Construction Of Plant Expression Vector Of Glycyrrhiza NCED4 Gene And Polymorphism Of β-AS Gene

Glycyrrhiza uralensis Fisch. is commonly used in China for its effects of nourishing qi, alleviating pain, eliminating phlegm, relieving coughin, tonifying spleen and stomach. In recent years, due to the over exploitation of wild Glycyrrhiza uralensis resources, G. uralensis cultivars have gradually become the mainstream commodity. Glycyrrhizic acid is the main active component of G. uralensis and its content is an important indicator of Chinese Pharmacopoeia. However, a large number of studies have shown that the glycyrrhizic acid content of cultivated G. uralensis is low, which is not up to the standard of Pharmacopoeia. The sustainable development of G. uralensis resources has been seriously restricted. Therefore, how to improve the level of glycyrrhizic acid content is the key problem to improve the quality of G. uralensis.Previous studies have indicated that the biosynthesis pathway of glycyrrhizic acid is not isolated, which is connected with the biosynthetic pathway of other secondary metabolites. The preliminary study in our laboratory has shown that appropriate concentration of exogenous abscisic acid (ABA) could improve the glycyrrhizic acid content in G. uralensis, but its mechanism was still unknown.9-cis-epoxycarotenoid dioxygenase (NCED) gene is one of the rate limiting enzyme gene in the synthesis pathway of ABA, and its expression is directly affected the synthesis of ABA. Therefore, according to glycyrrhizic acid biosynthesis network, we considered that the differences expression quantity of NCED gene would lead to the different content of ABA, which in turn may affect the activities of enzymes in glycyrrhizic acid biosynthetic pathway and finally regulating the level of glycyrrhizic acid content.The development of transgenic technology has provided a way to insert an objective gene into the genome of explants, which leads to stable expression in the progeny of specific tissues. Using this technology, we can investigate the expression of objective genes in plants and analyze the influence mechanism of different gene expression levels for the biosynthesis of secondary metabolites.In this paper we have constructed the root specific expression vectors containing NCED4 gene of G. uralensis. We have used 3 methods——gene-fusion method, multi-fragment-cloning method and root-specific-universal-vector method to contruct vectors containing NCED4 gene, and then made genetic transformation, and finally cultivated regenerated plants. This paper lays a foundation for further research on the regulation mechanism of NCED4 gene expression for the biosynthesis of glycyrrhizic acid, and the main results are as follows:(1)The vector pCA-TobRB7-NCED4-2 containing NCED4 from G. uralensis was successfully constructed by the multi-fragment-cloning method, and it has been successfully transfered into the Agrobacterium EHA105.(2) The root-specific-universal-vector pCA-TobRB7 was successfully constructed. On the basis, the vector pCA-TobRB7-NCED4-3 containing NCED4 from G. uralensis was then successfully constructed. We have transferred it into Agrobacterium tumefaciens strain EHA105.(3)We have tried to estimate 3 methods of vector from accuracy, efficiency and experiment period. The accuracy of multi-fragment-cloning method and root-specific-universal-vector method were higher, and the results of sequencing were right, but the accuracy rate of gene-fusion-method was low by repetitive experiments. From the point of view of connection efficiency, the positive rate to transforming into competent cells by multi-fragment-cloning method is only 50%;and the positive rate of transforming into competent cell by root-specific-universal-vector method was 90%. From the point of view of the experimental cycle length, the programs by multi-fragment-cloning method were shortest. While using Root-specific-universal-vector method, we needed two steps to complete, first we should conduct a universal vector, and then we can insert the objective gene into it. After successfully conducted, the universal vector can be frozen stocked. When we need to conduct a new root specific vector containing another objective gene, we may just choose other appropriate restriction enzyme cutting sites behind the TobRB7 promoter sequence to insert it. In the long run, root-specific-universal-vector method was more convenient, time-saving and labor-saving.(4) The recombinant EHA 105 containing NCED4 gene from G. uralensis were transformed into hypocotyls and cotyledonary node of G. uralensis, and transgenic callus were obtained.Glycyrrhiza uralensis is one of the most frequently used herbs in traditional Chinese medicine. In recent years, the G. uralensis cultivars have become the mainstream products of the market, but the content level of glycyrrhizic acid of G. uralensis is generally low, which fails to meet the requirement of Chinese pharmacopoeia standard. The sustainable development of G. uralensis is in a bottleneck. Therefore, it is an effective way to improve the glycyrrhizic acid content. Finding out molecular markers related to high level of glycyrrhizic acid content is an important foundation for excellent selection in the germplasm resources, and it has great importance to molecular breeding of G. uralensis.In this paper, we have treated P-amyrin synthase (β-AS) gene——one of the glycyrrhizic acid biosynthetic pathway downstream key enzyme gene as the research object. The polymorphisms in introns of β-AS gene were sequenced, and the correlationship between the polymorphisms and glycyrrhizic acid content were analyzed. We have tried to screen out the molecular markers related to high level ofglycyrrhizic acid content in order to lay a foundation for molecular breeding of G. uralensis. The following results were drawn in this paper:(1)The results indicated that there were different kinds of base mutations in β-AS gene: ① There were 3 SNPs sites, T-C transversion was at 1742 bp, T-C transversion at 1743 bp, T-G transversion was at 2216 bp. ② There were 3 hybrid allelic polymorphisms sites, C/G was at 506 bp, C/G was at 512 bp, T/G was at 2216 bp.②There were 2 indels sites, such as AT repeating at 1710-1716 bp,12 bases indel was at 2187-2198 bp.④ We have found that β-AS gene have copy number variations in G. uralensis. After removing all heterozygous polymorphism and copy number polymorphism samples, there are 138 homozygous samples.(2) Using the gray correlation analysis method, we have found that the base types of 1710 bp,1712 bp,1714 bp,2187 bp,2189 bp,2193 bp,2197 bp,2198 bp were significantly correlated with the glycyrrhizic acid content of G. uralensis. (r>0.65). According to the 8 highly correlated base mutations,138 homozygous samples were divided into type B1-B5, and the glycyrrhizic acid content of B5 (1710 bp A,1712 bp A,1714 bp A,2187 bp A,2189 bp A,2193 bp A,2197 bp A,2198 bp A) was highest. We have also constructed a system clustering tree of 138 homozygous samples based on the glycyrrhizic acid content. All of the samples were divided into a total of 4 branches which were named Ⅰ,Ⅱ,Ⅲ, Ⅳ. In accordance with the order of glycyrrhizic acid content, there were group Ⅰ (0.54%-0.92%), group Ⅱ (0.97%-1.26%), group Ⅲ (1.29%-2.46%) and group IV (2.54%-3.33%). The proportion of type B1-B5 in 4 groups of glycyrrhizic acid content was analyzed. It was found that the proportion of B5 in 4 groups was significantly different (PG5=0.002<0.05), and the proportion of B5 in the highest content group (IV group) was the highest (66.67%). Therefore, B5 was a molecular markers related to high level of glycyrrhizic acid content.(3) We have analyzed the correlationship between different genotypes of G. uralensis and glycyrrhizic acid content.138 homozygous samples were divided into 7 genotypes. The result has shown that glycyrrhizic acid content of G7 (1710-1715 bp ATATAT,1744-1745 bp TC,2187-2198 bp ATATTGACTTAA,2216 bp G) was the highest. The proportion of 7 genotypes in 4 groups of glycyrrhizic acid content was analyzed. It was found that the proportion of G7 in 4 groups were significantly different (PG7=0.0010.05). And the proportion of G7 in the highest content group (IV group) was the highest. Therefore, G7 was a genotype related to high level of glycyrrhizic acid content.(4) Through comprehensive analysis of the relationship among glycyrrhizic acid content and base mutations and genotypes, we have known that the bases of G7 were A at 1710 bp、 1712 bp、1714 bp、2187 bp、2189 bp、2193 bp、2197 bp, which were the same characterasitcs with B5.Therefore, we can draw a consitent conclusion that B5 was a molecular markers and G7 was a genotype related to high level of glycyrrhizic acid content. The molecular marker is located in the 6th-8th, which can be used as a candidate detection region to select high quality G. uralensis.