Functional Analysis Of Poplar KNAT7 And MYB002 Regulating Secondary Wall Formation And Drought Tolerance

Poplar is the main afforestation tree specie in northern China.It grows fast and needs more water.It has important ecological function and economic value.However,there is a shortage of wood and water in most parts of northern China,so it is of great significance to improve the wood modification and tolerance of poplar to drought stress.KNOXs transcription factors play an important role in the process of plant growth and development.Based on the previous research in the laboratory,we conducted in deep study on the KNOXs transcription factor family of poplar,and transformed PdKNAT7,which plays a role in xylem,into Arabidopsis thaliana by genetic engineering technology to explore its function in secondary wall development.MYBs transcription factors play an important role in plant growth and stress.In this study,PtrMYB002 gene was overexpressed in Arabidopsis thaliana to explore its function in secondary wall development and drought stress.The main results are as follows:1.In this study,PdKNAT7 gene was cloned from poplar NE19.qRT-PCR analysis showed that PdKNAT7 was mainly expressed in poplar stem.In addition,PtrMYB002 gene was cloned from Populus tomentosa.qRT-PCR analysis showed that PtrMYB002 was mainly expressed in stems and roots of poplar,and was up-regulated by drought and exogenous ABA treatment.Subcellular localization showed that PdKNAT7 and PtrMYB002 were located in the nucleus.2.PdKNAT7 gene was transformed into wild-type(Col-0)Arabidopsis thaliana by inflorescence infection,and overexpressed plants were obtained.The WT,overexpression line and mutant knat7 were observed by freehand slicing.The results showed that compared with wild-type plants,the fiber wall of overexpression plants was thinner,while that of mutant lines was thicker;qRT-PCR analysis of lignin,cellulose and hemicellulose synthesis related genes in different genotypes of Arabidopsis showed that the expression levels of 4CL1,C4H1 and IRX10 genes in overexpressed plants were down regulated,and the mutants were on the contrary;Meanwhile,yeast single hybridization experiment showed that PdKNAT7 could bind to the promoter of Pd IRX10.3.PtrMYB002 gene was transformed into wild-type(Col-0)Arabidopsis thaliana by inflorescence infection,and overexpressed plants were obtained.The WT and overexpression lines were observed by freehand slicing.The results showed that compared with wild-type plants,overexpression plants had thicker fiber wall between bundles,xylem fiber wall and vessel wall;qRT-PCR showed that the expression levels of KNAT7,4CL1,C4H1,CCR1 and Ces A8 were up-regulated.After drought treatment,compared with WT,overexpression plants didn’t wilt,and DAB and NBT staining showed stronger antioxidant capacity;At the same time,qRT-PCR results showed that the expression of LEA14 and RD29 B genes responding to drought was up-regulated.4.PdKNAT7 and PtrMYB002 genes were transiently transformed into 84 K poplar by vacuum infiltration method,respectively.qRT-PCR was used to detect the expression of lignin,cellulose and hemicellulose synthesis related genes and drought related genes in poplar,which was basically consistent with the results of Arabidopsis,indicating that the above two genes may have similar regulatory functions in PoplarIn conclusion,the genes PdKNAT7 and PtrMYB002 of poplar can affect the thickness of secondary cell wall of Arabidopsis by regulating the accumulation of lignin,cellulose and hemicellulose.In addition, PtrMYB002 gene can improve drought tolerance of Arabidopsis by regulating drought response genes. Moreover,MYB002 can affect the expression of KNAT7;PdKNAT7 may regulate its expression by combining with the promoter of Pd IRX10.The study revealed the molecular regulation mechanism of secondary wall formation and drought tolerance of poplar,which provided a new theoretical basis for improving the wood quality and drought tolerance of poplar.