Molecular Cloning Of MVD Gene And Essential Oil Analysis From Houttuynia Cordata Thunb

Houttuynia cordata Thunb is the whole body of Houttuynia. Due to the interference of polysaccharide, it is difficult to isolate RNA from Houttuynia cordata. The MVA way is exists in plastid of plant.The IPP is not only the product of the MVA way, but also the important precursor substances of essential oil synthesis. The MVD which catalyzed MVAPP to IPP was a key enzyme of the MVA pathway.Taking Xianghai Houttuynia cordata Thunb as materials, the main researches of this thesis were listed as follows:(1) Four extraction methods: Classic Total RNA Isolation Kit, Trizol A, Trizol B and Easy Way RNA Amylose Plant MiNi Kit were compared for extracting total RNA from Houttuynia cordata. The results showed that it was difficult to extract total RNA from Houttuynia cordata leaf by Trizol and Classic Total RNA Isolation Kit method. Total RNA extracted by EasyWay RNA Amylose Plant MiNi Kit method had high quality and could be use for the further research of molecular biology. The ordinary RNA isolation Kit was hard to extract high-efficiency total RNA from Houttuynia cordata. The EasyWay RNA Amylose Plant MiNi Kit was the best method of total RNA isolation.(2) RT-PCR was carried out with degenerate primer which designed by using some internet database and biological software such as NCBI、GENEDOC.EXE、Primer Premier 5.0. and a cDNA fragment with length of 514 bp was cloned from the Houttuynia cordata.Then, the 3’and 5’ends of the MVD cDNA was cloned by 3’RACE and GenomeWalker, respectively. The MVD cDNA include an open reading frame of 1266bp, which coding 421 amino acids. This sequence was submitted to NCBI and the accession number was JF430481.(3) MVD gene expression in different parts of Houttuynia showd that express quantity in root was very high, but were lower in stem, flower and leaf. The essential oil from Houttuynia cordata was obtained with steam distillation. Essential oil of flower, root, leaf and stem were decrease progressively.Qualitative and quantitative analysis was done by GC-MS with areas normalization method. 35 compounds were identified in root, stem, leaf and flower, among which 24 components simultaneously exist. The types of the components were similar while the contents were different. The differences and similarities among different parts were analyzed with Principal Component Analysis, Mahalanobis distance and Correlation Coefficient. The results showed that the difference between the stems and leaves was little, and difference between flowers and roots, stems, leaves was significant, and the differences between the root and the other three parts was large. There exists certain correlation between the essential oil components and MVD gene expression, but is various between different essential oil components and MVD gene expression.