| Leaf is the main organ of the plant for photosynthesis and respiration, which plays a vital role in the growth of plants. As an important aspect of plant morphogenesis, leaf development is closely related to the plant type and agricultural production. Rice is one of the most important crops in the world, especially in china, due to the contradiction between its limited cultivated land and huge population, to enhance the rice yield become particularly urgent. And rolled leaf is one important morphogenetic index for high yielding. Therefore, to explore the molecular mechanism of rolled leaf can not only help people better understand the mechanism of rice leaf development, but help experts to breed better plant types through molecular design.Using rolled leaf mutant discovered in progenies of transgenic rice as material, this study conducted leaf morphology analysis and genetic analysis, so as to explore the molecular mechanism of rolled leaf, based on differential proteins analysis by two-dimensional electrophoresis technique and digital gene expression profiling analysis by high throughput sequencing. The results are as below:1. The rolled leaf employed in this study was out-rolled leaf in half-barrel shape, getting rolled from the third leaf. The spreading leaf was prone to droop, while the rolled leaf could keep erect. Besides, the cause of the out-rolled leaf of GM-rice lies in the increase of bulliform cells in leaf epithelium.2. With the chlorphyll content in the leaves of out-rolled leaf mutant getting lower, leaf photosynthesis became less efficient, which caused some important agronomic traits lower than those of normal plants, such as plant height, ear length, set spikelets rate and 1000 grains weight. At the meantime, the status of rolled leaf also influences the quality of the rice grain, which reduces the amino acid contents.3. The result of genetic analysis showed that the ratio of flat leaf:rolled leaf in two kinds of hybridized combinations Rolled/WT and WT/Rolled both meet 3:1. Therefore, the trait of rolled leaf is determined by a single recessive gene. Real-time PCR was employed to analyze the copy member of foreign gene 3a-HSD. The result showed that foreign gene was single copy. The result of co-segregation detection of insertion element and mutant character showed that T-DNA and rolled-leaf character were co-segregation, which led to a presumption that rolled-leaf mutant was caused by the insertion of T-DNA. The result of segregation of sequences flanking the insertions showed that the insertion site located out of the gene eara in chromosome 9 of rice. The nearest gene away from the insertion site was Os09g0293400. There were 7 genes in 100kb back and forth the insertion site, which were all unknown genes.4. Total protein of WT-. Unrolled and Rolled in different periods was distracted to proceed two-dimensional electrophoresis.181 differential proteins were detected by analyzing electrophoretograms of two-dimensional electrophoresis. Further analysis of differential proteins of different materials in different periods found 34 differential proteins related to rolled-leaf character.28 differential proteins were successfully identified by MS/MS. There were 22 ennergy metabolism related differential proteins included 16 Ribulose bisphosphate carboxylase large chain family members, photosynthesis related protein putative transketolase 1 and phosphate dikinase 1, carbohydrate metabolic process related protein phosphohexose mutase and Beta-glucuronidase as well as 2 ATP synthase CF1 alpha chain (chloroplast); besides ennergy metabolism related proteins there were protein folding related protein peptidyl-prolyl cis-trans isomerase protein, cellular amino acid metabolic process related protein Aspartate aminotransferase, protein biosynthesis related protein elongation factor, protein transport related protein chloroplastic outer envelope membrane protein and lipid catabolic process related protein phosphoinositide phospholipase C.5. Picked up young leaves of WT% Rolled and Unrolled in tillering stage, then extracted total RNA for high throughput sequencing analysis base on illumina HiSeq2000 sequencing platform. Firstly, a series of quality evaluation was carried on raw datas in order to insure that data quality was good enough for follow-up analysis. Then consult sequence comparative analysis, gene expression level analysis and overall quality evaluation of RNA-seq were carried, in order to insure the veracity of gene diference expression analysis. The results of differential expressed genes selection and cluster analysis showed that the gene expression difference between WT and Rolled was smaller, while that between Unrolled and them was bigger, which showed that the influence of foreign gene on rice growth and development was stronger than that of rolled-leaf character, which is supported by Venn diagram of differentially expressed genes. The result of GO enrichment analysis of differentially expressed genes showed that the differentially expressed genes between WT and Rolled mainly enriched on biological progress included fatty acid metabolism, lipid metabolism etc, molecular function included Ester bond hydrolytic enzymes activity, oxidoreductase activity etc. the differentially expressed genes between Unrolled and Rolled mainly enriched on biological progress included cell component assembly or biosynthesis, organelles assembly etc, cellular Component included macromolecular complex, cell part etc, molecular function included structural constituent of ribosome, structural molecule activity. The result of KEGG enrichment analysis of differentially expressed genes showed that the differentially expressed genes between WT and Rolled mainly enriched on metabolic pathway, secondary metabolites synthesis etc. the differentially expressed genes Rolled and Unrolled mainly enriched on ribosome, Starch and sucrose metabolism etc. |
