Effects Of Fusion Gene 4CL1-CCR On Tobacco Cell Wall

4CL(4-coumarate coenzyme A ligase,4-coumarate: coenzyme A ligase;)is the branch-point enzyme connecting phenylpropane metabolism and lignin biosynthesis,CCR(cinnamoyl-Co A reductase)is the first ate-limiting enzyme of lignin-specific biosynthesis.Regulating the expression of these two genes will affect the flow of "carbon flow" in the plant.In previous research,these two enzymes were connected to form the bifunctional enzyme 4CL1-CCR,the catalytic activity of this bifunctional enzyme in vitro and in prokaryotes have been studied.However,the role of exogenous genes in plants is different from that in prokaryotes.Therefore,we analyzed the fusion gene 4CL1-CCR in tobacco in this study.The lignin deposition in tobacco plant and suspension cells were analyzed,the affection of 4CL1-CCR on the process of tobacco suspension cells was analyzed by using RNA-seq.The research results are mainly divided into the following five parts:(1)The analysis of the content of transgenic tobacco monolignols by thiosulfolysis and GC-MS found that overexpression of fusion gene 4CL1-CCR not only increased the total content of lignin in cell wall,but also changed the lignin monomer composition.The content of G-unit in transgenic tobacco was 14.34±0.52?g/mg,which was significantly higher than that of WT 10.51±0.03 ?g/mg,but the changes of S-unit lignin was not consistent in different transgenic plant.(2)Hydrochloric acid-phloroglucinol staining was performed on different parts of the transgenic tobacco stems.It was found that the lignified cells of the transgenic tobacco xylem(tender tissue)showed a red loop,while in the WT not all cells at thesame location exhibited a staining reaction.Observation of the mature tissue of transgenic tobacco revealed that the width of the xylem and the perimeter of the vessel inner of the transgenic tobacco increased significantly.(3)Using Raman spectroscopy,mapping analysis of lignified cells of transgenic tobacco stems revealed that the areas with higher lignin density were compound middle lamella and cell corners,while the lignin density was lower in the secondary cell wall.The area of high-density lignin in transgenic tobacco stems increased significantly compared with WT,while the increased lignin in the secondary wall of transgenic tobacco showed thickened of the secondary wall,and did not change the lignin density of secondary wall.(4)Subculture of transgenic tobacco suspension cells(MS+2,4-D 1.0mg/L),it was found that the 4CL1-CCR gene did not affect the cell growth,the transgenic suspension cells had the same growth curve as WT.However,the fusion gene4CL1-CCR caused the lignin accumulation of transgenic suspension cells to advance.On the 11 th day of subculture,the transgenic suspension cells were observed the reaction of lignin with hydrochloric acid-phloroglucinol.The transgenic suspension cells showed deepened staining on the 24 th day,but the WT cells were still not stained after 24-days subculture.It was detected that a small amount of G-unit lignin accumulated in the transgenic tobacco suspension cells after 24 days of subculture by using GC-MS.Through the analysis of lignin monomers in different stages of tobacco development,it was found that G-unit monolignol was accumulated during the early development of transgenic and WT tobacco,and then S-unit lignin began to deposit into the cell wall area as the tissue gradually matured.And finally make G:S approach to 1.(5)RNA-seq analysis of gene expression of WT and transgenic suspension cells at different subculture durations showed that the expression levels of multiple lignin biosynthesis genes were up-regulated to varying degree in transgenic tobacco suspension cells.The cellulose synthesis multienzyme complexes in the primary and secondary wall of tobacco were down-regulated to varying degrees,the synthesis ofcellulose was weakened.The up-regulation of pectin methyl esterase gene expression reduced the content of methylated pectin,which in turn affects the structure and hardness of the cell wall.Analysis of the expression of the auxin transporter family AUX1 members found that the expression of this family member genes in transgenic tobacco suspension cells was severely inhibited,which hindered the transmembrane transport of 2,4-D into the cell.The low concentration of auxin in cell cannot activate the auxin response factor,which ultimately affects the expression of target genes downstream of the auxin signaling pathway.The expression of lignin-synthesis gene related transcription factors myb-releated protein 306 and myb-releated protein 315 increased.Among them,myb-releated protein 306 may be involved in the transformation of cells from proliferation to differentiation,and myb-releated protein315 may be directly involved in regulating lignin monomers biosynthesis.The regulation of the expression of lignin-related genes eventually led to the early lignin deposition of transgenic suspension cells.In this study,the experimental data on the function of the fusion gene in the tobacco completed data of the fusion gene 4CL1-CCR in vitro,prokaryotes,and eukaryotes.Transcriptome analysis of suspension cells explained the effect of fusion gene 4CL1-CCR on tobacco cell morphology and lignification process from a molecular perspective.