| Lignin is an important component of wood,which mainly plays the role of mechanical support,water transport and insect defense.4CL enzyme is one of the key enzymes controlling lignin synthesis pathway.In this study,84K poplar was used as the experimental material to clone the cDNA sequence of Pag4CL3/4CL5 gene from 84K poplar cDNA using PCR technology.The GenBank registration Numbers were MK183033(Pag4CL3)and MK183034(Pag4CL5)respectively.The functions and characteristics of the two genes were analyzed by bioinformatics software.Overexpression and inhibition of plant expression vector was constructed,and agrobacterium-mediated genetic transformation of 84K poplar was conducted to obtain Pag4CL3 and Pag4CL5 overexpression of transgenic lines and the joint inhibition of expression of transgenic lines.The main research results are as follows:(1)By using the PCR cloning technology,the full length of the Pag4CL3 coding region and the full length of the Pag4CL5 coding region were determined to be 1623bp and 1632bp respectively by taking the 4CL3 and 4CL5 coding sequences of Populus trichocarpa as reference.In addition,SSGTTGLPKGV and GEICIRG were conserved motifs.Subcellular localization prediction showed that Pag4CL3/Pag4CL5 protein was mainly located in the endoplasmic reticulum,which may be a membrane protein.Protein hydrophilicity predicted Pag4CL3/Pag4CL5 are hydrophilic proteins.(2)The expression differences of Pag4CL3/Pag4CL5 in different tissue parts were analyzed by qRT-PCR,and the results showed that Pag4CL3 was highly expressed in leaves and stems,but low in roots and terminal buds.The expression of Pag4CL5 was higher in leaf and root,but lower in stem and terminal bud.It is speculated that Pag4CL3 and Pag4CL5 may function together in the leaf,Pag4CL3 in the stem and Pag4CL5 in the root.(3)Plant expression vectors pROKII-4CL3 and pROKII-4CL5 were constructed,and through agrobacterium-mediated genetic transformation of 84K poplar,6 transgenic lines were obtained,respectively.After PCR and qRT-PCR,the results showed that Pag4CL3 and Pag4CL5 genes were successfully transferred into 84K poplar genome and expressed.Compared with WT 84K poplar,the relative expression of Pag4CL3 overexpressed transgenic plants was the highest,which was 195 times higher than that of L11.The highest expression levels of Pag4CL5 overexpressed transgenic lines were L7 and L4,which were increased by 368 and 321 times,respectively.(4)The common sequence of Pag4CL3/4CL5 was designed as interference fragment and pRNAi-4CL3/4CL5 inhibitory plant expression vector was constructed.The common interference plant expression vector of Pag4CL3/4CL5 gene was constructed.Through genetic transformation of 84K poplar,7 transgenic lines were obtained.After PCR and qRT-PCR detection were performed on them to confirm that exogenous DNA had been integrated into the genome of 84K poplar.QRT-PCR results showed that the expression levels of all the 7 lines were reduced to varying degrees.When the expression level of Pag4CL3 gene interference was detected,the largest relative expression level was decreased by L1,which was 75%lower than that of WT 84K poplar.When the expression level of Pag4CL5 gene was detected,the relative expression of L1 was the most significant compared with WT 84K poplar,and the decrease was 80%larger than that of Pag4CL3.(5)Through the annual Pag4CL3/4CL5 expression and interfere with the expression of genetically modified 84K poplar cellulose,hemicellulose and lignin content determination,the results showed that:The comparison of the wild-type 84K poplar showed that the cellulose content in the over-expressed Pag4CL3 transgenic lines was significantly reduced by 2.65%at most(L11).The content of hemicellulose was different,except that there was no significant difference between L33 and WT.The lignin content increased by 20.11%(L11).The cellulose content of Pag4CL5 overexpressed transgenic lines was significantly reduced by 4.24%at most(L8).The content of hemicellulose was not significantly increased or decreased,except for L9 water with significant difference.The lignin content increased by 17.27%at most(L4).Except that there was no significant difference between L3 and wild type,the content of other transgenic lines was significantly higher than that of wild type.The content of cellulose in transgenic plants inhibited by pRNAi-4CL3/4CL5 increased,with the maximum increase of 2.39%(L1),in which all transgenic lines except L2,L14 and L39 increased significantly.Hemicellulose content increased except for the decrease of 0.82%in L12,with a maximum increase of 3.84%(L14),in which L33 and L44 reached a significant difference level.The lignin content was significantly reduced by 27.79%... |
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