The Content Evaluation And Key Genes Analysis Of The Condensed Tannin In Lotus Corniculatus Germplasm Resources

Lotus corniculatus is a perennial leguminous genus of the genus,its origin is the temperate zone of Eurasia,and now is widely distributed all over the world and China.L.corniculatus has many stems and leaves and with good palatability.Because of high nutrient content,it has high feeding value.In addition to this,the biggest feature of L.corniculatus is that the stems and leaves are rich in condensed tannins,which can effectively reduce the occurrence of bloating.In this study,we collected 235 L.corniculatus seeds from all over the world and established germplasm resources nursery.Then,the qualitative and quantitative determination of the content of condensed tannin in the leaves was measured and the relationship between CT content and 13 agronomic traits was analyzed.Besides,the Agrobacterium tumefaciens-infected Arabidopsis inflorescence transformation method was used to transfer the key enzyme(Anthocyanin reductase)gene ANR into Arabidopsis genome.In the transgenic plant,the content of condensed tannin were determined to lay the foundation for the breeding of varieties of L.corniculatus.The main findings are as follows:1.The variation of condensed tannin content in 235 leaves of L.corniculatus is 5~16 g/Kg DM,then it was divided into 3 grades: 41 parts with low content of CT(5~7 g/kg DM),122 parts with medium content of CT(7.1~9.5 g/kg DM)and 72 parts with high CT content(9.6~16 g/kg DM).2.The 13 agronomic traits of Lotus corniculatus germplasm resource nursery were observed,and the correlation between the condensed tannin content and the agronomic traits was analyzed.The results showed that the coefficient of variation among the 13 main agronomic traits fluctuated between 11.01% and 40.32%,indicating that the cultivars was rich in diversity.However,we have not found the relation between the content of condensed tannins and agronomic traits.3.The variety"SVW198"with high content of condensed tannin was used as experimental material,and the cDNA sequence of ANR gene was cloned by RT-PCR.The open reading frame was 1017 bp,encoding 338 amino acids.It was found that the homology of the ANR gene sequence between L.japonicus and L.corniculatus was as high as 99.70%,so it was named LcANR.4.Construct pCAMBIA3301-LcANR plant expression vector and infect inflorescence through agrobacterium,we obtain transgenic LcANR gene Arabidopsis thaliana.The progeny transgenic plants were identified by Basta screening and PCR to confirm that LcANR has been successfully integrated into the Arabidopsis genome.Quantitative determination of CT content in transgenic LcANR Arabidopsis plants revealed that CT content in transgenic Arabidopsis thaliana was1.45~1.58 times higher than wild-type Arabidopsis,indicating that ANR gene plays an indispensable role in the biosynthesis of condensed tannin.