The Cloning、Prokaryotic Expression And Its Influence Factors Of Chalcone Synthase Gene In Nephrolepis Auriculata

The fern is rich in bioactive flavonoids, which are important medicinal ingredients in ferns. The metabolic pathways of flavonoids ferns are still not clear. It is of great significance to make clear the mechanism of metabolic pathways for elucidating the metabolism of flavonoids, and regulating the expression of flavonoids in ferns;The fern Nephrolepis auriculatais is a traditional medicinal fern,which is rich in bioactive flavonoids.It has the function of treating the fever and cold, cough,dysentery and anticancer. Nephrolepis auriculata can be collected and cultured easily. In this paper, Nephrolepis auriculatawas as materials to determine the contentof flavonoids in it. Chalcone synthase gene(chalcone synthase CHS) was obtained through cloning and induced its prokaryotic expression. Then, the prokaryotic expression conditions were optimized and the function of the gene was analyzed. This study has some scientific significances in elucidating the mechanism of the metabolism and regulation of flavonoids in fern plants.Based on the above background, we obtained the following three aspects of the results.1. The total flavonoids in Rhizome, rachis, pinna of Nephrolepis auriculataare determined. Measurement results show that the content of total flavonoids was higher. The total flavonoids content in rachis and pinna was about 2%. The total flavonoids content in rhizome(about 6%) was much higher than that in rachis and pinna. So we choose Nephrolepis auriculata as the materials for studying the flavonoid metabolism pathway.2. The chalcone synthase gene(Na CHS) cloning and function analysis: The total RNA, DNA were extracted from leaves. Then the ORF sequence and genomic sequence of chalcone synthase were cloned by PCR. Bioinformatics analysis showed that the ORF of Na CHS gene sequence was 1209 bp and encoding 402 amino acids, which contain an intron and obey gt/ag rule. BLAST showed that the Na CHS gene is homogenous to other plants, phylogenetic analysis indicated that the Na CHS gene close evolutionary relationship with Cyclosorus acuminatus, Salvinia natans, Ceratopteris thalictroides and other ferns. Meanwhile,there are differences in the sequence of Na CHS. These differences are important to reveal the genetic relationships among species, or effect on gene function. Furthermore,the recombinant p ET32-Na CHS plasmids was transformed into host E. coli BL21 for expression. SDS-PAGE analyse showed that the fusion protein was highly expressed in E. coli BL21 with isopropyl-D-thiogalactoside(IPTG) induction, and the expressed Na CHS protein had a molecular weight of about 63 KD, a size matching that of the predicted protein by bioinformatic analysis. In vitro testing of enzyme activity verified that the Na CHS protein have activity, that could catalyte the substrates to generate the naringenin, which furtherly explained that the expression of the protein is the Na CHS.3. Influencing factors of prokaryotic expression.(1)IPTG was choosed as inducer to induce protein expression and the protein expressed was detected quantitatively. The amount of protein expression showed a trend of increase. And the variation was most obvious after 1h and cumulative protein reached maximum after 4 h inducing.(2)Set IPTG concentration gradient. After inducing expression of 4h,sampling and detection of bacterial concentration and quantitative detection of protein.With the increase of IPTG concentration,bacterial growth was inhibited. Addition of IPTG to 0.1-0.2 mmol/L,the protein expression was higher.(3) Set temperature gradient, including 25 ℃, 28 ℃, 31 ℃, 34 ℃, 37 ℃ and 40 ℃, the total amount of expressed protein increased with the increase of temperature.Studies have indicated that Bacteria grow fastest at 37 ℃ and the expression of Na CHS in a part of the soluble. The optimum expression temperature was 37 ℃.(4)Before the induction of expression, set bacterial concentration gradient. Concentrations were 0.224, 0.385, 0.621, 1.120. At a concentration of 0.621 and 1.120 have higher expressions.In summary, Nephrolepis auriculata is rich in flavonoids. Chalcone synthase is the first committed enzyme in the flavonoids biosynthetic pathway. Functional analysis showed that Na CHS protein has higher biological activity. Through experiment analysis,the optimal inducing condition of CHS is that the bacteria concentration should be about OD600=0.6,0.1mmol/LIPTG, 37 ℃ celsius inducing about 1 h. This research lay the foundation for studying the flavonoids biosynthetic pathway. It also has great significance for illustrating the regulatory mechanism of the biosynthesis of flavonoids in Nephrolepis auriculata, which providing the reference for domain development of Nephrolepis auriculata.