Cloning And Expression Analysis Of Two Key Enzyme Gene Of The Blinin Pathway From The Medicinal Plant Conyza Blinii H.

Conyza blinii H. Lev is a member of the family Compositae conyza. It is a traditional Chinese medicinal plant that is distributed mainly in southwestern Sichuan and northern Yunnan. It has effect of detoxify, antiasthmatic and expectorant. It contains flavonoids, saponins and blinin. Blinin only exists in conyza blinii that is its characteristic compounds which can be used as the quality control of this medicine. The level of this ingredient in the plant is low which is insufficient to meet demand for pharmaceutical preparations. Improving blinin content is the key to improve the quality of conyza blinii. Blinin biosynthetic pathway elucidation is the basis to improve its content, but currently has no research about blinin biosynthetic pathway. This study had been preliminary researched on conyza blinii germplasm resources and active ingredients. The project will use homologous cloning and RACE to clone the enzymes of blinin biosynthetic pathway. It will be the first time to explore the blinin biosynthetic pathway. Besides will use semi-quantitative and HPLC techniques to analyse time and spatial characteristics of enzymes activity and blinin content in conyza blinii whole growing season summarized their relationship. The results of this study are as follows:1. Homologous cloning and RACE were used to clone the housekeeping gene 3-glyceraldehyde phosphate dehydrogenase (GAPDH) from C.blinii. The gene full-length was 1020bp encoding 340 amino acids. Its GenBank accession number was KF027475. Compares with other plant GAPDH gene the similarity reach more than 93%. The semi-quantitative PCR results prove that glyceraldehyde-3-phosphate dehydrogenase gene is able to be reference gene for gene expression analysis. The results showed that CbGAPDH gene has stable expression in various parts and it can be used as internal reference.2. The DXS gene was successfully cloned and characterized from Cblinii. It was designated cbDXS and contains a 2190-bp open reading frame encoding 730 amino acids (aa), including a 17-aa signal peptide and a 713-aa mature protein. Its GenBank number is KJ155788. Compared with the sequence of GenBank database revealed that the gene has a TPP binding domain, and this DXS gene belongs to the plants DXS class Ⅱ. Semiquantitative PCR was taken out to inspect the expression levels of CbDXS gene in different types of tissues at different developmental stages. The results showed that at seedling-stage the expression of CbDXS in leaves, stems and roots are 72.67%,17.67% and 28.00%. At flowering stage the CbDXS gene has not expression in the flowers and roots, the expression of leaves and stems are:59.27% and 34.37%. The corresponding blinin concentrations were analyzed by high-performance liquid chromatography (HPLC). The results showed that both seedling stage and flowering stage the leaves have the highest blinin concentrations. At seedling stage blinin concentrations in leaves were 0.961%, while the content of the stems and roots was closely, the stem is 0.228% and 0.262% in the roots. At flowering stage blinin concentrations in stems and leaves were about 1.5 fold than seedling stage. It is 1.534% in leaves and 0.378% in stems. It’s not obvious improving in root which is 0.309%. The blinin concentration in flowers was 0.568%.It is higher than the content of stems and roots. Correlation analysis results: CbDXS gene expression in levels regardless of periods are positively correlated with blinin concentrations; Expression of CbDXS gene in stems and blinin concentrations were correlated most significant; While the correlation between expression of CbDXS gene in flowers and roots and blinin concentrations was small or irrelevant.3. Additional, cloned a GGPPS gene designated CbGGPPS was 1342bp contained a 1086bp open-reading-frame encoding a protein of 362 amino acids. It has a conserved aspartate-rich sequence belonging to GGPPS family. Its GenBank No. is KJ652907. The CbGAPDH is used as the internal control gene in this study. Semiquantitative PCR was taken out to inspect the expression levels of CbGGPPS genes in seedling-stage and flowering stage. The expression of CbGGPPS gene in seedling-stage leaves, stems and roots are 70.80%,66.03% and 17.50%. And at flowering stage CbGGPPS gene was expressed in various parts, showing strong tissue specificity. The expression in flowers was 3.37%, in leaves was 60.13% and stems of 15.20%, the smallest in roots of 2.50%. Correlation analysis found that, unlike CbDXS, the expression levels of CbGGPPS gene in all parts except flowers and blinin concentrations are significantly correlated; Expression of both CbGGPPS gene and CbDXS gene is significantly correlated. Additional, the CbGGPPS gene was cloned into pET30b(+) vector and transformed into Escherichia coil BL21. The gene was expressed fused with E.coil and had an approximate molecular mass of 40.0 kDa. Proved this study successfully obtained the enzyme gene.