Construction Of Rnai Expression Vector Of Dendranthema × Morifolium Met1 Gene And Establishment Of Regeneration System Of Artemisia Annua L.

Chrysanthemum(Dendranthema × morifolium) is a perennial herbaceous plant with perennial root, which is popular for its exceptionally diverse in varieties, flower shape and color. Sweet Wormwood Herb(Artemisia annua L.) is annual herbs and regarded as the rootstock used in the chrysanthemum grafting. As the most common epigenetic modification, DNA methylation plays an important role in plant. As a new technology for knocking out specific genes, RNAi(RNAi interference) is applied extensively to plant functional genomics, plant stress resistance, plant breeding. Based on the theory of epigenetics, morphological character can be changed for the reason of DNA methylation and small RNA which could transmitted through the vascular. The purpose of our research is construction of RNAi expression vector of Chrysanthemum MET1 gene and establishment of regeneration system of Artemisia annua L.. The RNAi vector knocked out the MET1 gene in Chrysanthemum through the method of RNAi interference which could interfere the MET1 gene in Chrysanthemum not Sweet Wormwood Herb. The results laid a foundation for the furture genetic transformation. The main results were described as follows.(1) The target gene sequence cloningThe 273 bp fragment of MET1 gene with different restriction enzyme cutting sites was cloned from Zi-jingling chrysanthemum. The MET1 gene fragment obtained was sequenced, and the DNA sequence indentity was 97 percent with MET1 gene in references. The sequence was confirmed as a part of Zi-jingling chrysanthemum MET1 gene.(2) The construction of chrysanthemum RNAi vectorRespectively, the sense and antisense gene target fragments were connected with RNAi vector DHpart27RNAiFADP1P4 which contained an intron, using the method of enzyme ligation-connecting. With PCR reation, restriction endonuclease digestion and gene sequencing, the target gene sequence was confirmed in RNAi vector DHpart27RNAiFADP1P4 and not gene mutation. The RNAi vector was transformed into Agrobacterium GV3101 by freeze-thawing.(3) Leaves and petioles of 40 days Artemisia annua L. were used as explants in order to establish the genetic regeneration system.The results showed as follow.① The best sterilization effect of leaves and Petioles were 8 min with 0.1 percent HgCl2, 10 mim with 0.1 percent HgCl2. The living rates of leaves and petioles were 92.1percent and 84.5 percent, respectively.② The optimal callus induction mediumExplants taken from leaves and petioles were cultured on MS medium supplement with 2.0 mg/L 6-BA + 0.5 mg/L 2, 4-D + 3 percent sugar + 0.7 percent agar. The induction frequency of petioles was 95.7 percent, far higher than the induction frequency of leaves(78 percent).③ The optimal differentiation mediumTransferred onto MS medium supplement with 1.0 mg/L 6-BA + 0.05 mg/L NAA + 3 percent sucrose + 3 percent agar powder, the callus of leaves differentiated at a frequency of 95.3 percent. Transferred onto MS medium supplement with 2.0 mg/L 6-BA + 0.5 mg/L 2, 4- D + 3 percent sucrose + 3 percent agar powder, petioles could differentiated one-step seedling, at a frequency of 11 percent.④ The optimum rooting mediumDifferentiation of Artemisia annua L. shoots were rooted on the medium of 1/2 MS + 1.0 mg/L NAA + 3 percent sucrose + 0.7 percent agar. After 6 days, white roots eyes were germinated fast and robustly, and the rooting induction rate was 98.9 percent. 21 days later, the survival rate of transplanted seedlings root was 90 percent.