Functional Analysis Of MtWRKY41 And MtCDF2 In Medicago Truncatula Under Salt Stress

Soil salinization has become an increasingly serious global problem.Salt stress hinders the growth and development of plants,reduces the quality and yield of crops,and largely restricts the stable development of agriculture.Alfalfa(Medicago sativa),a perennial high-quality legume forage,is one of the important forage crops in the world.Compared with other forage crops,alfalfa has the following advantages:(a)It is rich in nutrients,such as protein,vitamin and microelement.The crude protein content can account for up to 20%of the harvest dry weight.At the same time,the protein is easy to be absorbed,and the total digestibility can reach 65%;(b)As a deep root crop,it is widely planted due to its strong adaptability to the environment;(c)Nitrogen fixation by the nodules can reduce the application of nitrogenous fertilizer;(d)As a perennial forage,it has a longer supply time and higher yield.However,soil salinity is still an important environmental factor limiting the production of alfalfa,which seriously reduces the quality and yield of alfalfa.Therefore,cultivating new varieties of alfalfa with strong stress resistance traits has become one of the focuses of scientists.However,the tetraploid genome and outcross pollination of alfalfa hinder the generation of inbred lines,which complicates the breeding and makes it inconvenient to conduct genetic and genomics research directly.Model legume plant Medicago truncatula is a self-pollinated diploid species with a small genome and short growth cycle,which facilitates molecular mechanism research.Therefore,we can reveal the salt-tolerant molecular mechanism of alfalfa by studying Medicago truncatula,and accelerate the research process of genetic improvement and molecular breeding of alfalfa.This study consists of two parts.The first part of the research is as follows:By screening genes that specifically respond to salt stress from RNAseq data,the MtWRKY41 gene was identified.Subcellular localization result showed that MtWRKY41 is expressed in the nucleus.Expression pattern analysis revealed that MtWRKY41 is relative highly expressed in leaves and pods.We ordered two Tnt1 insertion mutants of this gene from the mutant library.RT-PCR analysis showed that MtWRKY41 transcript was absent in mtwrky41-1 and mtwrky41-2 alleles.Phenotype analysis demonstrated that mtwrky41-1 and mtwrky41-2 mutants were salt-tolerant.Measurement of physiological and biochemical indicators found that mtwrky41-1 and mtwrky41-2 mutants have lower MDA and H2O2 content,while chlorophyll content and SOD activity higher than the wild type.After salt treatment,the leaves of wild-type and mtwrky41 mutant were stained by DAB and NBT.Compared with the wild-type leaves,the mtwrky41 mutant leaves exhibit lower staining,indicating that the expression levels of ROS synthesis and antioxidant enzymes scavenging related genes are lower than that in wild-type.It is speculated that MtWRKY41 may participate in the ROS pathway as a negative regulator in response to salt stress.In addition,we constructed the overexpression vector 35S:MtWRKY41-GFP and transformed it into the wild-type,and the transgenic positive plants have been obtained.Later,the salt stress experiment was conducted on T1 generation overexpressing plants and the relative phenotype will be analyzed.Furthermore,we have transferred the constructed MsWRKY41 RNAi vector into alfalfa SY4D,and we are currently screening the transgenic positive lines.We currently screening the transgenic positive lines.After the positive lines are successfully obtained,we will conduct salt stress phenotypic observation.In the second part,we obtained a salt-sensitive mutant,which was identified as a transcriptional abnormality caused by Tnt1 insertion in the AtCDF2 homologous gene MtCDF2 by amplification,co-segregation and sequence alignment analysis of Tnt1 flanking sequences.RT-PCR analysis showed that the transcription of MtCDF2 was interrupted in mtcdf2 mutant.Subcellular localization showed that MtCDF2 was mainly expressed in the nucleus.Tissue expression patterns analysis found that the relative expression level of MtCDF2 was the highest in flowers and leaves.After salt stress treatment,the leaves of mtcdf2 mutant accumulated more MDA and H2O2,the SOD activity and chlorophyll content were significantly lower than those of the wild-type.DAB and NBT staining showed that the color of the leaves of the mtcdf2 mutant in the salt-treated group was much darker than that of the wild type.We speculated that MtCDF2 might be involved in the ROS pathway.The detection of ROS pathway-related genes under salt treatment revealed that the expression of ROS synthesis genes in the mtcdf2 mutant was highly up-regulated,while the expression of ROS scavenging genes MtSOD1 and MtSOD2 were significantly lower than those of the wild type.In order to determine the salt tolerance of MtCDF2,we overexpressed the gene in the wild type background of Medicago truncatula.We are currently screening positive plants,and then salt treatment will be carried out to observe the phenotype.At the same time,we overexpress the gene under the wild-type background of alfalfa and are in tissue culture.At this stage,we hope to reveal the salt tolerance mechanism of alfalfa by studying the response of Medicago truncatula to salt stress and speed up the research process of alfalfa salt tolerance.