| The berberine bridge enzyme(BBE)family is a subfamily of a typical bi-covalent flavin oxidase superfamily(SCOPe d.58.32).Our previous study found that number of BBE were developmental xylem glycoprotein of populus,but the biological function was unclear.We identified all members of populus BBE gene family,bioinformatically analyzed the information of PtBBE gene family,analyzed the expression characteristics of PtBBE9a and PtBBE18a,identified their overexpressed functions.The main results of this study are as follows:Sixty-nine members of the populus BBE family were identified and classified into six categories,A-F.Most PtBBEs are located on chromosome 1 or chromosome 11,the expansion of PtBBE gene family due to inter-chromosomes duplication,intra-chromosome duplication and gene duplication.According the published electronic data in phytozome and our RT-PCR result,we analyzed the tissue expression characteristics of PtBBE gene,PtBBE23a and PtBBE24a were mainly highly expressed in xylem;PtBBE6b expressed in each tissue.Then screened some BBE members may functionally involved in stem development and lignin biosynthesis:PtBBE9a and were expressed high levels in xylem,and the expression level gradually increased with the stem nodes number,similar was PtBBE18a gene.We constructed two plant expression vectors pGWB3-ProPtBBE9a and pGWB3-ProPtBBE18a.The expression patterns of PtBBE9a and PtBBE 18a gene promoter were detected by transgenic plants.GUS staining was mainly detected in immature apical meristem、mature capsule epidermis and mature roots of transgenetic ProPtBBE9a::GUS seedling,while GUS staining was detected in immature vascular tissue of ProPtBBE 18a::GUS seedling.The pGWB5 overexpression vectors of PtBBE9a and PtBBE18a were constructed.The overexpression functions of PtBBE9a and PtBBE18a genes were identi fied by transgenes.35S::PtBBE9a::GFP plants were early flower than WT,and rosette leaf number were fewer.Base stem of 35S::PtBBE9a::GFP plants became thicker.Phloroglucinol staining area became wider,and the staining became darker.Lignin content were increased.similar was PtBBE 18a gene.We constructed PtBBE23a/PtBBE6b/PtBBE24a CRISPR/Cas9 gene editing vector,which gene were all highly expressed in xylem.Successfully get three PtBBE23a/PtBBE6b/PtBBE 24a mutation genetic material,and several single or double gene mutation material. |
