Cloning Of A Ferritin Gene AsFer From Artemisia Sphaerocephala And Construction Of Its RNAi Vector

Artemisia sphaerocephala Krasch, also called seed Artemisia or yellow hair rapeseed, is not only the main constructive species and dominant species on the sandy vegetation, but also the ideal drought-resistance, sand-prevention and sand-fixation plants. Ferritins (iron-binding protein) are ubiquitous iron storage proteins that are present in plants, animals and micro-organisms, they are highly conservative, multi-functional and multi-subunit proteins. As improving iron content of organisms, regulating iron biological function, resisting the external environment stress, preventing the occurrence of iron-deficiency disease, Ferritins were highly concerned by the researchers. In this research, A. sphaerocephala was seclected as experimental material, a whole new Ferritin gene(AsFer) was cloned, and its RNAi plant expression vector was constructed. It has an important significance in pasture adverse resistance and pasture genetic engineering research.The main results are as follows:1. The degenerate primers which based on the conserved sequences of Ferritin genes from other plants were designed. A Ferritin gene was isolated from A. sphaerocephala. The leaves of A. sphaerocephala were selected as the material, using RT-PCR to gain the nuclear fragment, and then by using the method of RACE to gain the 3' and 5' sequence. Finally, we acquired the Ferritin gene's full length cDNNA sequence of A. sphaerocephala by sequence assembly and named as AsFer(GenBank No. GQ871941).The cDNA is 1113bp in full length including an open reading frame (ORF) of 741bp, with a 5'untranslated region (5'URT) of 57bp and a 3'untranslated region (3'URT) of 315bp. It encodes a protein of 247 amino acids, which contains ferritin-like diiron domain (94-247AA of the protein) and iron-binding regions signature (214-234 AA of the protein).The AsFer has a high homology with the other plant Ferritin gene relatively, it shares 88.2%,79.2%,77.4%,77.3%,76.5%,76% and 76% homology with the seven reported other Ferritins, namely Conyza canadensis, Glycine max, Vigna unguiculata, Nicotiana tabacum, Jatropha curcas, Solanum tuberosum respectively. Most of amino acids in ferritin gene of A. sphaerocephalaare hydrophilic amino acids, predicting its secondary structure contains 56.68% alpha helix,26.72% coil,12.55% extensions chains and 4.05% beta turn.2. Taking AsFer as the target gene, the gene silence specific RNAi plant expression vector was constructed. From the ORF of AsFer, we selected 370bp fragment to construct palindrome sequence, pHANNIBAL vector was used as basic skeleton, the expression vector function was guided by CAMV 35S promoter, sense fragment and anti-sense fragment were separated by PDK intron, and the terminator is from OCS sequence. Inserting the well-build middle vector AsFer (+)/pHANNIBAL/AsFer (-), which contains hairpin structure, into plant binary expression vector pART27, then finished the construction of RNAi silence vector prNAi/AsFer of A. sphaerocephala.By using freeze-thaw method, the restructured plasmid pRNAi/AsFer was imported into agrobacterium GV1301, and gained the pKNAi/AsFer agrobacterium GV1301 engineering bacterium..3. Constructed the tissue culture of A. sphaerocephala. RNAi expression vector pRNAi/AsFer was transformed into A. sphaerocephala by using Agrobacterium-mediated method, the positive transformants will be selected by using MS medium which contains spectinomycin.