Vector Construction Of OsPPDK Gene In Rice And Study On Its Correlation With Stay-Green Characters

Stay-green property refers to the characteristics of leaf senescence or yellowing retarded while maintaining the green color in the late stage of plant growth.It is closely related to plant anti-aging,antibiotic and abiotic stress,and increased yield.It is considered to be the ideal agronomic trait of the crop.The research and utilization of functional stay-green genes is a viable technical route to resolve the premature senescence of hybrid rice and break through the existing rice productivity.In this study,the rice OsPPDK gene was cloned and analyzed using bioinformatics,genetic engineering and molecular biology methods and using premature senescence indica rice Jin23B and stay green indica rice R287 as materials.The expression of OsPPDK gene was analyzed by real-time fluorescence quantitative PCR in the late growth stage of rice,and the expression of OsPPDK gene at different sites under different nitrogen concentrations was determined.At the same time,the ds1301-OsPPDK RNAi vector and pU1300-OsPPDKover-expression vector were successfully constructed,and the constructed expression vector was transferred to the premature senescence indica rice Jin23B and the stay green indica rice R287,and the expression of the OsPPDK gene in the transgenic rice plants was analyzed.The experimental results obtained are as follows:1.Designing primers of the TaNAM gene which find from NCBI database,rice OsPPDK sequences were obtained by PCR amplification of rice Jin 23B cDNA.The length of the target fragment amplified was 2844 bp.Bioinformatic analysis of the sequence revealed that the rice OsPPDK gene encodes 947 amino acids.The translated protein is a stable acidic hydrophilic protein located in the cytoplasm and its protein secondary structure is mainly composed of four structures:?-helix,?-turn,random curl,and extension chain.Among them,the highest percentage of helix is 41.29%,followed by irregular curl,accounting for 29.67%;The proportions of the ?-turn and the extended chain were 11.4%and 17.63%respectively.And the protein has no transmembrane structure and no signal peptide.2.Quantitative PCR was used to analyze the expression levels of OsPPDK in different tissue parts(leaves and leaf sheaths)at the heading stage,filling stage,milk ripening stage and ripening stage in Jin 23B and R287 rice.It was found that OsPPDK is expressed in four stages of Jin 23B and R287.In leaves and leaf sheaths,the expression levels at heading stage and filling stage were low,and the expression levels were not significantly different.However,the expression level was relatively high in the milk ripening stage and the ripening stage,and there was a big difference in expression.During the milk ripening stage,the expression level of OsPPDK in R287 leaves was 2.32 times that of Jin 23B,and 1.48 times that in leaf sheath.In the sallow phase,the expression of OsPPDK in leaves of Jin 23B was 7.34 times that of 8287,but there was no significant difference in the expression of OsPPDK in leaves.This suggests that OsPPDK gene expression may be associated with promoting aging.3?Based on the domain analysis of the OsPPDK gene,it was found that the OsPPDK gene has three nitrogen-binding domains,so the Jin 23B and R287 rice seedlings were treated with different nitrogen concentrations.The expression level of OsPPDK in different tissues was determined.The results of real-time quantitative PCR showed that OsPPDK gene was expressed in leaves grown at different nitrogen concentrations.The expression level of OsPPDK in Jin 23B was generally higher than that of R287.In both leaves and roots,OsPPDK expression increased with increasing nitrogen concentration,but the expression difference was opposite.Under half of normal nitrogen concentration in leaves,the expression level of Jin 23B was 2.86 times that of R287.At the two times normal nitrogen concentration,the basic expression level is equivalent;In the roots,on the contrary,at the half of normal nitrogen concentration,the expression level was the lowest and equivalent,and the expression difference was the highest at the two times normal nitrogen concentration,and the expression level of the Jin 23B was 4.63 times that of the R287.It shows that the expression of OsPPDK gene is affected by nitrogen concentration.4.The pU1300-OsPPDK over-expressed vector and ds1301-OsPPDK-RNAi vector of OsPPDK gene were successfully constructed.In addition,the genetic transformation of Jin 23 B and R287 rice Callus was induced by Agrobacterium-mediated transformation and the transformants were successfully obtained.Among them,there were 7 Jin 23B OsPPDK over-expressed plants,6 positive plants,3 Jin 23B OsPPDK-RNAi plants,1 positive plant,and 2 R287 OsPPDK-RNAi plants,all of which were positive.No R287 OsPPDK over-expressed plants were obtained.The expression level of transformants was analyzed.The results showed that the expression of OsPPDK was generally increased in 6 over-expressed positive transformants,with the lowest increase 2.4 times and the highest increase 6.43 times;Among the three RNAi-positive transformants,the expression level of the Jin 23B RNAi-positive transformants was down regulated by 48%,while the expression levels of the two R287 RNAi-positive transformants were down-regulated by 43%and 57%,respectively.It was demonstrated that the constructed vector was successfully transformed into the transformant and could have an effect on the expression of OsPPDK.