Cloning And Expression Analysis Of PPF-1 Homologue In Poncirus Trifoliata (L.) Raf.

Citrus is one of the most important cultivating fruits in China. Now, one of the main studies of citrus breed is to study resistant breeds on Poncirus trifoliatha (L.) Raf. PPF-1 has been showed to involve in the regulation of flowering, senescence and responses to stresses. In the present study, we cloned the homologue gene of PPF-1 and primarily analyzed the expression pattern in Poncirus trifoliate, establishing theory foundation of breeding well citrus species.Primers used for RT-PCR and 3'-RACE were designed according to conserved sequences of Pea PPF-1 and Arabidopsis ALBINO3 cDNA sequences. A cDNA sequence from P. trifoliate was designated as PtPPF-1. The nucleotide sequence of PtPPF-1 was 1493 bp and encoded a protein of 452 amino acids with a logical molecular weight of 125.1 kDa and an isoelectric point of 4.92. There was a chloroplast localization signal domain at the leading side and five inner transmembrane domains in the protein of PtPPF-1.PtPPF-1 was high sequence similar to PPF-lof Pea, ALBINO3 of Arabidopsis and PPF-1 of Oryza, and was lower sequence similar to some putative proteins from Chlamydomonas, Gonium, Bigelowiella.Phylogenetic analysis showed that PtPPF-1 has highest homology to pea PPF-1, secondly Arabidopsis ALBINOS and then Oryza PPF-1, indicating little variation of PPF-1 genes in the evolution.RT-PCR revealed the higher expression level of PtPPF-1 both in leaves and in apical buds but almost non-detectable level in roots of P. trifoliata. As to the expression level of PtPPF-1 in Citrus tangerine Hort.ex Tanaka, it were on differences between that in leaves and in stems, both were lower than that in apical buds, and non-visible expression in roots. We presumed that the chloroplast localization signal domain maybe contributed to the PtPPF-1 expression.The expression of PtPPF-1 was also studied in P. trifoliata seedlings, which were treated by phytohormone (GA₃, IAA, ABA, KT) . The results indicated that treatments (GA₃, IAA, ABA) increased the expression of PtPPF-1 in leaves, and that of KT decreased the expression. There were different mechanisms which acted on PtPPF-1 between GA₃, IAA, ABA, and KT. After abiotic stresses treatment (cold, drought, salt) , the expression of PtPPF-1 increased obviously. Activities of SOD, POD maintained high level and that of CAT was low level when stress treatments (cold, drought, salt) began. The content of proline kept increasing during the three treatments. The trends of PtPPF-1 expression, activities of SOD, POD and the content of proline were similar in the treatments, especially cold treatment. And we proposed that there is some correlation between PtPPF-1 expression and SOD, POD, proline, and PtPPF-1 could act as an important symbol of stresses, especially cold stress.