The Study Of C3H3 Gene's Function In Populus Tomentosa

Lignin,one of the three main components of the cell wall,on the one hand,plays an important role in plants for the mechanical support and water transport;on the other hand,because of its close integration with cellulose and hemicellulose,hinders the industrial use of cellulose.Poplar as a model creature of woody plant,with great ecological value and commercial value,although there are many reports on lignin in poplar,lignin biosynthetic pathways are complex and still not studied thoroughly,and the use of CRISPR/Cas9 technology to research lignin in Poplar is still uncommon,it must play a positive role in research of lignin in Poplar.C3H is a cytochrome P450 enzyme,which is a rate-limiting enzyme for the phenylpropane pathway in the lignin biosynthetic pathway,which determines the flow of carbon from the lignin monomer and is a very important component of the lignin synthesis pathway.Here,we took Populus tomentosa as our experimental material,then introduced the vector?C3H3-DN?into Populus tomentosa by transgenic technology,then the gene knockouted pla nts were obtained,and done a series of analysis on its growth and physiological characteristics.This study provides a new way for lignin content regulation and improvement in poplar.The main results are as follows:1.The phylogenetic tree analysis of C3H gene in six representative species show that it's barely exist in ancient creatures.2.There are four C3H homologous genes in Populus tomentosa?C3H1,C3H2,C3H3and C3H4?,we analyzed their expressions in four different tissues of stem tip,leaf,xyle m and phloem.We fing that the expression of C3H3 gene in xylem was the highest,and the other three genes were expressed in four different parts but the expression level was very low.Thus we selected the C3H3 gene as our knockout target gene.3.After a series of gradient experiments,the genetic transformation system of Populus tomentosa was gradually improved on the basis of stable regeneration system of Populus tomentosa.We chose leaf and stem as the explants,and the OD₆₀₀ was about 0.6,and the infection time was 20 min.The best time for co-culture was 3 d.On the basis of CRISPR/Cas9 related technique,the C3H3 gene was knocked out and the mutated loci were detected.The results showed that C3H3 gene were mutated and C3H3 gene mutant Poplar has been obtained.4.The growth trend of the wild type and mutant plants was studied.The results showed that the growth of the mutant plants was slow and the growth was stopped when the height was about 3 cm.The surface cells of the mutant plants were significantly smaller than those of the wild type.5.The photosynthetic characteristics analysis and hormone determination of wild type and mutant plants showed that the net photosynthetic rate index,stomatal conductance index and transpiration rate index of mutant plants were lower than wild type.The IAA content was higher than that of wild type and ABA content was lower than wild type.6.Sampling of the stems of wild-type and mutant plants revealed that the lignification of the mutant plants was low and the ductal cell deformation and secondary wall thickening were significantly reduced.Which indicated that C3H3 plays an important role in the secondary growth of poplar.