Molecular Cloning And Characterization Of SDD1 Gene Related To The Stomatal Density And Distribution Of Isatis Indigotica

SDD1 (Stomatal Density and Distribution), a member of subtilase superfamily, encodes a subtilisin-like serine protease which is one of the key components known to be involved in the regulatioin of plant stomatal development. The sdd1 mutant exhibits increase of stomatal density and formation of clustered stomata, whereas the internal leaf architecture is not altered. Arabidopsis thaliana whole genome Affymetrix gene chip revealed that the expression of SDD1 in autotetraploid Isatis indigotica Fort. (2n=28) was obviously higher than that in diploid progenitor (2n=14). It indicated tetraploid attain inhibitive expression of SDD1 after genome duplication. The result could probably effect the stomatal development and change the stomatal density which leads to the disparation of biomass and water use efficiency of the autotetraploid plant. And eventually it might cause increase of production and raise of drought tolerance.It is the first report that IiSDD1 gene (gene bank ID : DQ407741) was successfully coloned from diploid and autotetraploid I. indigotica by RACE (Rapid Amplification of cDNA Ends). The 5' and 3' flanking region of IiSDD1 were isolated by genome walking technique. Their bioinformation was basically analysed. Sequence alignment showed high homology (100%) of IiSDD1 cDNA and genomic DNA nucleotide sequence between diploid and tetraploid plants. The IiSDD1 had no intron. The predicted IiSDD1 polypeptide included the catalytic triad of aspartate, histidine and serine active sites that share the highly similar architecture with other subtilases.The expression profiles of IiSDD1 gene in different nutritional organs and various treatment were semi-quantified by RT-PCR. Its expression in roots of tetraploid was higher than that in stems and leaves, while the corresponding expression was the highest in diploid leaves then decreased progressively in stems and roots. The gene was characterized the negative feedback after induction by various outer stimuli such as ABA, GA3, darkness, drought, high salinity and low tempareture treatment, which had silencing tendency same with the multiploidy evolutionary process in tetraploidy plant.There was obvious different methylation degree in the promoter region of IiSDD1 in tetraploid and diploid plants. Epigenetic DNA methylation probably caused that the expression of IiSDD1 was significant higher in diploidy plant while lower in tetraploidy one. It might help to recognize IiSDD1 silencing in polyploidy plants and find the inner correlation with their good performance of higher yield and drought tolerance.