Creating Tiller Mutants And Cloning Tillering Genes And Verifying Their Function In Switchgrass

Switchgrass(Panicum virgatum L.), belonging to the Poaceae family, is a perennial warm season C4 grass. Switchgrass, as an bioenergy plant, has been usually used for soil and water conservation, livestock feeding, and bioethanol production. In recent years, more and more studies on switchgrass had been presented, but the studies on switchgrass tiller were few. So our experiment chosed one switchgrass variety which had the better resistance and stronger adaptability than others as the mutagenic material. Using the EMS(ethyl methyl sulfonate), the switchgrass seeds which came from one plant were induceed, and created switchgrass tillering mutants. Then tiller mutants were chosen and their traits were studied. Using the RNA-seq technology, some genes which were related to tillering and different expressed in the two mutants were cloned and their functions were verified.1. In order to select a better resistance and stronger adaptability variety for mutagenic material, different switchgrass varieties which were bred in our lab were studied for the their drought resistant, acid and alkali resistant and poor resistance. In the drought resistant test, with the drought stress time increased, the growth of switchgrass seedling was restrained. When the drought stress time was under 12 d and the Soil Moisture Content(SWC) was higher than 3.7%, most of switchgrass seedlings would die for water shortage. In acid and alkali resistant test, all the switchgrass would be died if the p H values were under 3.5 or over 11. Compared with the acid stress, the adaptation for switchgrass seedling in the alkaline condition was better. But in the poor eesistance test, switchgrass requirement for NPK did not need more to keep growth and to increase the nitrogenous fertilizer would increased the tiller number. To sum up, effects for adversities on switchgrass adaptations differed from different varieties(lines), and resistances of switchgrass varieties breeding from 'Alamo' coming from USA is better, especially‘Xiji 2'.2. Switchgrass seed from one plant of the‘xiji2'was induced using EMS and sowed in the flied. After treatment, the seedling surviving rate, physiological and biochemical indexes were studied, and the results showed that mutagenic conditions for switchgrass seed were 1.0% of EMS concentration and 4h of mutagenic time. Using vegetative propagation, switchgrass generations from mutants were obtained and their phenotypes, nitrogen content and endogenous hormone content were studied for testing the difference of switchgrass mutants. In the end, a pair of switchgrass mutants which had stable genetic and biggest difference in tillering, switchgrass L and switchgrass M, were chosen for further study.3. Using RNA-seq technology, switchgrass transcriptomes from the pair of mutants were analyzed and 2,877 different expressed genes(DEGs) were obtained. These DEGs were primarily involved in physiological processes, including phenylpropanoid biosynthesis, energy production, carbohydrate transport and metabolism, and plant hormone signal transduction. Using sequence homology analysis, we confirmed genes related to switchgrass tillering, including D3, D10, D14, PVtb1, and PIN1. Cloning and identification of the function of these genes will benefit future studies of tillering in switchgrass.4. Two genes were cloned which were related to switchgrass tiller and named Pv PIN1 gene and Pv TB1 gene, and their functions were identified. The first gene was Pv PIN1, a switchgrass PIN1-like gene, which is a putative auxin efflux carrier. Neighbor-joining analysis in NCBI databases showed that the Pv PIN1 gene belongs to the PIN1 family and is evolutionarily closer to the Oryza sativa japonica group. Using the transgenic technology, tiller number of transgenic plants in which the Pv PIN1 gene were underexpressed by RNA interference(RNAi) was significantly promoted, which presented a phenotype similar to that of TIBA(2,3,5-triiodobenzoic acid, an auxin transport inhibitor)-treated wild-type plants. Pv PIN1 gene overexpression or underexpression resulted in changes in tiller number and shoot/root ratio using the transgenic approach. The data suggested that Pv PIN1 played an important role in auxin-dependent adventitious root emergence and tillering.5. Moreover, Pv TB1 gene in switchgrass was firstly cloned and its fuction was identified, which was homologous with the teosinte branched1(TB1) gene in zea. Using the blast in NCBI datbase, the result showed that the Pv TB1 gene sequence was similarity with Os TB1 gene, which was involved in lateral branching in Oryza sativa. Like other TB1 genes, the Pv TB1 gene had a same conserved domain that encoded a putative transcription factors named TCP domain. The tiller emergence and development were significantly inhibited by the overexpression of Pv TB1 in transgenic plants, and these unusually phenotypes could be rescued by 6-benzylaminopurine. The overexpression or suppression of Pv TB1 through a transgenic technology resulted in changes in tiller numbers and height. Taken together, our results suggested that Pv TB1 negatively regulated tillering in switchgrass, presumably via its expression in axillary buds.6. In addition,comparing the domestic with the imported rupture disk in the particle bombardment of switchgrass calluses, the burst pressure, the hyg-resistance callus rate, the number of regeneration plants and the rate of transgenic plants were analyzed and the result showed that the stability of the domestic was poorer than the domestic, but the average pressures of the two disks were not significant difference; The others of the domestic and imported were also not remarkable except the resistance callus rate of the imported was significantly higher than the domestic. In conclusion, the domestic rupture disk could completely replace the imported and the result could provide a theoretical premise to save the scientific research funds in the particle bombardment.