| 1.[Objective]In this study,maize inbred Zheng 58 was transformed by the ultrasonication-assisted pollen-mediated transformation method with gene NtAn2 and the study is to optimize pollen-mediated genetic transformation system in maize using ultrasoncation treatment.[Method]In order to test the in vitro germination rate of fresh corn pollen in different concentrations of sucrose solution,three factors of ultrasonic treatment,treatment power,treatment duration and treatment times were optimized through an orthogonal design experiment.Through orthogonal test,the most effective combination of the four factors of the concentration of Agrobacterium,the concentration of ultrasonic treatment,the concentration of sucrose and the concentration of AS were optimized.Then NtAN2 was transformed into maize inbred lines by pollen mediated transformation.PCR detection,blot Dot blot analysis,blot Southern hybridization analysis,and determination of anthocyanin content in transgenic plants were compared and analyzed.[Result]The optimum concentration of sucrose solution was 20%.The optimal parameters of the ultrasonication treatment of pollen were determined as follows:ultrasonic treatment power 150W,processing duration 5s,and 6 with times.Considering the conversion efficiency and strong efficiency conversion peasants Bacillus solution and the optimum concentration OD600 for 0.6,AS the optimum concentration of 100 mol/L,sucrose solution the optimal concentration of 20%,ultrasonic processing parameters for processing power of 150W,processing duration 5s and processing times for 6 times.Two of the transgenic plants were confirmed by both color selection and PCR detection and Southern blot hybridization.It is verified that the anthocyanin gene was integrated into maize genome and could be used for visual selection of transformants Then the T2 plants on test results were positive,confirmed that the exogenous gene can be stably inherited.The optimized genetic transformation system of maize by pollen mediated-method assisted with ultrasonication treatment was established,which laid a foundation for improving the efficiency of the method.2.[Objective]Garlic is a now edible plants,concern,it in the plant,animal and human medicine have an important position,especially its bacteriostatic effect in plants,the use of this nature explore it on the corn germ inhibitory effect,and there are studies to indicate that cysteine synthase product is rich in sulfur amino acids in garlic is an important source of sulfur amino acids in plants,properties and biological functions of the garlic cysteine synthase to clarify its role in garlic sulfur metabolism.To study the properties of cysteine synthase and its expression in different tissues of quantity.[Methods]he use of coated perforated method comparative analysis of garlic crude enzyme liquid,allicin,garlic amino acid enzyme extraction of corn mold,silk smut fungi,black powder bacteria inhibition.Using NCBI database bioinformatics and RT-PCR,three cysteine synthase genes of garlic was found,ASGCS2,ASGCS3 and ASGCS4.[Results]The lengths of their open reading frames were1152bp,1296bp,and 1236bp,respectively.The theoritical relative molecular weights of their coded enzymes were 40.6KD,34.1KD,and 36.1 KD,respectively.ASGCS2 was localized in the chloroplasts,while ASGCS3 and ASGCS4 were localized in the cytoplasm.The results of the amino acid sequences revealed that the similarity of ASGCS2 with ASGCS3 was 70%,and that with ASGCS4 was 59%,while it was 68%between ASGCS3 and ASGCS4.Phylogenetic analysis showed that AsGCS2 belonged to Bsas2,AsGCS3 belonged to Bsas1,AsGCS4 was different from all known cysteine synthases,possibly belonging to Bsas6 or Bsas7.RT-PCR expression analysis indicated that the expression level of CSase varied in various tissues.The expression level of ASGCS2 was the highest in the leaves,the highest expression of ASGCS3 was in the root,and ASGCS4 expressed highly in both leaves and roots. |
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