low temperature but not salt. Overexpressed PtrMAPK tobacco showed elevated tolerance to dehydration and drought, lower water loss and ROS accumulation, higher anti-oxidant enzyme activities. Meanwhile, the expressions of ROS-scavenging and stress-related genes were higher in transgenic tobacco before and after drought stress.3. PtrbHLH was isolated using the same method as PtrMAPK, owning an ORF of1464bp, Mw53.6kD, pI5.30. It enjoyed high amino acid homology with bHLH of other species. The bHLH family of transcription factors is characterized by an acidic domain near the N terminus and a conserved near the C terminus. At the C terninus of the PtrbHLH there are one bHLH zipper domain and one SUMO conjugation motif. PtrHLH was localized in nuleus and has strong transcriptional activation which demonstrated that it is a TF. Its expression can be induced by low temperature, dehydration, salt and ABA.and the overexpression of PtrbHLH in tobacco resulted in cold tolerance. Overexpressing PtrbHLH in lemon enhanced its cold tolerance but the introduction of RNAi-PtrbHLH into Poncirus undermined its cold tolerance. Affymetrix chip data revealed that there were108up-regulated genes in the lemon overexpressing PtrbHLH, including POD gene which can scavenge H2O2. Further experiments using PtrbHLH-overexpressing transgenic materials (tobacco and lemon) showed that POD activities have been obviously elevated, so have the anti-oxidative ability. When challenged with POD inhibitor, the transgenic tobacco demonstrated lowered POD activity and cold tolerance. These results suggested that PtrbHLH may enhance cold tolerance via manipulating POD activity.4. PtrICE1was isolated same as PtrMAPK, owning an ORF of1680bp, Mw61kD, pI5.27. It enjoyed high amino acid homology with ICE1of other species. Two conserved bHLH motif and one SUMO binding domain were present in PtrICE1. Localized in nuleus, strong transcriptional activation and binding to cis element MYC demonstrated that it is a TF. Its expression can be induced by low temperature, dehydration, salt and ABA. And the overexpression of PtrICE1in tobacco resulted in cold tolerance.