| Lignins,some of the most abundant biopolymers on earth,accumulate mainly in the secondary cell walls of xylem cells.Lignins are crucial for the structural integrity of cell walls,and the stiffness and strength of stems,allowing plants to grow upward.The biosynthesis pathway of lignins is a branch of the phenylpropane metabolic pathway.Of all the enzymes involved in lignin biosynthesis pathway,cinnamoyl-CoA reductase(CCR,EC 1.2.1.44)is of particular interest as it converts cinnamoyl-CoAs into their corresponding cinnamaldehydes.In this study,we isolated and characterized the specific functions of a birch CCR1(which we named BpCCR1)in wood formation and growth.We modified BpCCR1 transcript levels in overexpression transgenic lines,35S::BpCCR1,and suppression transgenic lines,35S::anti-BpCCR1,and analyzed the effect of this ongrowth,lignin and cellulose content,the structure of secondary xylem and salt resistance.We analyzed the transcriptome sequence of WT(wild-type),overexpression transgenic lines and suppression transgenic lines to reveale the relationship between BpCCR1 gene and other genes Our result demonstrated that by modifying the BpCCR1 gene,it may be possible to develop sustainable trees that possess desirable lignin content,and appropriate wood properties for both pulping and conversion to clean bioenergy fuels.The results are as follows:(1)The overexpression transgenic lines,suppression transgenic lines and WT were screened by PCR to provided preliminarily evidence,and northern blotting and western blotting were used to further verify the transgene expression of overexpression transgenic lines,suppression transgenic lines and WT.The results showed that both sense and antisense constructs containing BpCCR1 were integrated into the genome of B.platyphylla × Betula pendula(WT),and they could transcription and translation normally.(2)Modification of BpCCR1 can cause the change in growth.We measured the height and ground diameter of 1-year-old and 2-year-old transgenic birches.We found the all of sixteen overexpression lines in 1-year-old decreased in heitht compared WT,68.75%overexpression transgenic lines decreased in ground diameter.In fifteen suppression lines in 1-year-old,three lines increased in heitht compared WT and five lines increased in ground diameter.In sixteen overexpression transgenic lines in 2-year-old,62.50%lines decreased in height but all of the lines increased in ground diameter.In fifteen suppression transgenic lines in 2-year-old,60%lines increased in heitht compared WT and all of the lines increased in ground diameter.Therefor,the overexpression of BpCCR1 genes could cause the birch to dwarf and suppression could cause to heighten.(3)Lignin and cellulose content was measured in WT plants,the three overexpression lines and three suppression lines.The lignin content in all the three overexpression lines was higher than that of WT.In two overexpression lines,C11 and C18,lignin contents were significantly increased by 8.4%,and 14.6%respectively.And the cellulose content in three overexpression lines was lower than that of WT.In two overexpression lines,C3 and C18.cellulose contents were significantly decreased 5.65%and 7.42%.In three suppression lines,lignin of contents of F1 and F11 were lower than that of WT and there was no obvious change in cellulose content.(4)Sections of stems were obtained from three different positions of birch:the top,the middle and bottom.For the secondary xylem,the number of xylem vessels in overexpression lines,C18,was more than that of the WT and the diameters of all xylem vessels in C18 were smaller.The xylem vessels of C18 were re-arranged in such a way that they appeared continuously in a straight line that was up to two times longer than that in WT plants.There were no obvious changes in the xylem vessel numbers insuppression lines,F11,as compared with the WT.However,we observed a much sparser distribution of xylem vessels in F11 in the middle of the stem as compared with the WT.Based on the above observation and the changed amounts of lignin contents,we chose overexpression lines of C11 and C18,and suppression lines of F1 and F11 for xylem wood ultra-microstructure analysis.We found that the cell walls of xylem fibers in C11(1.56 ?m)and C18(1.55 ?m)were significantly thicker than those in WT(1.13?m)plants,whereas the cell walls in F1(0.98 ?m)and F11(0.89 ?m),were significantly thinner than those in WT plants.The overexpression of BpCCR1 gene contributed significantly to thickening of the xylem fiber cells and the number of xylem vessels in birch.(5)In this study,salt tolerance and disease resistancehas been analyzedin transgenic birch.In the concentration of 1%NaCl,the salt tolerance ofC3 and C18 were stronger than that of WT in three three overexpression lines.Salt injury index of C3 and C18 were 29.64%and 19.54%lower than WT,respectively.That meaned the damage extent of C3 and C18 from salt was lower than WT.Salt injury index of all of the suppression transgenic lines,F1,F11 and F12,were 18.89%,9.12%and 37.46%higher than that of WT,respectively.It showed the damage extent of suppression lines from salt was stronger than WT.After inoculating ofleaf blight(Alternatia alternate)in transgenic birch,the disease resistance of C7,C8 and C18were stronger than that of WT in four overexpression lines.The disease index of C7,C8 and C18were83.56%,78.08%and 38.36%lower than WT,respectively.The disease index of all of the suppression transgenic lines,F1,F2,F11 and F12 were 35.62%,4.11%,12.33%and 17.81%higher than that of WT,respectively.These results showed overexpression of BpCCR1 gene could enhance salt tolerance and disease resistanceof birch and suppression could reduce the salt tolerance and disease resistance.(6)Overexpression lines(C18),suppression transgenic lines(F11)and WT were determined using Solexa technology.By transcriptome sequencing and assembly,we obtained 132,427 unigenes in total,and 47.61%of unigenes were annotated.There were 8243 differential unigenes between C18 and WT,which contained 3362 upregulated unigenes and 4881 downregulated unigenes.And there were4657 differential unigenes between F11 and WT,which contained 2270 upregulated unigenes and 2387 downregulated unigenes.GO analysis showed that significant differences between C18 and WT were existed in 42 parts,such as the plant cell wall,kinase activity,protein kinase activity,transferase activity and stress responses.Significant differences between F11 and WT were existed in 111 parts contained the cell wall and lignin decomposition and metabolic pathway,phenylalanine biosynthesis pathway.Pathway analysis showed 28 pathways were significant different in overexpression line and WT in overexpression database,and 36 were significant different in suppression line in suppression database(Q-value<0.05).And there were 23 pathways to be obtain together in two database,which was phenylpropanoid biosynthesis pathways,the structure of secondary xylem,and salt tolerance.The analysis of lignin biosynthesis pathway showed BpCCRl gene could cause significant changes on the genes from lignin biosynthesis pathway,CAD and HCT were the most obvious.These differential genes will help us to reveal the molecular mechanisms that how BpCCRl gene to work for lignin biosynthesis pathway... |
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