Construction Of RNAi Expression Vector Of CmMET1 Gene And Genetic Transformation Of Dendranthema × Morifolium And Artemisia Annua L.

With the development of society, people pay more attention to the economic, cultural and social values of ornamental plants. So they have higher requirement for breeding of ornamental plants. DNA methylation is vitally important for gene silencing and genome stability in the Chrysanthemum(Dendranthema × morifolium). In order to seek more exotically breeding targets, this research propose a new breeding idea which using RNAi to control the expression of DNA methyltransferases gene in Chrysanthemum, transferring the signals through grafting the Artemisia annua L., then changing the DNA methylation level of Chrysanthemum genome, finally inducing it to produce trait variation. Therefore, the aim of this research is to create new variation, through using RNAi to control the expression of DNA methyltransferases gene, which can lower the level of Chrysanthemum DNA methylation. The main results were as follow:(1)The diversity analysis of CmMET1 gene sequence in ChrysanthemumWith the help of the software DNAMAN, we analysed the cDNA nucleotide sequence of MET1 gene from Chrysanthemum and Artemisia annua L, analysed the diversity of 10 species of Chrysanthemum target sequences with 4 nucleotide sequences. Sequence alignment analysis showed that only by Chry2 primer can get a lower nucleotide sequence homology between Chrysanthemum varieties, and the highest similarity rate reached to 63.74%. The nucleotide sequences produed by the other three pairs of primers were highly homologous to the Chrysanthemum cultivars, with a similarity of 97.43%-100%.(2)RNAi vector constructionSelected two homologous sequences from different Chrysanthemum cultivars, which located between 2417 th to 2515 th bases as well as between 1601 th and 1767 th bases in Chrysanthemum CmMET1 gene. Designed primers on both sides of the position,and the amplified fragment length were 136 bp and 183 bp respectively. Used the method of "enzyme- links" to construct RNAi vector(DHpart27RNAiFADP1P4) successfully, and then transferred it into Agrobacterium GV3101.(3)The established system for callus induction and callus regeneration of Chrysanthemum cultivar "Zi Jing Ling" and "Guo Qing Hong"(1) Optimized callus induction and callus differentiation medium of "Zi Jing Ling" stem:MS+3% sucrose+0.7% agar+2.0 mg/L 6-BA+1.0 mg/L 2,4-D, induction rate was 76.7%;MS+3% sucrose+0.7% agar+1.0 mg/L 6-BA+0.8 mg/L NAA, differentiation rate was 75.3%;(2) Optimized callus induction and callus differentiation medium of "Guo Qing Hong" stem:MS+3% sucrose+0.7% agar+2.0 mg/L 6-BA+1.5 mg/L 2,4-D, induction rate was 63%;MS +3% sucrose+0.7% agar+1.0 mg/L 6-BA+0.6 mg/L NAA, differentiation rate was 63.8%;(3) Optimized rooting medium of "Zi Jing Ling" and "Guo Qing Hong" :MS + 3% sucrose + 0.7% agar, rooting rate achieves 100%.(4)Transferred RNAi vector into the ChrysanthemumTransferred RNAi vector of different nucleotide sequences into the callus of "Zi Jing Ling" ? "Guo Qing Hong", and finally got 16 "Zi Jing Ling" and 14 "Guo Qing Hong" in the condition of Agrobacterium OD600 reached to 0.4-0.5 and Kan concentration 10 mg/L. Many of the transformed plants showed a slow growth, shorter stem length, longer petiole, fewer leaves, leaf bleaching and death. Selected stronger 8 "Zi Jing Ling" and 6 "Guo Qing Hong" to screening Kan+ marker and obtained 6 and 5 positive transformation plant respectively. Then selected 2 strongest from the positive transformation plant to detect CmMET1 gene expression and the results showed that compared with controled group, the CmMET1 gene expression quantity were 54%, 52.5%, 53% and 54.5%, respectively in "Zi Jing Ling" and "Guo Qing Hong" transformed plant.(5)Transferred RNAi vector into Artemisia annua L.Transferred RNAi vector of different nucleotide sequences into the callus of Artemisia annua L. and finally got 8 thrasformed plants in the condition of Agrobacterium OD600 reached to 0.5-0.6 and Kan concentration 8 mg/L. The transgenic plant contained homologous RNAi nucleotide sequence showed a phenotype of longer petiole, lessleaf blade, less and shoal blade incision and death. The transgenic plant contained non homologous RNAi nucleotide sequence showed a phenotype of shorter stems, slower growth. Five stronger transformed plants from 8 were selected to be subjected to Kan+ screening and 3 positive transgenic plants were obtained. Two of positive transgenic plants were selected to detection of MET1 gene expression, and the results showed that compared with controled group, the MET1 express of transformation plant were 90.5% and 95.5%, respectively.