Research On The Function Of Berberine Bridging Enzyme Gene (PdPapBBE53) In Populus Davidiana× P.alba Var.Pyramidalis

Populus davidiana × P.alba var.pyramidalis(Shanxin poplar)is infertile and does not fly catkins.It is an artificial hybrid variety suitable for the cold area of Northeast China.The transgenic study of Populus sinense will provide theoretical basis for breeding poplar varieties with high resistance and rapid growth,which is of great significance in the field of landscaping.Berberine bridging enzyme is the key enzyme of berberine biosynthesis,which has typical characteristics of flavin protein oxidase.In this study,Shanxin poplar was used as experimental material.The transcriptome database of interaction between Shanxin poplar and Trichoderma asperellum and Shanxin poplar and Alternaria alternate had been constructed in our laboratory.The gene PdPapBBE53(Genbank ID: MN445357.1)of berberine bridging enzyme was screened and cloned based on the database.Bioinformatics analysis,tissue expression pattern and expression regulation in response to stress were performed for it.PROK2-BBE53 overexpression vector was constructed and successfully transferred into Shanxin poplar by Agrobacterium mediated method.Overexpressed lines(K2-5A and K2-8E)were obtained.Then,the gene function was studied.The results were as follows:1.The PdPapBBE53 gene was cloned and obtained.Bioinformatics analysis showed that:This gene belongs to the flavinase family,with a full length of 1593 bp and an ORF encoding530 amino acids.There are two conserved domains "FAD＿binding＿4" and "BBE".Subcellular localization predicts that PdPapBBE53 protein is most likely extracellular(including cell wall);PdPapBBE53 protein is predicted to be a stable hydrophilic,secreted protein with transmembrane structure;The secondary structure of PdPapBBE53 protein accounted for32.26% of α helix,23.96% of extended chain,5.47% of β folding and 38.30% of random curl.The tertiary structure predicted the highest consistency with the template 4UD8.1.A of Arabidopsis under the maximum GMQE value.Protein prediction showed that protein sequences with high homology to PdPapBBE53 all had three highly conserved motifs.The phylogenetic tree was constructed to show that: PdPapBBE53 is the closest relative to KAG6794189.1(Populus tomentosa).2.The tissue expression characteristics of PdPapBBE53 gene in 6-week-old Shanxin poplar tissue culture seedlings were studied by q RT-PCR.The results showed that: In untreated conditions,PdPapBBE53 is expressed in different degrees in shoot tips,leaves,stems and roots,with the highest expression in mature leaves;The expression levels of PdPapBBE53 in roots were up-regulated in all three abiotic stress environments,and there were significant differences under salt and alkali stress,suggesting that PdPapBBE53 was regulated by salt,alkali and high osmotic pressure stress.In the five biotic stress environments,the expression of PdPapBBE53 was down-regulated in leaves,and the expression of PdPapBBE53 was upregulated in roots except for Cytospora chrysosperma stress,speculating that PdPapBBE53 may play an important role in resisting biotic stress;In the three phytohormone treatments,the expression of PdPapBBE53 was significantly up-regulated only in roots induced by JA,and was significantly down-regulated in different tissues induced by ABA and SA,suggesting that PdPapBBE53 may be involved in the phytohormone signaling pathway.This study provides a theoretical basis for further study of PdPapBBE53 gene function.3.The PdPapBBE53 overexpression vector pROK2-BBE53 was reconstructed and constructed and infected into the leaves of wild Shanxin poplar by agrobacterium-mediated method,and the gene overexpressed Shanxin poplar was obtained(K2-5A and K2-8E).The growth index,photosynthetic characteristics,gene coexpression,biological and abiotic stress analysis of K2-5A and K2-8E lines of Shanxin poplar showed that compared with the control group,K2-5A and K2-8E lines had a greater growth advantage and enhanced photosynthetic capacity.The expression levels of PdPapBBE53,HRGP,WAK and WRKY22 in mature leaves(7 leaves)of K2-8E and K2-5A lines were 3.22 and 1.74 times,52.03 and 22.76 times,57.35 and 45.34 times,1.65 and 0.93 times,respectively.The expression of PdPapBBE53 was the highest in mature leaves,and it was co-expressed with HRGP,WAK,ERF34 and WRKY22 genes to cope with various stresses.In addition,the experimental results of responses of isolated leaves and adult seedlings to biological and abiotic stresses further proved that K2-5A and K2-8E lines had better resistance to biological and abiotic stresses than wild-type lines.Therefore,it is speculated that PdPapBBE53 gene has a regulatory function in response to stress.In conclusion,the berberine bridging enzyme gene(PdPapBBE53)in Shanxin poplar plays an important role in improving the growth characteristics of overexpressed plants and their ability to resist biological and abiotic stresses.