Comparative Transcriptome Analysis Of Alfalfa Responses To Salt Stress And The Investigation Of Mechanism Of Plant Salt Tolerance

The area of salinized land in the world has reached 9.8×108hm2,and the area of salinized land in China has reached about 200 million hectares.The aggravation of salinization seriously affects the production of agriculture and animal husbandry and has become a worldwide problem.Alfalfa,known as the "King of Forage",is the most widely cultivated legume grass with strong salt tolerance in the world.This study will be two kinds of alfalfa "Adrenalin"(salt-stress tolerant)and "Peru"(salt-stress sensitive)as experiment material.In this work,catalase(CAT)activity,peroxidase(POD)activity and malondialdehyde(MDA)content,proline(PRO)content,leaf relative water content(RWC),chlorophyll(Chl)content of the two alfalfa cultivars with different salt tolerance were determined before salt treatment(0 h)and after salt treatment(150mmol/L NaCl)2,4,6,8,and 16 h,respectively.According to the results of physiological and biochemical indexes,transcriptome sequencing was performed at two time points(2h and 6h),and transcriptome comparative analysis was conducted on its roots to explore the molecular law of alfalfa in response to salt stress.It provides theoretical support for the interpretation of the salt tolerance molecular regulation mechanism of alfalfa.The experimental results are as follows:1.Under the stress of 150 mmol/L NaCl,Under the stress of 150 mmol/L NaCl solution,catalase(CAT)activity,peroxidase(POD)activity,malondialdehyde(MDA)content,proline(PRO)content and relative water content(RWC)of the two alfalfa species were significantly increased or decreased at 2,4,6,8 and 16h compared with the control group(0h).This indicated that strong biochemical reactions occurred in two alfalfa within a short period of time after salt stress to cope with salt ion injury,so 2,4,6,8 and 16h could be used as important time nodes for exploration.2.In this study,179 million clean reads were obtained by ma-seq transcriptome sequencing,and 111,734 Unigenes were obtained by assembly with De novo,and 41,351 differentially expressed genes were screened.GO through enrichment analysis found that 5233,6614,4709,5314 DEGs were annotated intoA-2h/A-0h,A-6h/A-0h,P-2h/P-0h and P-6h/P-0h comparison group,the significantly enriched GO Term was mainly related to tryptophan and proline metabolism process,response to water,iron ion binding,heme binding,chalcone biosynthetic process,glutathione transferase activity and so on.Through KEGG enrichment analysis,it was found that 1715,2267,1404 and 1404 DEGs were annotated into A-2h/A-0h,A-6h/A-0h,P-2h/P-0h and P-6h/P-0h,respectively.And the significantly enriched KEGG Term was mainly related to phenylpropanoid biosynthesis,drug metabolism-cytochrome P450,metabolism of xenobiotics by cytochrome P450,glutathione metabolism,alpha-Linolenic acid metabolism,linoleic acid metabolism,plant hormone signal transduction,flavonoid biosynthesis,taurine and hypotaurine metabolism,ABC transporters,glucosinolate biosynthesis.3.In order to make the selected differential genes more reliable,the screening conditions were further set as p-value<0.01 and | log2foldchange|>1.A total of 166 differential genes related to salt stress response were obtained.We found that the differential expression multiple(log2FoldChange)of these genes in the A-2h/A-0h was 0.8-5.5 times than that in the P-2h/P-0h.In the same way,the differential expression multiple in the A-6h/A-0h was 0.9-2.8 times than that in the P-6h/P-0h.This indicated that the expression level of these genes in the salt-tolerant variety "Adrenalin" was much higher than that in the salt-sensitive variety "Peru" under the same salt stress.It is speculated that these genes may be the reason why Adrenalin is superior to Peru in salt resistance.We can screen these genes for further cloning and verification.In addition,it was also found that these differentially expressed genes were mainly involved in transcription factors,antioxidants,plant hormone signal transduction,signal sensor and transduction,transport proteins and so on.In addition,qPCR results also confirmed the reliability of ma-seq sequencing results.In addition,qRT-PCR results also confirmed the reliability of RNA-seq sequencing results.