The Study Of Germplasm And Molecular Cloning Of Chalcone Synthase Gene And β-amyin Synthase Gene From Conyza Blinii H. Lev. In Panxi Area

The two years old Conyza blinii H. Lev. (family Compositae) is distributed mainly in the Yunnan and Sichuan provinces of China. It is most abundant in the Panxi area of Sichuan with its arid, hot weather, high altitude and abundant sunlight, an environment that is best suited to C. blinii growth. The Panxi area, located in southwestern Sichuan,is part of the upper Yangtze River region,which comprises Panzhihua city and Liangshan prefecture.For years,the research of Conyza blinii H. Lev. has focused on the chemical composition and efficacy of the main active ingredient,but the study of the active ingredient content is scarcely,and the research based on molecular biology is belong to the blank fields.In this study,we first collected the distribution information of Conyza blinii H. Lev. in Panxi region,then sampled 16 C. blinii from different area in this region. Then we detect the content of inorganic element by ICP-AES, the content of total flavonoids and total saponins by ultraviolet spectrophotometry, the content of rutin, quercetin and blinii. Using RAPD, SRAP and SCAR markers to research the genetic diversity; With RACE technology, CbCHS and CbβAS genes are firstly cloned from this plant.1. The research of growth environment and plant morphology showed that the plant morphology of C. blinii changed greatly in different growth stages and the the comparison of ultra-microscopic result showed these leaf structure is not significant.2. The results of inorganic elements content test show that five different sources of C. Blinii are free of heavy metals Cr and Cd. The content of total flavonoids in 16 different sources with an amount from 4.660% to 7.530% and the rutin and quercet in leaf have the highest content than that in flowers, stems and roots.The content of total saponin in 16 different sources with an amount from 0.566% to 0.909% and the blinii amount from 0.560% to 1.185%, the content in leaf are both higher than that in other parts. As a reference of the ingredients, the 16 sources can be clustered into four categories. It means that the similarity of the active ingredient is not entirely related to its geographical location in the distance,but also with the growth phenology of them.3. A total of 147 bands were amplified by 15 RAPD primers; 97.14% of these were polymorphic, and 92.38% of the polymorphic bands were observed in 341 bands amplified by 12 SRAP primers. Combined RAPD-SRAP cluster analysis divided the 16 populations into three groups. One major group was formed by populations from Pzhihua city (nos.1 to 7) and another two populations from Liangshan prefecture (nos.8 and 9); two populations (nos.10 and 11) collected from Liangshan clustered into the second category, and the remaining populations made up cluster III. Results suggested that the genetic relationships among populations are strongly associated with their geographical distribution.The results of SCAR markers conversion from RAPD showed it is a co-dominant marker of C.blinii and the specific fragments of SCAR have high homologous(71.32%). It can distinguish C.blinii fromthe confusing medicinal plants such as Artemisia annua L..Artemisia argyi H. Lev. and Conyza muliensis quickly and clearly. The results shows that under ideal state the SCAR markers can discriminate C.blinii from A. annua in only 5%content.4. The full-length cDNA sequence of chalcone synthase (CHS) gene and P-amyrin synthase (PAS) gene are cloned firstly using RT-PCR and RACE technology, named CbCHS and CbβAS:Sequence analysis indicated that CbCHS(GenBank number KJ155749) was 1197 bp in opening reading reading frame (ORF) and the amino acids encoded has high homology with other plants. Intron analysis of nucleotide sequence shows a different result with other plants’CHS gene,the C.blinii has two introns which located in the first and second base of 63 cysteine and 294 glycine. It possessed the sequence of the family signature: GVLFGFGPGLand the conserved active sites for the CHS function:Cys (C) 167, His (H) 306 and Asn (N) 339.The RT-PCR result showed that CbCHS was highly expressed in stems, followed leaves and flowers, while lowest in roots; the correlation analysis showed that there was a significant positive correlation between the active ingredient amount and CbCHS expression in floral organs (r values are tended to 1).The cDNA sequence of CbCHS gene was firstly obtained and reported from Conyza blinii Levl. and compared with the reference gene (GAPDH) all tissues had a significant expression differences.The recombinant CbCHS protein was successfully expressed in Escherichia coli strain BL21 with pET-30b(+) vector. The amount of gene expression reached the maximum 4h after induction, and the inducing concentration of IPTG was 0.24mM.5.Sequence analysis indicated that CbβAS (GenBank number KJ650043) was 2151 bp in opening reading reading frame (ORF) and the amino acids encoded has high homology with other plants. Intron analysis of nucleotide sequence shows a different result with other plants’PAS gene,it possessed the sequence of the family signature:DCTAE and QXXXGXW, The RT-PCR result showed that CbβAS was highly expressed in leaves, followed stems and flowers, while lowest in roots; the triterpenoid saponins content was highest in leaves then flowers and roots, the lowest in stems, the correlation analysis showed that there was a significant positive correlation between the total saponin content and CbβAS expression in stems and roots (r values is tended to 1).The cDNA sequence of CbβAS gene was firstly obtained and reported from Conyza blinii Levl. and compared with the reference gene (GAPDH) all tissues had a significant expression differences.