Analyses Of Functional Complementation And Transcriptomic Changes In Rice Mutant Osmgd2-1?

In eukaryotes, the biomembrane system is essential for cell life. The two layers of lipid membrane separate the cell that the reaction can be organized in an orderly way. Monogalactosyldiacylglycerol?MGDG? and digalactosyldiacylglycerol?DGDG? are the main lipid components of plastid membrane. The biosynthesis of MGDG is catalyzed by MGDG synthase?MGD?, which transfers D-galactose from UDP-galactose to sn-1,2-diacylglycerol. MGDG is also the substrate for the synthesis of DGDG, so MGD is considered as a key enzyme in the biosynthesis of galactose and the formation of plastic membrane.The results from the pre-experiments of promoter-GUS expression and quantitative PCR showed that the Os MGD2 was preferentially expressed in pollens at the binuclear and mature developmental stages. The results of RNAi also suggested that Os MGD2 played an important role in the late stage of rice pollen development. On the basis of the above researches, a complementation experiment and a transcriptomic analysis by gene chip technology were carried out using a T-DNA insertion mutant?osmgd2-1??, in order to verify the function of Os MGD2. The main results in this study were as follows:1. The genotype of mutant osmgd2-1 were analyzed. The results showed that the heterozygous mutant?116 plants?: homozygous wild type?54 plants? = 2.15: 1, and no homozygous mutant was isolated.2. The Phenotypic analyses of mutant osmgd2-1??T₄-T5 generation? showed that both the pollen fertility and seed setting were reduced significantly.3. The results of gene chip indicated that 308 genes were up-regulated and 117 genes were down-regulated in mutant osmgd2-1? compared with the control ZH11. According to the results of gene clustering and enrichment analysis, the changed genes were mainly concentrated in the cell membrane components, RNA components, cell growth and development, nucleic acid transcription process and so on.4. A Os MGD2 functional complementation vector was introduced into mutant osmgd2-1? and resulted 4 transgenic lines?H1-1, H1-2, H9-1 and H9-2?. The results of quantitative PCR analysis showed that in addition to H1-1, the expression level of Os MGD2 was increased significantly in lines H1-2, H9-1 and H9-2. Furthermore, the pollen fertilities of the three lines H1-2, H9-1 and H9-2 in T₀ and T₁ were also increased significantly compated with the mutant osmgd2-1?. The results of the complemental experiments showed that the partial restoration of Os MGD2 expression level was positively correlated with the pollen fertility, and demostrated that the Os MGD2 plays an important role in rice pollen development.5. Over-expression of Os MGD2 was also carried out to understand the function of Os MGD2. The results showed that the expression level of Os MGD2 was significantly increased in the leaves and florets of transgenic plants, but there was no significant changes occured in the pollen fertility and seed setting rate. The results indicated that the over-expression of Os MGD2 had no effects on the fertility of rice plants.