Functional Divergence Of Key Genes Involved In Monolignol Biosynthesis In Populus Tomentosa

Lignin is widely present in vascular plants,and together with cellulose,hemicellulose constitutes the basic skeleton of plants.Achieving plants with directional modification of lignin composition and structure will not only reduce the cost for producuing the second generation of bioenergy energy but also lay the foundation for the deep processing of lignin.In this dissertation,the functional divergence and evolution of the key genes of monolignol biosynthesis were studied systematially combining with the current understanding of monolignol biosynthesis pathway with poplar as the basic research object.We tried to explore emergence and evolution of the process and also provide reliable toolbox for performing genetic engineering in order to modified the monolignol biosynthesis pathway in Populus tomentosa.The main contents and conclusions are as follows:1)First,using Populus trichocarpa genome,combined with phylogenetic analysis,expression profiling,promoter analysis,We screened candidate genes encoding caffeoyl shikimate esterase(CSE)or hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferase(HCT).The results showed that HCT1 and PtoCSE1 and 2 had corresponding catalytic activity in vitro,and it was speculated that PtoCSE1 was mainly involved in monolignol biosynthesis.The CSE orthologs could not be found in most monocots except Oryza sativa.This is the first time for systematic study on the CSE gene family since the discovery CSE.In addition,our results further confirmed the presence of CSEs involved in monolignol biosynthesis in Populus.2)Functional divegence studies of cinnamoyl coenzyme A ester reductase(CCR)and cinnamyl alcohol dehydrogenase(CAD)have shown that only PtoCAD1,2,8 and PtoCCR1 and 7 can be detected with corresponding activity in vitro.The enzymatic kinetics of PtoCAD2 further confirmed that the so-called SAD should be only a branch of CAD.Site-directed mutagenesis studies for PtoCCR7,as well as molecular modeling,revealed the key residues associated with CCR activity.H208 and R259 are considered to be two important signature sites for distinguishing CCR and CCR-like proteins.3)Based on the study of CCRs from different phylogenetic plants,we found that the consevative domain NWYCY which was reported as a CCR-specific recognition was not universal,while the signature sites H208 and R259 we reported remains available in all tested plants.The study of CCR in Selaginella moellendorfii has revealed the possibility of a new CCR subfamily.And Based on our established criteria,the screening of genuine CCRs in Physcomitrella patens and several algae genomes has shown that genuine CCR or genuine CCR precursors are likely to occur in moss,rather than the algae as reported.4)We cloned and obtained the complete molecular toolbox of the key genes involved in monolignol biosynthesis in Populus tomentosa.Using the four-season expression data of xylem and phloem,we found that the upstream genes in monolignol biosynthesis pathway led the process in spring and summer while the downstream CAD and CCR dominant monolignol biosynthesis pathway in the autumn and winter and the efficiency of lignification will decline.In summary,I have studied the key enzymes and genes of the whole lmonolignol biosynthetic pathway,and provided a complete molecular toolbox for genetic engineering in order to recognize lignin improvement.Our results also reveals the structure and evolution of some key enzymes in monolignol biosynthesis.