The Research For Flavone SynthasesⅡ Variation Features Of Lonicera Japonica And Mignonette Glycosides Content Changes

Objective:First of all, compare the part of the FS II gene sequence which is the key gene of biosynthetic pathway of galuteolin, among different cultivated varieties of Lonicera Japonica, among different varieties of honeysuckle, and among different traditional Chinese medicine, to summarize its variation characteristics; then, determine the content of galuteolin in different cultivated varieties of Lonicera Japonica, to analysis the law of content change; finally, combine the molecular biology experimental results with the assaying experiment results, to analysis whether there is a certain correlation between the FS Ⅱ gene polymorphism and the expression quantity of galuteolin.Method:Design specific amplification primer based on the part of FS Ⅱ gene encoding area sequence of Lonicera Japonica which have been reported on NCBI, extract total RNA of each sample, acquire part of FS Ⅱ gene coding sequence of Lonicera Japonica through RT-PCR and sequencing, make a comprehensive comparative analysis using MEGA5.0software and Blast function of NCBI. Extract effective components by ultrasonic, establish the condition of high performance liquid chromatography, determine the content of galuteolin in different cultivated varieties of Lonicera Japonica after the methodology validation, and do some preliminary comparison and simple induction.Result:1. Acquire part of FS Ⅱ gene coding sequence of Lonicera Japonica:The integrity, purity, concentration of all total RNA meet the requirements. PCR products are in line with expectations, with high specificity. After sequence similarity comparison Using NCBI BLAST function, identified the part of cDNA fragments is which of Lonicera Japonica.2. Intraspecific variations of Lonicera Japonica:There is no differences among the20cultivated varieties of Lonicera Japonica. But we found a missense mutation happened on one abasic site of the21and22samples.3. Intraspecific variations of Honeysuckle:The two bases of the above two sequences are in line with most varieties of honeysuckle. Mutation happened in multiple sites of Red glandular honeysuckle, there are there missense mutations, the other two are same sense mutations without changes of amino acids. There is a genetic relationship of relative distance among different varieties of honeysuckle, the genetic relationship of Lonicera macranthoides and Lonicera Japonica is close, red glandular honeysuckle is far away from others. In addition, there is no mutation at Heme combination zone of FS II amino acid sequence of all varieties of honeysuckle.4. Intraspecific variations of different genera:There are obvious differences of FS Ⅱ gene coding sequence among different genera, but they follow the genetic relationship. 5. determine the content of galuteolin:Established the HPLC experiment parameters of Lonicera Japonica determination(the preparation method of samples, the selection of moving phase, The choice of detection wavelength and so on). Determine the content of galuteolin in22different cultivated varieties of Lonicera Japonica. The pharmacopoeia has a regulation about content of galuteolin in Lonicera Japonica, so we classify them to three categories according to the numerical sizes of the experiment result.Conclusion:1. There are a certain degree polymorphisms of the FSⅡ gene coding region sequence in the part analyzed among different cultivated varieties of Lonicera Japonica among different varieties of honeysuckle, and among different traditional Chinese medicine.2. There is a big difference among the contents of different cultivated varieties of Lonicera Japonica, but the law is not very clear.3. It is not showed obvious correlation between the FSⅡ gene polymorphism and the expression quantity of galuteolin by the experimental result of this article, but we detailed analyzed the influence factors of the content differences.