Study On The Role Of The Phospholipase PPLAII? In Regulating Mucilage Synthesis And Fatty Acid Content In Seeds Of Arabidopsis

As an important renewable energy source, the rational use of the plant oil can reduce the excessive use of non-renewable energy sources such as coal, oil in a large. At the same time, the plant oil plays an important role in plant growth, development, and the process of abiotic-stresses and biotic-stresses. Arabidopsis thaliana is a model organism to study oil crops, investigate the functional genes in the process of oil metabolism including lipid biosynthesis. The researches of functional genes in Arabidopsis provide the basis to the similar genes researches in the other plants.This study is about to the Arabidopsis phospholipase gene pPLA??, and we do some researches on its expression location, expression stage, metabolic mechanism of oil and mucilage. The results are as follows:1. Analyze the expression of pPLAIIa gene in various tissues of ArabidopsisConstruct the pPLAIIa gene promoter to GUS fusion expression vector, the pPLAIIa promoter::GUS transgenic Arabidopsis was obtained by the floral dip method. We found that the GUS signals were significantly detected in the mature leaf and the top of mature embryo, where was the most important position of oil synthesis. Therefore, pPLAIIa maybe participates in the pathway of Arabidopsis oil synthesis.2. pPLAIIa increases the Arabidopsis seed oil contentTo investigate the function of pPLA?? in seed oil production, we obtained the pPLAIIa overexpression plant pPLA??-OE and isolated transfer DNA (T-DNA) insertional pPLA??-KO mutant.We measured the seed oil contents by gas chromatography. The results showed that pPLA??-OE seeds displayed a significant increase in oil content compared with wild-type seeds; the oil contents of pPLA??-OE and wild-type seeds were 39.6% and 37.8% of the seed weight, respectively. Taken together, these datas indicate that pPLA?? plays a positive role in seed oil accumulation.3. pPLAIIa involves the Arabidopsis seed mucilage synthesisWe analyzed the mucilage of the WT, pPLA??-OE and pPLA??-KO by Ruthenium red staining. The mucilage layer of pPLAIIa-KO was thicker than wild-type plants, while the pPLA??-OEwas thinner. These results indicate that pPLA?? involves the Arabidopsis seed mucilage synthesis.The results of RT-PCR and quantitative real-time PCR (qRT-PCR) showed that pPLAIIa can express in seeds at different developmental stages (4,7 and 10 DPA), the pPLAIIa gene was expressed throughout the seed coat differentiation process, which expressed the highest level at 10 DPA, and this stage was the key period of the degradation of starch granules and the forming of columnar structurein seed mucilage. Above all, we can infer that pPLAIIa influences the Arabidopsis seed mucilage.4. GL2 regulates the expression of pPLA?? directly in the seed developmentResearches have shown that GL2 is an important transcription factor regulating the seed mucilage synthesis and the seed oil content of the gl2 mutant increased. To test whether thepPLA?? gene was the target of GL2 in seeds, we detected the expression levels of pPLA?? gene in developing seeds of the gl2-2 mutant by using quantitative real-time PCR. The results showed that pPLA?? expression levels in the gl2-2 mutant seeds were down-regulated compared with wild-type in all these development stages of seeds development. These results suggest that the pPLA?? gene islocated in the downstream of GL2 and may regulate directly by GL2.We analyzed the promoter of pPLAIIa and found two L1-Like boxes (named L1-1 box and L1-2 box),which may be the binding elements, so we proved it by yeast one-hybrid assay and Gel mobility shift assay.The full-long promoter of pPLA?? was cloned and it was bound by GL2 as revealed from growth of yeast transformants harboring pAD-GL2 and pAbAi-pPLA??pro in selection medium SD/-Leu/+AbA. Moreover, we truncated the promoter into 365bp, which included two L1-Like boxes. At the same time, the GL2 was truncated into the GL2HD-ZIPdomain, which was the DNA binding domain. The result also proves that GL2HD-ZIPdomain can bind to the pPLAIIa shorter promoter.Then we made the two L1-Like boxes probes with biotin labeling. The increased concentrations of the non-labeled competitors significantly reduced the band intensity of the DNA-protein complexes, which indicated that the GL2HD-ZIP domain binding to these elements specially. These results indicate that GL2 specifically binds to two elements in pPLA?? promoter regions and regulates the expression of pPLA??.In addition, the pAbAi-pPLA??vector was constructed, and a transcription factors library in Arabidopsis thaliana was used to be screened. We obtained an important C2H2 transcription factor--At5g60470, Which could bind to pPLAIIa promoter as well as GL2. Logically, we attempted to verify whether they can interact with each other by yeast two-hybrid, but the result was not.5.PA binds to GL2PA(Phosphatidic acid)is an important signal molecule in the plant oil metabolize. Above all, to investigate whether the GL2START domain and PA can interact with each other, we used Fat Western blot experiment. The results showed that with concentration of PA increased, the signal strengthened gradually, so GL2START domain specifically combined with PA.