Activity Analysis Of Plasma Membrane H~+-Atpase Gene Promoter And Function Identification Of Interactive Transcription Factors In Blueberry

Blueberry(Vaccinium spp.)is suitable to grow in acidic soil with a p H value of 4.0-5.5,and has obvious"eosinophilic"to rhizosphere soil.However,at present,the soil p H of many blueberry planting areas in China is higher than 5.5,which is not suitable for blueberry growth and limits the development of blueberry industry to a certain extent.Root PM H~+-ATPase(Plasma Membrane H~+-ATPase,HA)can secrete intracellular H~+to extracellular,thus acidifying rhizosphere soil to adapt to non-acid rhizosphere stress.However,the expression of blueberry Vc HAs was not adaptively up-regulated in non-acid rhizosphere environment,and its related regulatory mechanism has not been reported,which shows that it is of great significance to analyze the transcriptional regulation mechanism of blueberry Vc HAs from the perspective of promoters and transcription factors.In this study,the response region of blueberry Vc HAs gene promoter to non-acid rhizosphere stress was explored by full-length promoter activity analysis and 5’terminal deletion analysis.Meanwhile,the transcriptional factors were screened and their function were further verified by bioinformatics prediction,yeast one-hybrid,Dual-Glo?Luciferase Assay System and genetic transformation in Arabidopsis thaliana,so as to explore the transcriptional regulation mechanism of blueberry in response to non-acid rhizosphere stress.It aimed to unveil the mechanisms involved in the cause of"eosinophilia"of blueberry and further improving the resistance of blueberry under non-acid rhizosphere stress.The specific results are as follows:Nine Vc HAs promoters(p Vc HAs),were isolated and cloned.Bioinformatics analysis showed that these promoters are distributed with cis-acting elements related to abiotic stress,such as light response,drought response,low temperature response and hormone response,indicating that Vc HAs was regulated by a variety of environmental factors.The transient expression of p Vc HAs fusion LUC reporter gene in tobacco leaves showed that all nine Vc HAs promoters were active,but the activity was different.The deletion analysis of the 5’end of the key gene Vc HA11.1 promoter showed that the activity of the deleted promoter was significantly up-regulated during p H 7.0 compared with that of p H 5.0,indicating that the promoter activity could respond to non-acid rhizosphere stress,but the promoter activity decreased when p H increased to 8.0,even lower than that of p H5.0,which may be due to the fact that too high p H was not suitable for tobacco growth,resulting in low overall expression level.Under the treatment of p H 5.0 and p H 7.0,there was no significant difference in p Vc HA11.1,p Vc HA11.1-A and p Vc HA11.1-B activity,but when the promoter fragment was shortened from p Vc HA11.1-B to p Vc HA11.1-C,the promoter activity decreased significantly,with a maximum decrease of 32.0%,indicating that the deletion of-712～-438 bp on p Vc HA11.1 led to a significant decrease in transcription level.In addition,the activity of p Vc HA11.1-C(-438～+124 bp)under p H 7.0 treatment was significantly higher than that of p H 5.0,suggesting that there may also be undiscovered non-acid rhizosphere stress response elements.Vc Dof2.1 and Vcb HLH104,the possible regulatory transcription factors of Vc HA11.1were predicted by bioinformatics.Yeast one-hybrid and tobacco Dual-Glo?Luciferase Assay System experiments confirmed that Vc Dof2.1 and Vcb HLH104 transcription factors could directly act on p Vc HA11.1-B.After blueberry was treated with different p H for 30days,the phenotype of blueberry was significantly different.Real-time fluorescence quantitative PCR showed that the expression of Vcb HLH104 in blueberry root was the highest at p H 7.0.In contrast to control,its transcript level was increased by 242.0%,but decreased significantly when p H increased to 8.0.The expression trend of Vcb HLH104 was consistent with that of its target gene Vc HA11.1,indicating that the expression of Vc HA11.1and Vcb HLH104 could be induced by non-acid rhizosphere stress.It is speculated that Vcb HLH104 positively regulates the expression of Vc HA11.1.The expression of Vc Dof2.1in roots decreased with the increase of p H,indicating that non-acid rhizosphere environment could inhibit the expression of Vc Dof2.1 in roots.The full-length c DNA sequences of 951 bp Vc Dof2.1 and 672 bp Vcb HLH104 genes were amplified by PCR,encoding 317,224 amino acids,respectively.Homology analysis and phylogenetic tree construction showed that blueberry Vc Dof2.1 and Vcb HLH104 had high homology and close relationship with Camellia sinensis and Actinidia chinensis Planch,respectively.After genetic transformation of Arabidopsis thaliana,it was found that Vcb HLH104 gene could improve the resistance of plants to non-acid rhizosphere stress.To sum up,the expression of blueberry plasma membrane H~+-ATPase gene Vc HA11.1can respond to non-acidic rhizosphere stress,Vc Dof2.1 and Vcb HLH104 transcription factors directly bind to the core response region p Vc HA11.1-B(-712～+124 bp).Vcb HLH104positively regulates Vc HA11.1 expression,and Vcb HLH104 can promote the resistance of Arabidopsis thaliana under non-acid rhizosphere stress to some extent,which preliminarily reveals the response mechanism of blueberry to non-acid rhizosphere stress at the transcriptional level.