| The flower of O.fragrans Semperflorens Hort is quietly elegant, it is light yellow, and the flowers bloomed all the year. But the flower quantity is little, even in full bloom period. The fragrance of O.fragrans is the weakest among various osmanthus, the flowers’ fragrance almost can’t be smelled, this limits its ornamental and commercial applications. So while the essential oil of osmanthus is widely used in medicine and food industry, but few commercial application of O.fragrans Semperflorens Hort essential oil is reported.The research is to improve the fragrance of O.fragrans Semperflorens Hort and the spatial and temporal expression differences of it, and explore the best technology of oil extracted from O.fragrans. The extraction capacity was used as measurement index. The extraction process of extraction with petroleum benzene was optimized, and the best extraction conditions were gained. To enhance the fragrance of O. fragrans, this essay cloned and carried on the bioinformatics analysis of the AACT gene, and tried to build AACT plant expression vector, to explore a feasible way for improvement fragrance of O.fragrans and spatial and temporal expression difference. This essay also studied the characteristics of difference of spatial and temporal expression, providing theoretical support for plant expression vector of transformation receptor. The main result of the experiment are as follows:1. The best extraction technology of essential oil by petroleum ether was:crush to 20 mesh, the solid-liquid ratio of 1:5 m/v, the extraction temperature of 60℃, the extraction time of 4 h. The GC-MS analysis showed that the aroma components included alcohol, esters, acids and other substances.The terpenoid was accounted for more than 85% in the compositions of O.fragrans. And the squalene was the highest content in those compositions, it was counted for 11.5986%. The single terpenoid substrances such as α-pinen, β-ocimene, phellandrene, acmphene and linalool were about 16.5957% in essential oil. And the content of ionone was 12.1328%.2. We gained the full-length sequences of OsAACT cDNA by RACE technology. The results of bioinformatics analysis showed that the open reading frame of OsAACT was between 80～1297 bp, encoding 405 amino acids, relative molecular mass was 41347.89D, PI was 5.66. It was the stability of hydrophobic protein,belonged to the Cond-enzymes super family. And both the amino acid sequence alignment and the evolutionary tree analysis could prove that the conservatism of the OsAACT was high, the AACT gene was reliable.3 The semi-quantitative analysis was used to observe the expression differences of tissue and time. It was to analyze the expression value of AACT gene in different quarters and periods of petals and leaves. The expression value was represented by the AA CT value/ 18S value. The petals’ expression levels of OsAACT were similar at different seasons, the similarity was 99%, the values were around 1.0241, the deviation was less than 0.0201. But the leaves’ expression levels of OsAACT were quantity differences in different seasons, it was gradually reduced with the season transformation, the value was reduced from 0.7740 to 0.1200. Meantime, the expression level of OsAACT in petals was much higher than that in leaves, the expression quantity gap between flowers and leaves of May, June, September and December was 0.3,0.4,0.6,0.9 respectively. The significant was obvious. The expression level of OsAACT was the highest in the early flowering petals, the value was about 0.9, and it was the least in the fade period petals, the value was about 0.4.4.The OsAACT cDNA and the pCMBIA2301 vector were digested by the BamHI enzyme and the KpnI enzyme. The digestion fragment were linked together by the T4 DNA ligase.The AACT gene with 1218bp was inserted to the pCMBIA2301 vector with 11633bp. We gained the plant expression vector with the CaMV35s promoter and the purpose gene-OsAACT. The full length of the new vector was about 12848bp. |
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