Transformation Of PtMADS5 Gene In Populus Tomentosa And Study On Knockout Plant By CRISPR-Cas9

Populus is a widely cultivated species in the world. They are viewed asmodel systems in plant biology. Populus is the third plant species which is sequenced after Arabidopsis and rice. And it is also the first complete sequenced wood species. The sequencing of the Populus genome promises to enrich the biology knowledge of energy sources. The plan shows important practice value.In order to study the roles of MADS-box genes in poplar development, one MADS-box gene was isolated from cambium materials using the PCR-based strategy combined with RACE methods. The gene was named as Pt MADS5. This gene‘s full-length was 1029 bp, and ORF was 660 bp(133-792), which was registrated as DQ646480 in Gen Bank. Sequence analysis demonstrates the gene are highly conformed in MADS domain with other MADS-box genes. They have typical structure of MADS-box genes. Homology tree analysis indicated that Pt MADS5 share high homology with SOC1, and belongs to the SOC1/AGL20 subfamily.RT-PCR was carried out to examine the expression patterns of the gene. The reasult shows that Pt MADS5 gene is expressed in roots, stem and flower, and didn‘t express in leaf.The 35S::Pt MADS5 expression was constructed, and which was transformed into plant by Agrobacterium. The transgenic plant of 35S::Pt MADS5 gene is much dwarf than the wide type. The shapes of leaves are also changed.Clustered regularly interspaced short palindromic repeats(CRISPRs) is Widely distributed in the genome of bacteria and archaea. The CRISPR-Cas system is a kind of adaptive immune system, which was formed in the evolutionary process of bacteria and archaea, gradually. The CRISPR-Cas system positions to the targeted point with sg RNA,and the DNA was double-strand break by Cas enzyme. CRISPR-Cas system as a new gene fixed-point editing techniques has been successful applied in many plants. Targeted genes can be targeting knock-out and knock-in by the technology of CRISPRs.The research was systematic on study of callus induction of Arabidopsis, and using the vaccine leaf as explant. A vector was constructed which knockout AG gene by CRISPR-Cas9 system, and then the Agrobacterium-mediated genetic transformation of CRISPR-Cas9 system was studied. The high efficient genetic transformation system was expected. The mutants of knockout were obtained. The main results were as follows:1.1/2 MS mediuinm( 2, 4-D 0.5 mg/L + 6-BA 0.5 mg/L) was the optimum for Arabidops is callus induction. Concentrations of antibiotics screening was Kan 50 mg/L, Concentrations of Cef 500 mg/L was as optimal screening concentration. The optimum conditions for genetic transformation was that precultured for 5 d, used agro bacterium liquid concentration for OD 600 = 0.5 to infect Arabidopsis callus for 20 min, and cultured 2d.2.Genes was fixed-point knocked out by the system of CRISPR-Cas9. In the study, we o btained mutations of the knockout length of 63-257 bp. The result showed that both mutations of fragments missing and additional sequence insertion appeared.