Cloning And Expression Analysis Of PnsGA20ox1 Gene From Populus Simonii × P. Nigra

Gibberellis (GAs) consist of an important kind of plant growth regulators, which play important roles in many aspects of plant growth and development. Gibberellin 20-oxidase (GA20ox) is a key enzyme that normally catalyzes the penultimate steps in GA biosynthesis. To generate a good pulpwood with fast growth rate and long fiber length by moleculer breeding methods, we did some research about the GA20ox gene from Populus simonii×P. mgra.Using degenerate primers based on GA20ox gene secquences of different species, one novel fragment of GA20ox gene was obtained from Populus simonii×P. nigra. A 1678 bp cDNA sequence encoding gibberellin 20-oxidase was cloned by RACE method and named as PnsGA20oxl (JF767015), including 140 bp of the 5'untranslated region and 380 bp of the 3' untranslated region. The open reading frame (ORF) is 1158 bp in length, encoding a deduced amino acid sequence of 385 residues, with a molecular weight of 40.04 kDa and an isoelectric point of 5.97. Analysis of amino acid sequence showed high homology of PnsGA20oxl and GA20ox from other plants.Real-time fluorescent quantitative PCR was used to investigate the PnsGA20oxl gene expression pattern in different tissues. The results showed that the highest expression of PnsGA20oxl gene occured in mature xylem and strong expression was also found in tender leaves. The expression in mature leaf, apical meristem, inmature xylem and phloem was higher compared with petiole, while the lowest expression was found in internode.To determine the physiological function of PnsGA20oxl, we constructed the excessive expression vector of PnsGA20oxl and expressed this gene in Nicotiana benthamiana by the Agrobacterium mediated method. Fourteen transgenic lines were selected for the test of holocellulose content, lignin content and fiber length. The results showed that holocellulose content in all transgenic lines was higher than that in the wild tobacco, while there were no significant changes in the lignin content between the transgenic and wild tobaccos. Longer fiber length was also found in the transgenic lines.