Analysis Of Biological Function Of PtrHAT22 In Populus Secondary Cell Wall Formation

HD-Zip is a plant-specific transcription factor family involved in many biological processes such as plant photosynthesis,lignin synthesis and morphological development.Previous bioinformatics analysis found that HAT22 of this gene family may be involved in the formation of secondary cell walls.In order to verify whether HAT22 is involved in the formation of plant secondary cell wall,the homologous gene of HAT22 was cloned from the model plant Populus trichocarpa in woody transgenic plants,and its tissue expression specificity,transcription factor of the basic characteristics and biological functions in the formation of secondary cell wall of poplar were studied.The main findings are as follows.1.The CDS sequence of 822 bp full length of HAT22 has been cloned and named as PtrHAT22.PtrHAT22 encodes 273 amino acids with a family of typical HD and Zip domains and an LxLxL inhibitory domain at the 17-21 amino acid sequence.Phylogenetic tree analysis of the HD-Zip transcription factor family of Populus trichocarpa showed that there is a high similarity among most members of the HD-Zip transcription factor family.2.qRT-PCR analysis of different tissues showed that PtrHAT22 can be expressed in the roots of Populus trichocarpa;primary leaves;transition leaves;secondary leaves;transitional phloem;secondary phloem;primary xylem;transitional xylem;this results indicating its involvement in the development of multiple organs and tissues of plants.It was also found that PtrHAT22 expressed the highest amount in the xylem in all tissues involved,suggesting that the main function of PtrHAT22 may be involved in secondary cell wall synthesis.Staining analysis of transient infection of PtrHAT22 promoter plus GUS gene showed that the secondary stem,root and vein were stained deeply,indicating that the gene mainly expressed in the secondary cell wall of the plant,which is related to the results of qRT-PCR.basically consistent.3.Subcellular localization studies indicate that PtrHAT22 is localized to the nucleus.Transcriptional activation studies found that PtrHAT22 has no self-activating,Yeast hybridization experiments revealed that PtrHAT22 was able to bind to the previously predicted downstream regulatory target genes MYB46,MYB58 and C2H2 promoters.By analyzing the promoter elements of other target genes downstream,the high frequency components of the above genes and the elements related to lignin synthesis were selected and subjected to yeast hybridization experiments.The results showed that PtrHAT22 can interact with AC-?,Box4,TATA-box and the G-Box element specifically binds.4.The growth phenotype of PtrHAT22 overexpressing Populus trichocarpa showed that the transgenic plants OE-2,OE-3 and OE-6 respectively decreased by 9.37%,14.9%and 28.67%compared with wild type Populus trichocarpa;The number of stem segments respectively decreased by 23.61%,12.50%and 34.72%;the dry weight respectively decreased by 36.79%,34.28%,and 48.06%;the fresh weight respectively decreased by 24.51%21.88,and 39.21%;leaf length respectively decreased by 40.00%,46.67%,57.78%,;leaf width respectively decreased by 50.48%,47.62%,64.76%,plant height,ground diameter,stem number,dry weight fresh weight and the blade size is significantly reduced.The above results preliminarily showed that PtrHAT22 can inhibit the vegetative growth of poplar.5.Determination of the main components of PtrHAT22 overexpressing the cell wall of Populus trichocarpa.It was found that the lignin content decreased by 17.15%,the cellulose content decreased by 10.95%,and the hemicellulose content increased by 14.68%.At the same time,the length of the fiber decreased by 2.46%and the width decreased by 13.01%.Scanning electron microscopy analysis showed that the secondary cell wall thickness of the transgenic lines decreased by 36.29%.qRT-PCR analysis of genes related to secondary cell wall formation in overexpressing transgenic lines showed that the expression levels of cellulose synthesis genes CesA4,CesA 7,and CesA 8 were significantly decreased,the expression of hemicellulose synthesis gene IRX9 was significantly decreased,and the expression of IRX10 was increased.The expression level of lignin synthesis gene PALI was decreased,and the expression levels of PtrPAL4,HCT1,CSE2,C4H2,COMT2,CAD1,CCR2 and CCoAOMT1 were significantly decreased.The expression level of downstream gene MYB46.MYB58,C2H2 was significantly decreased.The expression changes of the above genes were basically consistent with the changes of secondary cell wall component content,indicating that PtrHAT22 passed.Direct or indirect regulation of the expression of these genes affects the formation of secondary cell walls in plants.