Regulatory Analysis Of Cytokinin Response And Function Characterization Of IPT Genes In Medicago Truncatula

Medicago truncatula is an important legume,as well as a model plant for the study of other legume plants.At present,the response mechanism of plant to cytokinin is mainly performed in traditional model plants such as Arabidopsis and rice,and the mechanism explore of the response of the hormone in Medicago truncatula remains unknown.At present,researches of cytokinin response were focus on the effect of direct induction by cytokinin,while few work is involved in cytokinin inhibitor.Meanwhile,the research on cytokinin is mainly focused on short-term response,and few studies were reported on the effects of long-term use of cytokinin.This study was mainly focused on the long-term response mechanism of M.truncatula to cytokinin and cytokinin inhibitor.Two compounds,6-benzylaminopurine(6-BA)and lovastatin were used to treat the seedlings for a long time as the induction or inhibition of cytokinin,respectively.Differential expressed gene or transcripts were screened by through high-throughput sequencing technology.Furthermore,a cytokinin biosynthesis key regulatory gene,IPT was cloned and selected for further function analysis.The results were shown below:1.After 6-BA or lovastatin treatments,the M.truncatula samples were used for transcriptome sequencing.Nine sequencing libraries were constructed and approximately 67.3Gb clean reads were generated.A total of 33,782 genes with 55,558 transcripts were detected in the three samples and 22,407 transcripts and 1,079 genes were identified as novel for further analysis.2.The experimental samples were divided into two groups: cytokinin/control(Cyto/Ctrl)and inhibitor/control(Inh/Ctrl).By DET analysis,there were 3,627 and 3,093 DETs in the Cyto/Ctrl and Inh/Ctrl groups,respectively.The results of function annotation and blast with plant hormone database showed that there existed lots of DETs involved in biosynthesis,metabolism or signal transduction.3.Using RT-PCR technology,the M.truncatula IPT gene was cloned,and the whole coding domain sequence was 900-bp in length,encoding a protein with 300 amino acids.With the E.coli expression system,the recombination protein of IPT was expressed successfully,and the polyclonal antibody of IPT protein was prepared by injecting Mus musculus with recombination protein.4.The plant expression vector of IPT gene was constructed and transgenic alfalfa were obtained with Agrobacterium mediated transformation.Those with high expression levels of IPT were selected and the phenotype identification showed that transgenic plants produced more branches and delayed senescence.Enzyme-linked immunosorbent assay displayed the levels of cytokinin in transgenic plants were much higher than those in wild type plants.Drought tolerances in transgenic plants were significantly higher than those of WT plants under continuous drought conditions.In this study,cytokinin and cytokinin inhibitor were respectively applied to M.truncatula for longterm treatments.A critical set of key regulatory genes involved in cytokinin response was selected from M.truncatula by transcriptome sequencing and analysis,which made a preliminary analysis of cytokinin regulatory and response mechanism in M.truncatula.One of potential key genes involved in the cytokinin biosynthesis was cloned and characterized.The results showed that IPT gene plays an important role in cytokinin biosynthesis,and high expression levels of IPT gene also can enhanced tolerances to drought and delayed senescence.This research provides abundant data for study of cytokinin response in M.truncatula,brings a theoretical reference for analysis of the long-term effects of cytokinin on plants,and lays the foundation for analysis of IPT gene function in M.truncatula.