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Chinese pharmacopoeia stipulates that the content of liquiritin in licorice slices should be no less than 0.5%. However, there are lots of unqualified licorice slices in the herbal medicine markets. Due to the important role of functional gene polymorphism in secondary metabolism, this study attempts to analyze the influence of chalcone synthase (CHS) gene polymorphism on liquiritin biosynthesis and find out the unique haplotypes in licorice samples with high or low content of liquiritin, and to provide a basis for further analysis of molecular mechanism in flavonoid biosynthetic pathway. The contents of the 4 main flavonoids (liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin) in 60 licorice samples were assayed by HPLC and the results were analyzed by Spearman and χ2 tests. The contents of the 4 main flavonoids were related to each other and obviously different in different original plants. They were highest in Glycyrrhiza uralensis samples and lowest in Glycyrriza inflate samples. Five G. uralensis samples with the highest liquiritin contents and five G. inflate samples with the lowest liquiritin contents were selected to clone the CHS cDNA sequences. 336 CHS cDNA sequences with a full length of 1 175 bp were obtained, 249 variable sites (141 missense mutation sites) were found, and 137 haplotypes were determined. 130 variable sites were found in the 336 CHS amino acid sequences and 102 types were determined. AA-3 is the major type of CHS in licorice, AA-35 is the special major type of CHS in the group with high flavonoids contents and AA-36 is the special major type of CHS in the group with low flavonoids contents. The mutation sites between AA-35 and AA-36 are I/V at 193 and V/T at 229. Discovery Studio 2.5 analysis of the three-dimensional structure of the CHS protein shows that the valine at site 229 of AA-35 is combined with malonyl-CoA. Homology analysis indicates that the homology of CHS among different species is low. This study is significant for identification of the unique haplotypes in licorices with high or low content of liquiritin and guiding the further molecular breeding of high-quantity licorice.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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