| Legume plants can interact with rhizobia,and form nodules on its root.In recent years,the symbiotic network related genes were being found,but little was known about how the immune system was controlled during the process of symbiosis.Some scientist analyzed the transcriptome data when the rhizobias were released into the plant’s cell,found that the genes that related to immunity response were up regulate temporal and then reduced;And DNF2,RSD,Sym CRK,etc were found to regulate the immunity response after rhizobia were released into the plant.All of this phenomenon draw more attention than before and more research about this had been done.Our laboratory also obtained a gene which was named as NAD1 was related to immune regulation by reverse genetics.Their research showed that the NAD1 gene was specifically expressed in the nodules of legume plants and the level was high.The mutation of NAD1 in Medicago truncatula showed the accumulation of phenolic compounds and nodules were necrosis,but didn’t show any different with wild nodules in Lotus japonicus.To understand the function of the gene in both Lotus japonicus and Medicago truncatula,we focused on the expression regulation of NAD1.In this study,we used Lotus japonicus and Medicago truncatulaas research material,applied the hair root transformation technique to study the core promoter of NAD1.And yeast one-hybrid was used to study the transcription factor of Lotus japonicus,we also union Gibson technique and SMART c DNA library construction technique to construct a AD-c DNA library of Medicago truncatula nodule.The main findings are as follows:1.Analysis the expression model and core region of Box1 of Lotus japonicus NAD1.Based on the research of our Laboratory,the region that regulated the expression of Lj NAD1 was(-400～-200)(the initial codon was+1).Gathered(-400～-200)sequences of different legumes and did sequence alignment.The region had been part into three sections,and were named as Box1(-391～-363),Box2(-331～-308)and Box3(-296～-269).The hair root transformation results showed that the Box1 and Box3 were important for the expression of NAD1 gene,none of them could be omitted;Box2 had no big function on the expression of NAD1;The core promoter of Box1 was the region(-381～-370);2.The research of transcription factor that activate the expression of Lotus japonicus.Used p Lj NAD1pro(-382～-276)-Ab Ai/Y1HGold as the bait strain to screen the Lotus japonicus c DNA library.We obtained 77 positive clone.Analyzed the fragment of the positive clone and finally got 39 genes.6 of them were selected as candidate genes:Sphingomyelin synthase-like domain,F-box/Leucine rich repeat protein,AHL23,AHL20,PLATZ,MULE transposase.We verified that the AHL20 and MULE transposase had interaction with the promoter,and there had no interaction between PLATZ and bait sequence;the F-box/Leucine rich repeat protein and Sphingomyelin synthase-like domain because of lacking sufficient information,so could not be sure whether interact with the bait sequence;3.Analysis the expression model and core region of Medicago truncatula NAD1.Our Laboratory research found that Lotus japonicus and Medicago truncatula share the same regulation region,but this region still unknown.The region(-512～-374)of NAD1promoter sequence in Medicago truncatula had a similarity with the region(-400～-200)of Lotus japonicus.The hair root transformation result showed that there were at least three cis-element in the NAD1 promoter of Medicago truncatula,which were locate at(-912～-483),(-446～-424),(-402～-374)region;single region(-402～-374)could initiate the expression of NAD1 gene,The expression level of NAD1 was increased when both region(-466～-424),(-402～-374)were present;4.Construct a AD-c DNA library of Medicago truncatula nodule.The AD-c DNA library constructed by using Medicago truncatula nodule as material,the transformation efficiency was 7.74×10~5 transformants/3μg p GADT7-Rec-New.The results of random colony PCR showed that the size of inserted DNA fragment was between0.5kb to 2 kb,the fragment that was larger than 0.75kb in all fragments was 45.6%,and the recombination efficiency was 91.2%.The AD-c DNA library was saved as plasmid. |
