Licorice Breeding Base And Hmgr Gene Cloning Research

The medicinal composition content,such as glycyrrhizin,in liquorice is the limited factor of medicinal material quality.So,to obtain superior quality cultivation liquorice,it is necessary to perform the high medicinal composition content breeding including glycyrrhizin and establish the good variety propagation system.Therefore,a study was performed using the base of good variety breeding of liquorice as the main line,the Hangjinqi liquorice from inner Mongolia as the major research materials.The study was conducted by morphology observation,paraffin section making and microscopic observation,the content determination of major medicinal composition and gene cloning to study the three parts of contents,including of sexual reproduction properties,germplasm resources evaluation and the gene clone and expression characteristics of the HMGR,which is the key enzyme in the metabolic pathway of liquorice.The main results were as follows:1.Observing and analyzing the floral characteristics,the development process of a single floral,flowering dynamics of a single floral and the quantitative characteristics of inflorescence and pod seeds.The variation of flower color in liquorice colony was found,this text named them as modena type and white type.The flowering rate was 38.9%from 10:00 to 16:00,and it was 35.3%from 20:00 to 6:00 the next day.The study determined finally the breeding system of liquorice,which was cleistogamy by various ways,simultaneity,the stigma could accept other pollens such as the pollen which was given by artificial pollination.2.The study filtered the detection method of the pollen viability and the stigma activity.Finally,the experiment of hand-pollination in the field was adopted.The pollen viability was the highest at 12:00 in daytime,the stigma had no activity in the fifth day after artificial emasculation.The results about microsporogenesis and male gametophyte development showed that it would estimate the development degree of stamen according to the external morphology of the flower buds.The development degree of pistil before fertilization were observed in the study.The fluorescence microscope observation results about the pollinated material in the different time after emasculation showed that the pollen tube entered into the ovule to fertilize 6h after pollination.3.The results of correlation analysis between agronomic characteristics and the content of glycyrrhizin and liquiritin content showed that the relationship of parietal and the leaflets width and the glycyrrhizin and liquiritin content was significant or had the trend in the four years transplanted liquorice,the analysis result between stem bark color and stem appendants(chaeta numbers) with the glycyrrhizin content showed that the stem bark color was deeper and the chaeta number was much more, the glycyrrhizin content was higher to some extent.The analysis result about the one year planting liquorice showed that the correlation of tap root length with liquiritin content was positive significantly,the correlation of root weight and the glycyrrhizin and liquiritin content was positive to some extent.The correlation of glycyrrhizin and liquiritin was positive significantly,which indicated that the accumulation rate of two medicinal composition content had a certain coordination.4.Analyzing and comparing the glycyrrhizin,liquiritin and isoliquiritigenin content among the modena,white flower type and commen flower color type,the white type was the selected better type.The Gaoshawo(from Gansu province) provenience showed a better growth status in plain region by comparing with the Hangjinqi(from inner Mongolia) provenience of the seedling physiological characterization and the medicinal composition content of the annual root.Comparing the major medicinal composition content among four proveniences in Hangjinqi(from inner Mongolia),the provenience 46 was the better breeding material.5.The anatomical structure of leaves of liquorice was observed to reveal the drought-resistant mechanism of Glycyrrhiza L.A selected way of high medicinal composition content was put forward which was the quantities of secretory tissues in the internal structure of the leaves.Considering the aerial parts as the flavonoids production materials,the selected order was G.glabra L.> G.inflate Batat.> G. uralensis Fisch.among the Glycyrrhiza L.,while,the selected order among the G. uralensis Fisch.was provenience 41> provenience 14> provenience 46.5.A full length DNA encoding HMGR(designated as GuHMGR1) was isolated from liquorice(G.uralensis Fisch.) based on degenerated PCR and genome walking approach.The comparison between the GuHMGR1 genomic sequence with the cDNA of Pisum sativum showed that the genomic DNA contained two introns.The lengths of three exons were 1206 bp,181 bp and 943 bp,respectively.The lengths of the two introns were 87 bp and 245 bp,respectively.The cDNA of GuHMGR1 was 2330 bp in length and contained a 1518 bp ORF,311bp 5' untranslated region and 501bp 3' untranslated region,encoding a protein of 505 amino acids.The analyzed result of conserved domain of GuHMGR1 found that the GuHMGR1 was ascribed to the I family of reductase super family in structure.6.The result of GuHMGR1 expression pattern in different tissues showed that GuHMGR1 was expressed in different growth period of roots and leaves,but the expression level was different in the different transcripts,the expression level in the roots was higher than in the leaves,the expression level in the taproots and old leaves was higher than in the lateral roots and new leaves.The results of GuHMGR1 characteristics in the liquorice with different glycyrrhizin content,one belonged to white flower type,and the other belonged to modena flower type,showed that the two type had base differences in the part DNA sequence.