CDNA Cloning Of GES And ADH Aroma Genes From Osmanthus Frangrans Var. And Transformation Research Of Loropetalum Chinense Var. Rubrum

Osmanthus fragrans is a kind of ornamental tree with strong frangrance, and planted widely in our country. Loropetalum chinense var. rubrum is another kind of ornamental tree cultivated widely in southern China,which has strong ecological adaptability and bright color but lack aroma. Here, we using petals of O. fragrans var. semperflorens as materials, the full-length cDNA cloning of GES and ADH genes were carried out by homologous cloning and RACE, then, bioinformatics analysis of the two genes was followed. The expression pattern of GES and ADH genes were analyzed by semi-quantitative RT-PCR using petals and leaves from different seasons, tissues and blossoms as materials. Meanwhile, the high-efficient regeneration system of L. chinense var. rubrum was established using stems as explants, and then, the agrobacterium mediated transformation system of L. chinense var.rubrum was also established. The results are as follows:1. The full-length cDNA cloning of GES and ADH genes from O. fragrans var. semperflorens. Using petals of O. fragrans var. semperflorens as materials, the total RNA of petals were extracted, then reverse transcripted to produce first strand cDNA. Using the cDNA as template, the conservative fragment was obtained by RT-PCR. Basing on the conservative cDNA sequence, the full-length cDNAs of GES and ADH genes were finally cloned by 3’RACE and 5’RACE, respectively.2. The bioinformatics analysis of O. fragrans var. semperflorens GES and ADH genes. The analytic results showed that GES cDNA is 1900bp in length; the ORF has 1745bp, which codes a 583 amino acid protein with 146839.3Da and 4.92 isoelectric point. The protein structure prediction showed that GES doesn’t have transmembrane helical region, contain many a-helics and a little β-helix regions, and has many amphiphilic helix regions. The cDNA of ADH is 1223 bp in lenghth, and contain a 1005bp ORF, which codes a 334 amino acids protein with 36105.99Da and 5.76 isoelectric point. The protein structure prediction showed that ADH also has no transmembrane helical region, contain many β-helics and a little a-helix regions, and has some amphiphilic helix regions.3. The expression pattern of O.fragrans var. semperflorens GES and ADH. Using leaves and petals collecting in March, June, September and December as materials, their total RNAs were extracted and then reverse transcripted into first strand cDNAs, respectively. The results of semi-quantitative RT-PCR indicated that both GES and ADH genes have higher expression levels in petals than in leaves, especially in full-blossom period.Then we indicate that the expression levels may have an important correlation with aroma volumes of O.fragrans var. semperflorens.4. Establishing of high-efficient regeneration system of L. chinense var. rubrum. Using stem tips as explants, to optimize the medium type and hormone combination in induction and multiplication of callus, induction and rooting of adventitious buds. The results showed that the optimal sterilizing condition is two steps of 0.1% HgCl2 sterilization during 3 minutes, the optimal mediums for callus induction and multiplication are MS + 6-BA l.Omg/L + NAA1.7mg/L and MS + 6-BA 1.0mg/L + IBA 0.06-0.08mg/L, respectively. By introducing AgNO3 firstly, the optimal medium for adventitious bud induction is confirmed as MS + 6-BA 1.5mg/L + NAA 0.01mg/L + AgNO3 1mg/L. The optimal medium of adventitious bud multiplication is MS + 6-BA 1.0mg/L + IBA 0.05mg/L + Vc 5.0mg/L. The optimal program for rooting of adventitious bud is:choose 2.1-3.0 cm tall buds with 2-4 leaves and inoculate to the medium 1/2 MS + IBA 4.5mg/L.5. Transformation of O. fragrans var. semperflorens ADH gene into L. chinense var. rubrum. Basing on the cDNA cloning of O. fragrans var. semperflorens ADH gene and the high-efficient regeneration system of L. chinense var. rubrum, using full-length cDNA of ADH gene as template, the ORF of ADH gene was inserted into plasmid pCAMBIA2301 by PCR and double enzyme digestion. The constructed over expression vector with ADH gene was successfully transformed into L. chinense var. rubrum by mediated agrobacterium. By resistance screening and molecular detection, the transgenic individuals of L. chinense var. rubrum were obtained finally.