Genes Amplification And Analysis Of Chalcone Synthase Core Fragment From Anemone Obtusiloba

Anemone obtusiloba is one of the perennial herbaceous plants within the family Anemone L. of Ranunculus subfamilies in Ranunculaceae and presents three kinds of color:yellow, light yellow and white. At present, the study of Anemone obtusiloba are mainly concentrated in its medicinal value, distribution, reproduction, reproductive characteristics and resource allocation, etc., at the molecular level, only Anemone obtusiloba of ISSR genetic diversity analysis under different environmental conditions are reported. Combined with the specific research conditions, this study uses the total RNA extracted from the petals of Anemone obtusiloba, RT-PCR method is applied to clone the first key enzymes, chalcone synthase after reverse transcription. And the results are as follows:1.This experiment built a method that verifies the extracted RNA whether can be used for subsequent experimental technology without RNA electrophoresis:Design a pair of primers of a skeleton protein or a constituted protein for gene amplification, then use the genomic DNA as the template for PCR amplification, and sequence the gene fragment. If the sequence is the designed gene fragment, it proved that the primers are designed right. And then use the primers on the extraction of total RNA transcription product amplification. if the gene sequence can also be obtained, then it certified that the total RNA extracted and reversed transcription product purity is high, can be used for RT-PCR reaction;2.A pair of primers of Actin, a kind of skeleton proteins was designed in this experiment. With leaf genomic DNA from Anemone obtusiloba as a template for PCR amplification, the Actin gene sequence fragment was obtained, and then use the primers with petals extracted total RNA reverse transcription product as a template for PCR, still got Actin gene fragments, and proved that the amplification product of DNA as a template contains a introns;3.Degenerate primers are designed according to the chalcone synthase (CHS) gene sequences logged in GenBank. Then RT-PCR were conducted to the extracted total RNA from petals of Anemone obtusiloba, and three CHS gene fragments of Anemone obtusiloba were obtained. After analysis, there are77bases overlapping between the3’ end of380bases gene fragments and the5’ends of the other583bases fragments. Presumably, they are the same sequence of two parts. Compared with some other plants CHS sequences after integrated, findings confirm that they are the same sequence of two parts.The two sequences encoded the same amino acid sequence.