The Functional Characterization Of Leucoanthocyanidin Reductases From Lotus Corniculatus

Leucocyanidin reductase(LAR)is the important enzymes in flavonoid biosynthetic terminal pathway,which determins the biosynthesis of catechins.But people still have doubts about the function of the enzyme.For example,the recombinant LARs from different species can catalyze leucocyanidin to produce catechin in vitro.Howerver,epicatechin was abundantly accumulated in LAR-overexpressed Nicotiana tabacum.Lotus corniculatus rich in catechin and corresponding proanthocyanidins(PAs),can be used as a model plant for studying the biosynthesis of catechin and epicatechin,as well as tobacco and Arabidopsis thaliana.In the study,the regeneration system of Lotus corniculatus was firstly established for the genetic transformation.Then,LcLAR1 gene was cloned from Lotus corniculatus,and the role of LcLARl gene in plants was validated using the genetic transformation system of A.thaliana,tobacco and Lotus corniculatus.The main conclusions were as follows:1.The regeneration system of Lotus corniculatus was established.MS+0.1 mg/L NAA+1.0 mg/L 6-BA was optimal medium for induction of callus and adventitious buds induction.The medium of MS+0.01 mg/L NAA could efficiently induce root growth.2.The result of qRT-PCR showed that the transcription level of LcLAR1 gene was higher in buds than that in leaves and roots,with the lowest level in stems.The contents of catechins were detected in different tissues,and the results showed that accumulation pattern of catechin and epicatechin were consistent with the expression profile of LcLAR1 gene.3.Compared to control,the contents of EC,EC-type PA dimer,EC-type PA trimer(EC-EC-EC)and the EC-glucoside were increased in the seed coats of LcLAR1 transgenic A.thaliana.But the contents of C and the C-glucoside were higher in seed coats of LcLAR1 transgenic ans.Interestingly,the contents of C,EC and the EC-glucoside were higher in LcLAR1 transgenic tobacco than those in wild type plants.The above results suggested that LcLAR1 was involved in the biosynthesis of catechins and epicatechins.4.CRISPR/Cas9 technology was used to knock out LcLAR1 gene in genome of L.corniculatus,and the results showed that the T-DNA vector has been integrated into the genome of L.corniculatus.However,the sequence of target gene was not changed,CRISPR/Cas9 technology will be continued to verify the function of LcLAR1 in subsequent experiments.