Constrution Of Rice Tissue-specific Synthetic Promoters, Identification Of Bidirectional Promoters And Functional Analysis Of OsPGIP1 In Sheath Blight Resistance

Plant architecture, development and interaction with environment are controlled by the expression of a series of genes. The diversity of gene expression pattern depends on the regulation at different levels from transcription to translation. As a key regulator in transcription level, the promoter is a “pioneer” of gene expression. Promoter is critical to start, close and level of the expression of target genes and is also crucial for exploring the mechanism of transcriptional regulation. Therefore, studies on promoter have shown great application potentials and theoretical values.Rice is one of the most important food crops in the world and a model plant for functional genomic research in cereals. More complete genomic information and more explicit gene expression information greatly facilitate the studies of tissue-specific synthetic promoters, cis-regulatory elements and bidirectional promoters.Sheath blight causes particularly severe damage in growth, development and production of rice. However, no great break-through has been made in the breeding progress for sheath blight resistance for a long time. The major reasons could be:(1) no rice variety which is completely immune to Rhizoctonia solani has been found;(2) no major resistance QTL against sheath blight has been identified;(3) limitation of candidate genes for transgenic breeding of rice varieties with sheath blight resistance. In recent years, it has been reported that polygalacturonase inhibiting proteins(PGIP) can inhibit the degradation of the plant cell wall by polygalacturonases(PG) from pathogens, resulting in the improvement of plant resistance to pathogens.The main work and results in this study are as follows:1. We first assembled seven regulatory sequences related to tissue-specific expression in different combinations and obtained five novel green tissue-specific synthetic promoters which are GSSP1, GSSP3, GSSP5, GSSP6 and GSSP7. Subsequently, we scanned and counted the cis-elements in different tissue-specific promoters based on the plant cis-elements database PLACE and the rice c DNA microarray database CREP, resulting in 10 cis-elements whose frequencies in green tissue-specific promoters were relatively higher. Finally, we identified a general regulatory sequence 5’-AAAATATTTAT-3’(the underlined sequence indicates the core element GEAT) which can be applied in the synthesis of green tissue-specific promoters and further analyzed the function of the flanking sequence in detail. These results provide an example for developing tissue-specific synthetic promoters in rice, and propose a feasible method for screening and functional identification of tissue-specific cis-elements with their flanking sequences at the genome-wide level in rice.2. We combined RNA-seq and c DNA microarray data to select four candidate bidirectional promoters BIP1, BIP2, BIP3 and BIP4. All of them showed bidirectional expression activity in the transgenic plants. Through 5’ and 3’ deletion analysis of one of the above bidirectional promoters, BIP1, we identified the enhancing region which sharply increased its bidirectional expression efficiency and the essential regions respectively responsible for its 5’/3’ basic expression activity. The bidirectional arrangements of the four gene pairs in six gramineous plants were also analyzed, which showed conserved characteristics of the four bidirectional promoters identified in our study. In addition, two novel cis-sequences conserved in the four bidirectional promoters were discovered by bioinformatic identification. Our study proposes a feasible method for selecting, cloning and functionally identifying bidirectional promoters as well as for the discovery of their bidirectional expression regulatory regions and conserved sequences in rice.3. In this work, we prokaryotically expressed and purified Os PGIP1 protein. The activity assay confirmed a inhibitory activity of Os PGIP1 against the PGase from Rhizoctonia solani. In addition, the location of Os PGIP1 was determined by subcellular localization. Subsequently, after obtaining homozygous Os PGIP1 transgenic lines with single-copy insertion, the expression levels of Os PGIP1 in these lines were analyzed by quantitative real-time PCR. The field inoculation of Rhizoctonia solani showed that the sheath blight resistance of the transgenic rice was significantly improved. Furthermore, the levels of sheath blight resistance were in accordance with expression levels of Os PGIP1 in the transgenic lines. Our results not only reveal the function of Os PGIP1 and its resistance mechanism to rice sheath blight, but also provide a novel approach for rice breeding against sheath blight.