| As an important crop,rice provides food rations for more than half of the world’s population.The production and safety of rice are often subjected to some biotic and abiotic stresses,resulting in the loss of food production worldwide and threatening global food security.Therefore,the molecular mechanism and genetic improvement of rice disease resistance and stress resistance have become an important topic.Phenylalanine ammonia-lyase(PAL)is a conserved homologous tetrameric protein formed by the polymerization of 77-83 k Da subunit.It catalyzes the phenylalanine to form carbon-carbon double bond to produce trans-cinnamic acid(t-CA)while releasing NH3.t-CA is the precursor of the biosynthesis of a variety of phenylpropanoid compounds,such as lignin,salicylic acid(SA),anthocyanin and flavonoid phytoantitoxin.Although many studies have investigated the expression characteristics of individual subtypes of Arabidopsis At PAL gene family and analyzed its function in different stresses,there is little information on the expression pattern of rice OsPAL family so far.In this thesis,the expression pattern and biological function of rice OsPAL family were studied.The main contents are as follows:(1)We used the genevestigator database to analyze the expression patterns of the OsPALs genes in rice growth and development.The results showed that OsPAL1-9genes were expressed in each development stage of rice,and their expression levels were different.Among them,OsPAL1 and OsPAL2 were highly expressed.(2)The content of total SA and the expression of OsPALs in young and aging leaves were detected respectively.The results showed that the expression of OsPALs in rice was related to SA biosynthesis.In addition,we also detected the expression OsPALs in different parts of rice seedlings.The results showed that there was no tissue-specific expression of OsPALs genes.(3)35S:e GFP-OsPAL1、35S:e GFP-OsPAL2、35S:e GFP-OsPAL3、35S:e GFPOsPAL4 、 35S:e GFP-OsPAL5 、 35S:e GFP-OsPAL6 、 35S:e GFP-OsPAL7 和35S:e GFP-OsPAL9 vectors were constructed and transformed into plasma cells of rice.The subcellular localization of OsPALs was observed by laser confocal microscope.It was found that OsPAL1 、 OsPAL2 、 OsPAL6 、 OsPAL7 were located on the endoplasmic reticulum,while.OsPAL3、OsPAL4、OsPAL5、OsPAL9 were located in the cytoplasm.(4)t-CA was detected in the reaction system of recombinant protein OsPAL1-9,and the results of enzyme activity experiment in vivo showed that the PAL enzyme activity decreased after OsPALs mutation in rice.Therefore,we believe that PALs in rice have the activity of catalyzing phenylalanine to produce t-CA.(5)The determination of SA and lignin content showed that the content of SA increased and the content of lignin decreased after OsPAL1 and OsPAL2 mutations;while OsPAL4 mutation resulted in the decrease of SA content and the increase of lignin content.It suggests that OsPAL1 and OsPAL2 are mainly involved in the lignin synthesis in rice,while OsPAL4 is primarily engaged in the synthesis of SA.In addition,there is an antagonistic relationship between SA and lignin biosynthesis in rice.(6)When the ospals mutants were treated with P6,the pal1pal2 mutant showed an obvious disease resistance phenotype.In contrast,the pal4 mutant showed a disease susceptibility phenotype,suggesting that the increase of SA positively regulates the disease resistance of rice.(7)The statistical results of agronomic characters of ospals mutants showed that the mutations of OsPAL1 and OsPAL2 in rice resulted in elevated SA levels,while SA accumulation in plants affected plant branching and resulted in a crop-yield reduction.The decrease in SA content caused by the OsPAL4 mutation did not affect the crop yield,while the increase in its lignin content regulated the mechanical strength of the plant stem,which may affect the lodging resistance of the plant.Based on the above conclusions,we found differences in the gene expression levels of 9 members of the rice OsPAL family,in which the expression levels of OsPAL1 and OsPAL2 were the highest.In contrast,the expression of OsPAL1-9 was not tissue-specific.OsPAL proteins can catalyze phenylalanine dehydrogenation to tCA in vivo and in vitro,and OsPAL1、OsPAL2、OsPAL6、OsPAL7 were located on the endoplasmic reticulum,while.OsPAL3、OsPAL4、OsPAL5、OsPAL9 were sublocated in the cytoplasm.Based on the further study of the biological function of OsPALs,we draw the following model: OsPAL family in rice can be roughly divided into three categories.The t-CA catalyzed by OsPAL1 and OsPAL2 enters the lignin synthesis pathway for cell wall biosynthesis.In contrast,the t-CA catalyzed by OsPAL4 is used in the biosynthesis of SA and regulates the disease resistance process of plants.Other members of the OsPALs may act on the synthesis of lignin and SA simultaneously.We speculated an antagonistic relationship between lignin synthesis and SA synthesis in rice. |
