The Screening Of Setaria Viridis Mutants And Gaining The Plants Cell-specific Express Promoters

Plants can be classified as C₃、C₄and CAM species, the efficiency of C₄photosynthes is higher than the efficiency of C₃photosynthes. In most C₄plants, twocell types, bundle sheath and mesophyll form the Kranz anatomy.This anatomycomes into being with denser vein spacing, favoring the exchange of sugars and C₄acids. BSC and MC also cooperate with each other form the “CO₂pump”. The pumpconcentrates CO₂in BSC, impels RubsiCO to play the roles of carboxylase in fixingCO₂and keeping away from O₂. This can alleviate photorespiration the losses of, raisethe efficiency of photosynthesis and increase production. As the increase ofpopulation, the crops can not provide enough food for human. So people may applyC₄Photosynthesis to C₃rice to raise the efficiency of photosynthes and to increaseproduction.Setaria viridis is one of NADP-ME subtypes，its survival ability is very strong. S.viridis with a small genome （510Mb）, dwarf stature （10–15cm）, rapid life cycle（about6weeks）,high seed production. The most important aspect is we can useAgrobacterium tumefaciens to transform S. viridis, this method is high-efficient andstable. So S. viridis has great potential to serve as a model for the genetic dissection ofC₄photosynthesis. It helps us to understand the molecular mechanisms of C₄photosynthesis and accelerate the pace of engineering C₄traits into the rice in order tosubstantially increase production.In this study, a series of mutants in morphology were screened and three cell-specific promoters were cloned. After obtaining a number of S. viridis seeds, seedswere induced with0.2﹪and0.4﹪EMS for24h. Lastly,12,000M1seeds weregained. Seeds were induced with γ-rays150Gy,200Gy and300Gy.Lastly,13000M1seeds were gained. We have grown6,000lines induced by EMS and3700linesinduced by γ-rays respectively. The deformed,etiolated,dwarf,more-tillering plants inmorphology, and premature、infertile mutants were found. The S. viridis genome is being sequenced, so we refer to the homologoussequences of Setaria italica to clone the promoters of PEPC,PPDK and PCKrespectively, which cell-specific expressed in Zea mays. The promoters of S. viridisare different from promoters of S. italica by ClustalX2.0analyse. These differencesmay affect the expression patterns of promoters and expression activity. By PLACE,the cis-acting elements such as TATA-box、CAAT-box are confirmed, the bindingelements of transcription factor DOF and GT1were also confirmed.These results area basis of researches on cell-specific promoters.The fragments of promoters are ligated into pCAMBIA3301, replaced35Spromoters which drove GUS express. The reconstruction vectors were transformedthe callus and multiple bud dumps of S. viridis.The seeds without episperm wereinduced to generate the callus after sterilize, however, it is difficult to obtain thehealing callus.Embryogenic callus are more likely to die in the subculturingmedium.The callus are easily to be hydrated, these regenerative power is gettingworse. Meanwhile, the multiple bud dumps were transformed. The shoot tipsdeveloping from sterile seeds were used as explants. The medium with2,4-D0.5mg/L,6-BA2.0mg/L induced multiple bud dumps, and the multiple bud dumps wereregenerated. The multiple bud dumps were well-differentiated, but so far, thecondition of culture has not been mastered and herbicide-resistant plants have notbeen gained.The innovations in the dissertation:1. S. viridis mutants were preliminarily screened,which is the basis of furtherscreening the genetic stability of S. viridis C₄defect mutants.It also helps people tobetter study genetic mechanisms of C₄photosynthesis.It was the first time we clonedthe S. viridis promoters of PEPC、 PPDK、PCK specifically expressed in cell, thenthe cis-acting elements such as TATA-box、CAAT-box are confirmed, the bindingelements of transcription factor DOF and GT1were also confirmed.These results area basis of researches on cell-specific promoters functions. LBA4404mediated 2. The fragments of promoters were ligated into pCAMBIA3301, replacing35Spromoters which drove GUS express. Lastly, LBA4404infected the callus andmultiple bud dumps of S. viridis respectively to gain the resistant plants, which is thebasis of researches on the role of promoters specifically expressed in cell.