| Seed oil is one of the most important characters in oilseed crops research. Triacylglycerol (TAG) is the main form of stored lipid in oilseed. TAG is formed by glycerol-3-phosphate (G3P) as carbon skeleton, and gradually combination with three molecular fatty acids. Previous study about regulation of seed oil focus on genes involved in fatty acid synthesis and utilization, there is little known about the effect of glycerol metabolism related gene on seed oil. Plants can use two different routes to synthesize Gly3P. In the first route, G3P is phosphorylated formed directly from glycerol via glycerol kinase (GLI1). In the second route, G3P is formed from dihydroxyacetone phosphate (DHAP) via glycerol-3-phosphate dehydrogenase (GPDH). Recent studies have shown that seed-specific over-expression of a yeast glycerol-3-phosphate dehydrogenase gene increase seed oil content in Brassica napus. Glycerol is a common metabolite, which widely spread in microorganisms, animals and plants. The mechanism of glycerol metabolism is relatively conservative, which have been numerous reported in microorganisms and alga, but in higher plants, the effect of glycerol metabolism on plant growth and development is little known. Root is vital organs in higher plants, also is one of the model organs in research on plant developmental biology. The effect of glycerol on Arabidopsis root growth and development would be helpful to comprehensively understand glycerol metabolism mechanismThis study consists of two parts, the first part is the effect of over-expression of BnGPDH genes on some traits such as seed oil; the second part is the effect of exogenous glycerol on root development using glycerol metabolism related mutants and over-expression lines of glycerol-3-phosphate dehydrogenase genes, the main results are as follows:1、 Using homologous gene cloning method, two BnGPDHs fragments were cloned from Brassica napus. The amino acid sequences have highly similarity with homologous in Arabidopsis thaliana, Brassica oleracea, Brassica rapa, which contains two typical domains and locates in plastids and cytoplasm revealed by bioinformatics analysis. Southern Blot analysis revealed that the BnGPDHp1fragment had two, two, four copies in Brassica oleracea, Brassica rapa and Brassica napus.2、Gene expression analysis showed the transcript of BnGPDHs expressed high in flower and bud, low in root and stem, also expressed in developing seeds. Functional complementation of an E.coli mutant auxotrophic of G3P revealed that the strain harbouring the BnGPDHp1construct grew well on basal medium in absent of glycerol.3、Constructs of35S::BnGPDHs were introduced into Arabidopsis, and the transgenic lines were screened by analysis of Southern bolt and RNA gene expression, five lines were chose for further study. The seed weight and seed oil content of the overexpression lines is insignificantly different as compared with wild type. Construct of Dsred-BnGPDHp1seed-specific overexpression was transformed into Arabidopsis, several positive lines and their negative controls were used and no significant difference exists between these lines. Analysis of the fresh weight and seed germination rate of the overexpression lines under various stresses as compared with wild-type control, overexpression of BnGPDHpl increased the resistant of transgenic plants in response to NaC1stress.4、We identified two glycerol metabolism homozygous mutant gpdhpl and gpdhcl, and obtained the double mutant gpdhcl gpdhpl. Analysis of RNA expression showed the mutants were both knockout. Compared with wild-type, the seed weight was higher in gpdhcl, not changed in gpdhpl and lower in double mutant gpdhcl gpdhpl. The seed oil content of gpdhpl was higher than that of wild-type, but the oil content of gpdhcl and double mutant were not obviously different from wild-type. The oil per seed was increased in single mutants, but no obvious change in the double mutants as compared with wild-type.5、Upon exogenous glycerol treatment, the primary root length of Arabidopsis seedling was gradually decreased along with the increased gradient concentrations of glycerol. The root length was significantly inhibited, and the number of lateral roots increased significantly under1mM glycerol or above. The root length is no significantly change in glycerol kinase mutant glil, but more serious damage in gpdhcl and fad-gpdh mutants than wild-type under1mM glycerol treatment. The primary root length of overexpression of FAD-GPDH lines is unchanged or slightly reduced under glycerol treatment.6^The aboved phenotypes in roots appear concurrently with increased endogenous glycerol-3-phosphate (G3P) levels and decreased phosphate levels. G3P and phosphate levels were unchanged in the glycerol kinase mutant glil. Increased G3P raised the H2O2content in wild-type and the gpdhcl and fad-gpdh mutants. Overexpression of the FAD-GPDH gene attenuated the alterations in G3P, phosphate and H2O2, resulting in increased tolerance to exogenous glycerol. These results demonstrate that impaired root development caused by exogenous glycerol is associated with altered endogenous G3P, phosphate and ROS levels and showed that FAD-GPDH plays an important role in modulating this response.7、Upon glycerol treatment, free indole-3-acetic acid (IAA) content increased by46%, and DR5pro::GUS staining increased in the stele cells of the root meristem, suggesting that glycerol likely alters normal auxin distribution. Decreases in PIN1and PIN7expression,β-glucuronidase (GUS) staining in plants expressing PIN7pro::GUS and green fluorescent protein (GFP) signals in plants expressing PIN7pro::PIN7-GFP were observed, indicating that polar auxin transport in the root was downregulated under glycerol treatment. Analyses with auxin-related mutants showed that TIR1and ARF7regulate root growth under glycerol treatment.8、Glycerol-treated plants showed significant reductions in root meristem size and cell number as revealed by CYCB1;1pro::GUS staining. Furthermore, the expression of CDKA and CYCB1decreased significantly in treated plants compared with control plants, implying possible alterations in cell cycle progression. |
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