Cloning And Sequence Analysis Of Adventitious Rooting Related Gene PSARRO-1 Of Tree Peony

A cDNA clone encoding a novel 2-oxoacid-dependent dioxygenase (adventitious rooting related oxygenase, ARR0-1) is up-regulated during the early phases of adventitious root formation in Malus domestica'Jork9'.Current date suggest ARRO-1 may prove a reliable molecular marker for adventitious root formation in woody plants. At present, Gene expression during to merisem formation in woody plants is poorly understood. In this study,PSARRO-1 gene was cloned by RT-PCR and RACE from the leaf and root of Paeonia suffruticosa. and its structure and function was analyzed by bioinformatics. This will help improve the use of molecular methods for the formation of peony root tissue culture and transgenic studies further Peony basis. The main results of this study were as follows:1. The optimizing of the extraction methods for total RNA from Paeonia suffruticosa. The roots of Paeonia suffruticosa'wu long peng sheng'was used to extract total RNA, and extracting effect of four methods, modified Trizol method, modified guanidinium thiocyanae method, modified CTAB method and Column Plant RNAout kit method was studied in this paper. The results showed that modified Trizol method or modified guanidinium thiocyanae method was not right for extracting the high-quality RNA from the root of Paeonia suffruticosa;The complete 28S rRNA, 18S rRNA and 5S rRNA bands could be seen clearly on the electrophoretogram of total root RNA extracted by modified CTAB method and Column Plant RNAout kit method, the brightness ratio of 28S rRNA and 18S rRNA were about 2:1; detection for concentration and purity showed that total RNA extracted by these two methods were higher purity. But the concentration of total RNA extracted by the modified CTAB method is as three times as that of the kit method. Considering total RNA purity and reagents cost, the modified CTAB method is more suitable for extracting total RNA from the root of Paeonia suffruticosa among four extracting methods.2. Cloning of the middle nucleotide sequence of PSARRO-1. With the conserved sequence of 2-oxoacid-dependent dioxygenase amino acid,we designed specific primers of TMW-1 and TMW-2,and cloned the middle nucleotide sequence of PSARRO-1 from the root of Paeonia suffruticosa'wu long peng sheng'.It was 300 bp in length and encoding 99 amino acids, GenBank accession number for the GQ330644. 3. Cloning of the3 'end of PSARRO-1 .On the basis of the middle segment,we designed specific primers of TMW-3, using TMW-3 and the anchor primer B26 for 3'-RACE PCR, approximately 328 bp DNA fragment was cloned. The sequence had high homology with other plant 2-oxoacid-dependent dioxygenase gene .and identified the position of termination codon of PSARRO -1.4. Cloning of the3 'end of PSARRO-1. With the basis of known sequence,we designed specific primers of TMW-4 and TMW-5, in accordance with the TaKaRa Biotechnology (Dalian) Co., Ltd. 5'-Full RACE Kit Kit for 5 'RACE-PCR. After nested amplification, by a treaty the size of 328 bp DNA fragment. Comparison of the sequence of Malus domestica, prunus domestica, Populus trichocarpa and other species of 2-oxoacid-dependent dioxygenase gene,they has high homology of 69%, 71% and 70%; and identified identified the position of initiation codon of PSARRO -1.5. Cloning of full-length nucleotide sequence of PSARRO-1.With the deduced full-length nucleotide sequence of PSARRO-1 gene of the peony,we we designed specific primers of PS-1 and PS-2 ,and cloned the full-length nucleotide sequence of PSARRO-1. It was 926 bp in length.6. Bioinformatics analysis of PSARRO-1.With the DNAman software,the nucleotides and amino acids sequence of PSARRO-1 were analyzed.The gene was 1135 bp in length, including 93 bp of 5'-non-coding region (UTR, untranslated region) and 145 bp of the 3'-non-coding region, 20 bp of the polyA tail and a 897 bp open reading frame (ORF, open reading frame), encoding 298 amino acids. Comparison of the PSARRO-1 amino acid sequences encoded by the 2-oxoacid-dependent dioxygenase genes of Arabidopsis thalian(AT2G25450), Populus trichocarpa(XP₀02298993),Malus domestica(AJ225045) and Prunus mume(BAE48659).The results showed that they have high homology of were 96%, 96%, 96% and 97%. Conserved domain analysis showed that there are existed a kind of domain:2OG-FeII_Oxy super family [cl01206].The phylogenetic trees were constructed according to the homologous analytic results,and it was found that the peony has the closest genetic relationship with the woody plant.