The Evolutionary Analysis Of Expansin Gene Families In Three Basic Species Of Brassica And Studies On Functional Differentiation Of Expansin Genes Related To Pollen Development

Pollen development and the stage of pollination and fertilization are important links for flowering plants to complete sexual reproduction.Abnormal pollen development prevents the formation of functional pollens,which seriously affects the reproduction of plants.The smooth progress of pollination and fertilization is inseparable from the normal germination and elongation of pollen tubes.The germination of pollen tubes usually starts from the intine of mature pollen grains.In the process of rapid elongation of the pollen tube,the rapid expansion of the cell wall is particularly necessary,and many cell-wall synthesis and remodeling proteins are involved.Plant expansins are an important type of cell wall remodeling protein that can bind to the polysaccharide components of cell walls,and cause the relaxation of plant cell walls by destroying the non-covalent bonds between cell wall components,and promote the growth of plant cells.The expansin gene family is subdivided into four subfamilies,namely EXPA,EXPB,EXLA and EXLB.Although the functions of expansin genes have been extensively studied in recent years,there are still few reports on the functions of expansin genes in plant reproductive development and the coordination of EXPA and EXPB subfamily genes.In this study,ajhGMS ‘Bcajh97-01A/B'of Brssica rapa syn.B.campestris,‘Z1411-02' of Brssica nigra,and ‘Jingfeng 1' of Brassica oleracea were used as plant materials.We have identified evolution and expression of expansin gene families in the three basic diploid species of Brassica,and clarified their evolutionary characteristics.So as to provide a relevant basis for exploring the function of expansin genes in the development of pollen and pollen tube.Two genes,BrEXPB4 and BrEXPB9,which are highly expressed in the late stage of the pollen development and in the pollen tube of Brassica rapa were screened by the expansin gene family analysis.The function of BrEXPB4 and BrEXPB9 in the development of Brassica rapa pollen and pollen tube was further studied by artificial microRNA technology.Moreover,the biological function of AtEXPB5,which is the homologous gene of BrEXPB4 and BrEXPB9 in the model plant Arabidopsis thaliana,was analyzed by CRISPR/Cas9 technology and overexpression technology.We also performed functional verification on AtEXPA4,which has redundant functions during the development of pollen and pollen tubes.Finally,the possible involvement of AtEXPB5 and BrEXPB4/9 in transcriptional regulation pathways were explored by yeast one-hybrid and dual-luciferase experiments.The main results are summarized as following:(1)The expansin gene families of the three basic species of Brassica(B.rapa,B.oleracea and B.nigra)were identified by the three-step analysis method,and compared with the model plant Arabidopsis thaliana expansin family on the phylogeny,gene structure.We also studied physical and chemical characteristics of their expansion patterns and evolutionary details have been studied,and it is found that after independent evolution,expansin gene families have more similarities in these three basic species,but less divergence.By studying the differences between promoter sequences and coding sequences of the orthologs,it is found that there are significant positive correlations between Arabidopsis thaliana and three basic species.Subsequent expression analysis showed that there are extensive functional differences in expansin gene families of these three species.In particular,BrEXPB4 and BrEXPB9,which are strongly expressed during the development of pollen and pollen tube,were screened during reproductive development.(2)The results of subcellular localization by the tobacco and onion transient expression system showed that BrEXPB4 and BrEXPB9 were both localized on the cell wall,which was consistent with the predicted results that they both have signal peptides.The spatiotemporal expression analysis by fluorescence quantitative PCR and GUS gene reporter system showed that the expression patterns of BrEXPB4 and BrEXPB9 were also very similar.They were all expressed from the late uninucleate stage,and with pollen development,expression levels were gradually increased,and could continue to pollen tubes.(3)The expression of BrEXPB4 and BrEXPB9 were inhibited separately or simultaneously by artificial microRNA technology and then to identify their biological functions.When they were inhibited separately,there were no significant impact on the growth and development of transgenic plants.However,when their expressions were suppressed simultaneously,about 50% of pollens were aborted and failed to germinate.Careful observation of aborted pollens showed that the abortion of pollens started at the late uninucleate stage,and the microspore cytoplasm and pollen intine were further abnormally degraded,which eventually led to the abortion and abnormal germination of pollens.(4)AtEXPB5 was located on the cell wall and its coding gene was also strongly expressed in mature pollen grains and pollen tubes by subcellular localization and spatio-temporal expression analysis.However,neither knockout of AtEXPB5 nor overexpression of AtEXPB5 had a significant impact on the growth and development of Arabidopsis thaliana.Combined with previous studies of related electronic expression profiles,it was found that only AtEXPA4 had a similar expression pattern to AtEXPB5 in reproductive development.Therefore,the knockout and overexpression materials of AtEXPA4 were constructed,and the double mutants of AtEXPA4 and AtEXPB5 were obtained by crossing.In the double mutant,the rate of pollen tube elongation slowed down significantly,and both AtEXPB5 and AtEXPA4 could restore the phenotype of reduced pollen tube elongation in the double mutant.This showed that AtEXPA4 and AtEXPB5 are redundantly involved in the development of pollen tubes.(5)AtEXPA4 was located on the cell wall and its coding gene was not only expressed in the reproductive development stage,but also predominantly expressed in roots of Arabidopsis thaliana by subcellular localization and spatiotemporal expression analysis.The measurement results of primary root length and root meristem showed that AtEXPA4 could promote the growth of primary roots.(6)Yeast one-hybrid and dual-luciferase experiments showed that the expression of AtEXPB5 could be co-activated by AtTDF1 and AtAMS,and this regulatory pathway was not conserved in Brassica rapa.This showed that the biological functions of AtEXPB5 and BrEXPB4/9 were differentiated,and they participated in different transcriptional regulatory pathways.BrEXPB4/9 and its homologous gene AtEXPB5 showed huge differences in expression patterns,functions and regulatory networks,indicating that in the course of evolution,homologous genes produced functional and regulatory changes.