Functional Analysis Of Dark-green Leaf Gene And Resistance Genes Against Soft Rot At EMS Mutants In Chinese Cabbage

Chinese cabbage?Brassica rapa.ssp.Pekinensis?is Brassica genus originated in central China,is the most widely grown vegetable crop.EMS as mutagens is the most widely used to create mutants.In our study,we screened a leaf dark green mutant dg from the mutant library by EMS in Chinese cabbage.We studied the physiological characteristics of the mutant dg,analyzed the genetic characteristics of the mutant gene,and the mutant site was located using MutMap.Overexpressed the mutant gene to WT using transgenic technology,and observed the phenotype of transgenic plants.The expressed regulation mechanism of mutanted gene was all-sided revealed by transcriptome sequencing?RNA-Seq?and RT-qPCR validation.To elucidate the transcriptional and translational expressed regulation mechanism of mutant gene and propose the hypothesis mechanism of mutant gene affected leaf color,we analyzed the level of chloroplast protein,and detected the catalytic efficiency of thylakoid membrane protein and prokaryotic expression protein in WT and mutant dg.In our study,a reliable inoculated Pcc method and evaluation system of soft rot resistance was established to accurately screen the resistant mutant from EMS mutant library in Chinese cabbage.We compared the transcript profiles of resistant mutant sr plants to the susceptible WT plants in response to Pcc using RNA-Seq,to elucidate putative resistance molecular mechanism operating against Pcc,including the infection process and recognition of the pathogen,signals transduction and synthesized secondary metabolites functioning in the immune system.The main research results are as follows:1.The mutant dg in reproductive stage and vegetative stage perfomed dark-green leaf color,the growth rate was faster than WT,and the content of chlorophyll a and chlorophyll b were significantly increased,the chlorophyll a/b ratio was decreased.The photosynthesis characteristic was showed that the mutant dg enhanced the ability of capture and absorpt light under the weak light.The maximum conversion efficiency of light energy was lower than WT,but the actual light energy conversion efficiency in photosynthetic apparatus was higher than WT.2.The mutated gene in dark green leaf mutant dg was controlled by a pair of recessive genes.The mutated base was located at 1391bp of the mutant gene CDS?BrFC2?by MutMap method,which was mutated from G into A,corresponding to arginine?R?changed to lysine?K?at 509^th amino acids.The sequencing results of BrFC2 and KASP method were confirmed this mutated base as a candidate mutated site.3.Overexpression mutated gene BrFC2-dg was transferred into WT,whose phenotype was significantly changed.The transgenic plant leaves showed dark green leaf color,which was same to the mutant dg.However,overexpression WT gene BrFC2-WT was transferred into WT,the phenotype of transgenic plant leaves was not changed.That result was further verified that the mutated site at BrFC2 was the reason that the phenotype was dark green leaf color in mutant dg.4.The heme of dark green mutant dg was significantly higher than WT.In the process of chlorophyll synthesis,the content of five important precursors?ALA,PBG,ProtoIX,Mg-ProtoIX and Chlide?in mutant dg were increased,which may be closely related to the increase of chlorophyll content.Transcriptome analysis showed that BrFC2 expression was unchanged in WT and mutant dg.Nine genes were up-regulated and one gene was down-regulated in the chlorophyll and heme synthesis pathway.At the same time,multiple photooxidation-related,photosystem subunit and photoprotein complex of photosynthetic system I and II were down-regulated in dark green mutant dg.5.The content of BrFC2 protein was no significant difference in WT and mutant dg.The two proteins were down-regulated in porphyrin and chlorophyll metabolism pathway.Four proteins in the photosynthetic-antenna protein pathway were up-regulated.The conserve motifs analysis of FC2 protein showed that the mutated site of dark green leaf was in conserve motif consisting of 50 amino acids at C-terminal.The results of secondary structure analysis of BrFC2-WT and BrFC2-dg gene showed that BrFC2-dg had one more amino acid to form random coil than BrFC2-WT,and one less amino acid to form extended strand.6.The results of the fusion protein subcellular localization showed that the BrFC2-dg-GFP fusion protein was located in the chloroplast.The catalytic efficiency of thylakoid membrane protein?containing FC2?and prokaryotic expression BrFC2-dg in mutant dg was significantly higher than WT.7.We screened a resistant mutant against soft rot sr from 800 plants of EMS mutant library in Chinese cabbage by inoculated Pcc method in vitro and in vivo.The disease severity of WT plants inoculated with Pcc was scored as 9 by both inoculation methods.The disease severity of mutant sr was scored as 1.8.To compare the transcript profiles of resistant mutant sr plants to the susceptible WT plants at 0,6,12 and 24hpi?hours post-inoculation?in response to Pcc using RNA-Seq.Approximately 512 differentially expressed genes?DEGs,421 up-regulated genes,91 down-regulated genes?between sr and WT plants were identified and occurred between 6 to 12hpi,which corresponded to the important defense regulation period?resistance?to Pcc in Chinese cabbage.Pathogen pattern-triggered immunity?PTI?appears to be central to plant defense against Pcc triggered by M/PAMPs or DAMPs through pattern recognition receptors?PRRs?on the surface of cell membranes,which then caused a series of immune responses.Inoculated Pcc could stimulate increase the content of endogenous jasmonic acid?JAs?in host plants,and the accumulation of JAs was involved in immune response to Pcc.JA/ET as a positive regulated signal could enhance resistance to Pcc.SA-mediated hormone signal pathways involved in defense response may inhibit the IAA-mediated signal to activate the defense response to Pcc.As well as spraying exogenous auxins,MEJA and BTH were also verified as functioning in the immune system.9.Concurrently,after inoculated Pcc,the expression of glucosinolates and lignin synthesis genes was up-regulated,the cotent of glucosinolates and lignin were higher,the accumulation of glucosinolates and lignin have a functional defensive role against Pcc.