Reduction Of Lignin Content In Alfalfa(Medicago Sativa L.) By RNAi Technique

Alfalfa(Medicago sativa L.)is one of the most important leguminous forage,which has the reputation of "Queen of forage".Its can adapt to various stresses,including drought,low temperature,high temperature,salt and alkali stress.It is the largest leguminous pasture planted in the world.Lignin is an important component of secondary cell wall in higher plants,but lignin reduces the digestibility of forage grass.The higher lignin content,the lower utilization rate of ruminants.Therefore,the quality of alfalfa can be improved by reducing the lignin content.SND1(Secondary wall-associated NAC domain protein 1)is a secondary wall-related NAC domain transcription factor found in Arabidopsis thaliana,which regulates stem secondary wall synthesis.Medicagotruncatula NST1(NAC secondary wall thickness promotion factor 1)is a homologous gene of SND1 and a key point for regulating the synthesis pathway of secondary wall.However,it has not been reported whether the lignin synthesis of alfalfa can be reduced by regulating the expression of NST1.In this study,the RNAi-MsNST1 interference expression vector was constructed,and the transgenic lines of alfalfa were obtained by Agrobacterium-mediated transformation.The expression level of MsNST1 was down-regulated by RNAi technology in order to reduce lignin accumulation.The results are as follows:Total RNA was extracted from alfalfa and reverse transcripted to cDNA.According to the gene sequence of MtNST1 on NCBI,primers were designed and CDS fragment of MsNST1 were obtained by PCR.A sequence of 230 bp from the 3’ end of the MsNST1 coding region was chose as the RNAi interference region.The RNAi-MsNST1 expression vector was constructed by ligating the sequence forward and backward to pZG103-RNAi vector.RNAi-MsNST1 plasmid was transformed into Agrobacterium strain EHA105,and positive clones were obtained.The mature leaves of alfalfa were used as explant and transformed by leaf disc method.304 regenerated plants were obtained throughmore than 200 calli induction,glufosinate resistance screening and differentiation induction.In order to screen the positive transgenic lines,the lethal concentration of glufosinate in wild-type alfalfa was determined.Ten gradients(100,200,300,400,500,600,700,800,900,1000 mg/L)were set and sprayed mature leaves of wild-type alfalfa at the same leaf position.One week later,dry water loss was observed in leaves coated with a concentration greater than 200 mg/L,while slight dry spots were observed in leaves coated with 100 mg/L.Therefore,a 200 mg/L concentration of glufosinate was determined as the lethal concentration of alfalfa,and the transgenic lines against glufosinate were obtained by smearing the regenerated lines with this concentration.PCR amplification was further applied to detect transgenic plants,and 58 transgenic lines were obtained containing RNAi fragment.The transgenic lines under the same status and growth were selected,and the regenerated plants identified as non-transgenic plants were used as control.The lignin content was measured by acetyl bromide method,and the transgenic lines 10 and 22 had lower lignin contents than WT-like line 9.Lines 10 and 22 were reduced by 27.01%and 22.91%.Decreasions of interfascicular fibersand the xylem cell layers in line10 was observed by cross-cutting stems.The number of xylem in line22 was decreased.There was no significant difference in crude protein contents between lines 10,22 and wild type.In conclusion,we achieved two alfalfa transgenic lines with reduced lignin contents through down-regulation of alfalfa MsNST1 expression.This study provided reliable materials for improving alfalfa quality.