Cloning And Analysis Of Cytochrome P450 And Small G Protein Ppran Gene From P.patens

The moss (P. patens) is the only plant that has been reported to have high frequency of homologous recombination. With its accurate and predictable result, gene targeting became one of the normal technologies being used in genomics study. And the core technology of gene targeting is the homologous recombination. The moss entitles many characteristics such as shorten life cycle, easy culturing, small body size, easy transforming (protoplast transformation with PEG) and strong regeneration ability. These characteristics are very important for producing transgenic moss mutant lines with the high homologous recombination technique on a large scale. There is alternative of generations in the lifecycle of moss, with the gametophyte generation being dominant. This makes the moss an ideal system to construct mutant and to analyze the genetic characters. It is well known that the moss has been a model plant in functional genomics research. The moss gene cytochrome P450 and P. pRan were cloned and analyzed with the moss homologous recombination technique in this study.Cytochrome P450 is a family of hemachrome binding oxido-reductase with complex functions. In plant, cytochrome P450 participated in multiple metabolic process, not only in the primary one, as well as in the secondary one with more effects. Four cytochrome P450 genes of moss were cloned and their loss-of-function vectors were constructed successfully with homologous recombination technique. At the same time, the P450promoter::cDNA::GFP fusion protein vector was constructed for gene subcellular localization. After transformation with these vectors, some transgenic moss lines of CYP73A49 loss-of-function were obtained. However, these transgenic lines showed no distinct phenotypic variation. The further physiological and biochemical studies on these transgenic lines are still on going. The subcellular localization of P450promoter::cDNA::GFP fusion protein experiments are undertaking.Ran, distributing abundantly in the nucleus, is a small molecule GTPase that take an important role in the transport between cytoplasm and nucleus. In addition, Ran is able to regulate the cell division and be involved in signal transduction. In this paper, Genomic and cDNA sequences of PpRan were cloned and the conservation of the PpRan encoded a protein was analyzed by means of bioinformatics tools. Aside from this, gene expression of PpRan in different tissue and in the condition of phosphorus deficiency was studied by semi-quantitative PCR and PpRan showed a tissue-specific expression pattern with higher expressed level in protonema and cormophyte but lower in Rhizoids. The long time subjected to phosphorus deficiency, the more and more highly transcripted level of this gene was detected with semi-quantitative PCR.