Study On The Function Of PagD53/SMXL6a,a Key Gene Of Signal Transduction In Poplar Witchweed Lactone

As an important tree species widely cultivated in China,poplar plays an important role in agricultural protective forests,intercropping,and urban greening.The selection of Populus species with a good crown type is the development direction of Agroforestry Management,in which the crown type structure and density of Populus species are correlated with branch development.Plant hormones are important regulators of branching development,and strigolactones（SLs）,as a novel class of plant endogenous hormones,are involved in regulating diverse biological processes including plant branching,reproductive development,and leaf senescence.D53/SMXLs is a suppressor in the carlactone signaling pathway and plays an important role in the signaling of strigolactones.In this study,we located the Populus PagD53/SMXLs gene family by homologous sequence alignment between rice and Arabidopsis D53/SMXLs genes,and performed a series of experiments including bioinformatics analysis,tissue expression pattern analysis,genetic transformation experiments as well as transcriptome sequencing analysis to explore the regulatory role of PagD53/SMXL6a gene in strigolactone signaling,and the obtained conclusions are as follows:（1）Bioinformatics analysis of PagD53/SMXLs gene family:Using BLAST analysis of the rice Os D53 and Arabidopsis At SMXLs gene sequences,the screen revealed four D53/SMXLs genes in Populus（Potri.009G046700;Potri.001G252500;Potri.010G188200;Potri.008G069100）,named PagD53/SMXL6a、PagD53/SMXL6b、PagD53/SMXL8a、PagD53/SMXL8b,respectively,which are distributed on chromosomes chr01,chr08,chr09 and chr10.Analysis of their domains revealed that the D53/SMXLs genes all contain Clp A and the P-loop＿NTPase conserved domain.The PagD53/SMXLs gene family was analyzed for promoter cis elements and found to contain action elements related to hormone response,biotic stress response,as well as plant growth regulation.（2）Expression pattern analysis of PagD53/SMXLs gene family:Expression patterns of the PagD53/SMXLs gene family were analyzed using microarrays of different tissues of Populus trichocarpa,and Heatmap results showed that PagD53/SMXL6a was highly expressed in seedlings after 43 hr of germination,PagD53/SMXL6b was highly expressed in interlobar stages,PagD53/SMXL8a was highly expressed in primary flower buds,chloroplasts,and shoot apex,and PagD53/SMXL8b was most highly expressed in pistil and stamen.To quantify the expression pattern of D53/SMXLs genes in various tissues,PagD53/SMXL6a、PagD53/SMXL6b were most highly expressed in the apical meristem;PagD53/SMXL8a、PagD53/SMXL8b were most highly expressed in mature leaves.The results suggest that the PagD53/SMXLs gene family may be involved in the developmental regulation of different tissues in Populus.（3）Response of PagD53/SMXLs gene family to treatment with monopodial lactone analogues:Wild type glandular aspen were treated with various concentrations of strigolactones configured for expression analysis of the PagD53/SMXLs gene family.The results showed that PagD53/SMXL6a、PagD53/SMXL8a were most highly expressed at a strigolactone concentration of 10 mol·L^-1;PagD53/SMXL6b、PagD53/SMXL8b were most highly expressed at a strigolactone concentration of 2 mol·L^-1.The overall expression of the four genes in the PagD53/SMXLs gene family was higher than that in the control group,indicating that all of the Populus PagD53/SMXLs gene families respond to treatment with strigolactones.（4）Creation and phenotyping of PagD53/SMXL6a-OE and PagD53/SMXL6a-ANTI:Construction of overexpression and suppression expression vectors for the PagD53/SMXL6a gene in Populus,transgenic plants were obtained by genetic transformation of Populus spp.Phenotypic analysis of the transgenic plants showed that the plant height of the PagD53/SMXL6a-OE transgenics was lower than the control,the ground diameter was smaller than the control,and the plant height of the PagD53/SMXL6a-ANTI transgenics was higher than the control,and the ground diameter was larger than the control.Observation at 7d after depilation treatment revealed that the number of axillary buds increased in PagD53/SMXL6a-OE and decreased in PagD53/SMXL6a-ANTI compared to the control.This suggests that the PagD53/SMXL6a gene affects axillary bud initiation and plays a negative regulatory role in Populus development.（5）Analysis of possible downstream genes regulated by PagD53/SMXL6a:RNA from control glandular aspen and PagD53/SMXL6a overexpression plants was extracted for transcriptome analysis.The analysis resulted in 177 differentially expressed genes between OE-2、OE-3 amd OE-23.In GO functional enrichment analysis,the differential genes were enriched in catalytic activity,metabolic process and binding.In KEGG enrichment analysis,the differential gene functions were mainly enriched in the top five categories,which were metabolic pathways,biosynthesis of secondary metabolites,phenylpropanoid biosynthesis,plant pathogen interaction,cyanoamino acid metabolism.The analysis is aware that these differential genes mainly exist in related functions such as plant branching development and hormone signaling.q RT-PCR experiments were used to verify that the transcriptome data were consistent with the transcriptome results.In summary,this study focused on the function of PagD53/SMXL6a gene during Populus growth and development,and the results showed that PagD53/SMXL6a gene could affect plant growth and development by acting as a repressor in the strigolactone signaling pathway,thus providing a clue to the signaling pathway of D53/SMXLs gene in woody plant growth especially strigolactones and a theoretical reference for tree genetic breeding.