Origin Of The BSP Gene In Plants And Preliminary Study On Function Of The OsBSP3 Gene In Rice

Bark storage proteins (BSPs), which are storage proteins of barks, play an important role in nitrogen metabolism of temperate deciduous plants. In autumn, the proteins in the leaves are degraded during leaf senescence, the released amino acids are transported into barks, then the BSPs are synthesized by phloem cells. When spring is coming, similar to storage proteins of seeds, BSPs are degraded into amino acids, then transferred into new buds and leaves to provide plants with sufficient nitrogen source. Although previous studies showed that the Populus BSP genes participated in storage and transportation of nitrogen, the physiological and morphological function of BSP gene family in Monocots was still undefined.In order to further study the origin of BSP gene, methods of bioinformatics were taken to do systematic identification, phylogeny evolution and functional divergence analysis of BSP gene in some representative plant species, and explored the study of BSP gene. And the way of reverse genetics was used to do the preliminary study of the function of BSP gene in rice. The main results were as follows:1. The BSP gene first appeared in Charophyta and was widespread in the genome of vasvular plants. The sequence of BSP gene in Oryza sativa was used as a query to search the NCBI nr protein database and Phytozome database.The results indicated there was at least one homologous gene in completed sequence of vascular plant genome. Evidence revealed that land plants originated from Charophyta. Charophyta and land plants were collectively called the streptophyte. Charophyta genome contained many genes which were typical in land plants. Klebsormidium flaccidum genome was further searched and we identified one gene encoding BSP protein in its genome. But when we searched in other completed sequence of Chlorophyta algae, BSP gene was not found in Chlorophyta algae. It indicates that BSP gene of green plants initially originated from Charophyta. While universal research of Physcomitrella patens genome, we found that there are no genes encoding BSP protein in its genome and probably it was lost in this genome.2. Plant BSP genes have two obvious functional divergence subfamilies. Using maximum likelihood and distance method to build a phylogenetic tree, the results showed BSP gene family can be divided into two subfamilies, named as A and B subfamilies. Wherein A subfamily includes genes from Charophyta alga, Pteridophyte, gymnosperm, angiosperm and B subfamily contains genes from gymnosperm and angiosperm. These results suggests that A subfamily was more ancient subfamily, while B subfamily was produced by gene duplication of A subfamily and had new sequence character because of undergoing smaller selection pressure.3. Functional divergence analysis between plant BSP gene subfamilies based on ML, result suggests that evolutionary rates have obvious difference between them. And nine amino acids are screened which are playing an important role in functional divergence between subfamilies.4. Rice OsBSP3 gene was regulated by temperature and 6-BA abiotic stresses. Techniques of reverse genetics and molecular biology were utilized to study the function of OsBSP3 gene. Our results revealed that the expression of the rice OsBSP3 gene was significantly repressed by 6-BA. Moreover, germination rate of different transgenic plants had significant differences at different temperatures. The germination rate of wild-type and over-expression seeds were significant lower than mutants at low temperatures, while the data showed opposite trend at high temperature.5. Rice OsBSP3 gene associated with the development of vascular bundles. Observation from the resin slice of rice stem and sheath shows that there were significant difference between wide-type, over-expression and mutant in terms of cell area and width. The largest in Wild-type, followed by over-expression and mutant. The number of big and small vascular bundles in stem also presented similar rules simultaneously. Therefore, we estimate that over-expression or mutation of OsBSP3 gene repressed the growth and development of vascular bundles. Only the gene normal expression could promote the growth of vascular bundles in rice.The results indicate that the origin of the plant BSP gene was the result of an ancient HGT event from bacteria. The origin time may before the land plants produced, but after the divergence of charophyta and chlorophyte alga. At the same time the rice OsBSP3 gene is likely to participate in the growth of vascular bundles and the 6-BA abiotic stress response.