Regulation Of Vacuolar Invertase In Arabidopsis Stomatal Movement And Its Effect On Drought Resistance

Stomatal movement plays an important role in plant drought resistance. Stomatal aperture was affected by endogenous concentration of Abscisic acid (ABA) and osmotic pressure in guard cells. Sucrose breaks down into glucose and fructose catalyzed by invertase. Therefore, change of invertase activity might play an important role in osmotic pressure and stomatal aperture, which could adjust stomatal movement in plant. Tobacco vacuolar invertase inhibitor gene Nt-inhh and vacuole specific promoter AtRabl8 which is sensitive to ABA were cloned to build the binary vector. Using a tissue or specific and inducible promoter AtRabl8 is helpful to generate a transgenic plant whose normal growth and development is not affected. The aim of this research is to study the effects of vacuolar invertase activity of the transgenic A. thaliana plants on drought resistance. Results of the present study were followed below:Genomic DNA was extracted from A. thaliana. rosette leaf to amplify AtRab18 promoter. The amplified DNA fragments (1600bp and 820bp) were then ligated into pBI101-1 to construct plant expression vector pBI101-1-AtRab-GUS.To gain tobacco vacuolar invertase gene(Nt-inhh), RNA was extracted from tobacco leaf to transcript and amplify the full length cDNA (529bp). The amplified Nt-inhh cDNA fragment was then ligated into plant expression vector pBI101-1-AtRab-GUS by restriction enzyme cutting site SalI/XbaI and the GUS reporter gene was replaced to construct binary vector, pBI101-1-AtRab-Nt-inhh.The plant expression vector of pBI101-1-AtRab-GUS and pBI101-1-AtRab-Nt-inhh wer e then transformed A. thaliana mediated by Agrobacterium. The transgenic plants transformed by pBI101-1-AtRab-GUS and pBI101-1-AtRab-Nt-inhh was obtained by Kanamycin screening followed by confirming by genomic PCR reaction. Semi-quantitative and quantity PCR examination was performed to detect foreign gene expression in transgenic A. thaliana.It was reported that AtRabl8 is an ABA sensitive and guard cell-specific promoter. Histochemical GUS staining analysis of the transgenic plants with pBI101-1-AtRab-GUS vector showed that the GUS signal was sensitive to exogenous ABA application and specifically located in guard cells, indicating that the promoter region amplified in the present experiments is long enough.Stomatal aperture and vacuole invertase activity of transgenic Arabidopsis plants expressing Nt-inhh driven by AtRabl8 promoter were both lower than that of the wild-type plants.Water loss of detached leaf, relative water content of rosette leaf and growing statues after drought treatment were determined in transgenic Arabidopsis plants expressing Nt-inhh driven by AtRab promoter and wild type plants. The present results showed that transgenic pants confers drought resistance.With all the aforementioned data, the present study elucidated the relationship between plant vacular invertase activity and drought resistance. Decline of vacuolar invertase activity resulted in a relative small stomatal aperture, which is sensitive to endogenous ABA concentration. And the ability of drought resistance of transgenic Arabidopsis enhanced.