Screening Of Setaria Viridis Mutants And The Identification Of Guard Cell-specific Promoters

C₄photosynthesis is the main way to fix and access the carbon source to most of food andenergy crops on earth. Studies have found that C₄plants have50%higher radiation useefficiency than those of C₃plants. Introducing C₄photosynthesis traits into rice, can make theradiation use efficiency and the yield of rice increased by50%, and can potentially improve thenitrogen and water use efficiency.Setaria viridis, Poaceae, Panicoideae clade, annual herbaceous plant, is typically C₄plants.S.viridis is a diploid plant,2n=36; its genome is small, about510M; its stature is small and itsgrowing conditions required is simple what is easy to control; its life cycle is short needing onlyabout40days, from breeding to mature; The photosynthetic pathway is the NADP-ME, a typicalC₄photosynthesis pathway. Based on the above advantages, S.viridis is expected to become aclassical model plant about C₄photosynthesis.Stomata is the channel of moisture and gas entering the plant leaves, to maintain the balanceof the plant transpiration and photosynthesis. In Arabidopsis, overexpression of carbonicanhydrase gene in the guard cells can make the transgenic plants appeared the following changes:stomatal conductance decreased; instantaneous water use efficiency increased; CO2fixed ratehas not changed; fresh weight of leaf in vitro lost; stomatal density and stomatal index decreased.This result provides a new way to increase the water use efficiency of plant.The research mainly includes the following works:1. Screening S.viridis mutants: We mutagenized S.viridis seeds by physical and chemicalmethods, to observe S.viridis phenotype, focusing on looking for the C₄mutation phenotype. Inthis part of the experiment, we took the method of EMS mutagenesis and γ-ray mutagenesis tomutagenized S.viridis seeds,and then we received the M2generation seeds per plant. Thetreatment concentration of EMS mutagenic was0.4%and0.2%; and the dose of γ-raymutagenesis was300,200,150Gy. After we got the M2generation seeds, we took the fieldplanting to screen the M2generation mutants. So far, we have carried out two large-scalescreening: EMS mutagenesis have been investigated6,000seedlings; γ-ray mutagenesis havebeen investigated3700seedlings, and we have received a large number of mutants. In thesemutants, we have filtered60mutants about veins morphological and32mutants about thenumber of veins. Veins morphology mutations included: major vein branch, secondary veinsclearly, partial veins. At the same time, we fixed the number of leaves veins changed to make into paraffin sections to observe the Kranz structure. The results show that, despite a change inthe number of plant veins, their Kranz structure hasn,t changed. This means that other reasonscause the change in the number of the veins, such as the cell volume increased.2. Cloning and identification of guard cell-specific promoters: According to the gene chipresults of Nelson issued in2009about gene expression in rice,we selected genes specificallyexpressed in guard cells as our research object:Os05g47630, Os09g15720, Os10g31320,Os04g57030, Os09g15670, Os08g02210, Os08g31810, Os04g57020.According to the existinggene sequences, we found the regulatory regions of the genes, and analysed the regulatoryregions, in which we focused on the number and location of guard cell-specific motifs occured,and then cloned the promoter of genes we selected. Then we connected the cloned fragment tothe vector pCAMBIA1301to construct an expression vector. We used the method ofAgrobacterium-mediated transformation to transform the expression vector we had built into therice calli induced. According to the resistance screening, regeneration and rooting stages, thecalli grew into the transgenic plants,we got transgenic plants: pCAMBIA1301-pOs08g31810::GUS、 pCAMBIA1301-pOs08g02210::GUS、 pCAMBIA1301-pOs04g57020::GUS、pCAMBIA1301-pOs09g15670::GUS、pCAMBIA1301-pOs10g31320::GUS15、11、12、11、10lines respectively. After that,we indentified the transgenic plants by PCR,and the resultshowed that we got28lines true transgenic plants.And then we made the transgenic plantsprocessed GUS staining, the result showed that the GUS reporter gene havn’t expressed in thetransgenic plants.