| Physcomitrella patens is a potential model plant for researching plant functional gene. Physcomitrella patens has high homologous recombination rate and the determination to complete genome sequences has already completed, which is possible to make use of gene targeting technology on its specific gene function research. The earlier research in our lab found that PpCalS12 gene and NPR1 gene may be associated with Physcomitrella patens resistance to grey mildew infections.In order to confirm the mechanism of PpCalS12 gene and NPR1 gene furtherly, this research has mainly carried out the following work: 1、(1) Filtering of CaCl2 can get higher transformation rate than high-press sterilizering of CaCl2.(2) Under the same conditions, and long-term preservation of E. Coli activating more than three times can get the highest transformation rate.(3) Under the same conditions, the highest transformation rate conditions are as follows :4℃,4000 g centrifugated for 15 minutes, then 4℃, 4000 g centrifugated for 5 minutes. 2, The test based on pTN182,the upstream and downstream homologous arm of Pp CalS12 and NPR1, through methods such as enzyme digestion and connection build PpCalS12.1-pTN182-PpCalS12.2 and NPR1.1-pTN182-NPR1.2 knockout vector. The enzyme digestion result is the expected fragment size. PpCalS12.1-pTN182-PpCalS12.2 target fragment sequencing results showed that the similarity rate is 99.10%.,and NPR1.1-pTN182-NPR1.2 is 99.10%. 3, Using the method of PEG mediated transformation PpCalS12 gene, two G418 resistance screened 8 plant regeneration plant.Through this experiment: 1, master the conversion rate of optimization conditions with competent cells of E.coliDH5α. 2, Successfully built knockout vectors of Physcomitrella patens PpCalS12 and NPR1 gene. 3, Get mutant plants of Physcomitrella patens, which may contain missing PpCalS12 gene. These create conditions for further studying PpCalS12 and NPR1 gene, and laid the foundation for Physcomitrella patens resistance mechanism. |
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