Cloning And Preliminary Functional Analysis Of MAX1 Family And D53 Family From Strigolactone Pathway In Brassica Napus And Its Parent Species

Brassica oilseeds constitute one of the most important oil crops in most parts of the world,and more than 90% of the area of Brassica oilseeds grown in China and the world is Brassica napus(rapeseed or oilseed rape).B.napus is an allotetraploid species formed by natural hybridization and chromosome doubling of B.rapa and B.oleracea.In order to meet the growing demand for vegetable oils,it is necessary to maximize the yield potential of the rapeseed crop.The plant branching pattern is an important part of plant architecture.The number of branches or tillers of the crop affects the plant architecture,yield factors and adaptability,then ultimately affects the yield,even has some influence on quality.Plant branching traits are regulated by a complex network of hormones,signaling pathways and transcription factors.Among them,strigolactones(SLs)constitute the most important type of hormones that affect branching traits and it is even called as branching hormone.MAX1(MORE AXILLARY GROWTH1)is an early-stage enzyme in the biosynthetic pathway of SLs,and D53 in rice is a repressor of SLs signaling.The orthologous genes of D53 in Arabidopsis thaliana are SMXL6,SMXL7 and SMXL8.MAX1 and D53 play an important role in the biosynthesis and signal transduction of SLs,respectively.In this study,the nucleic acid sequences of MAX1 family and D53 family from B.napus,B.rapa and B.oleracea were cloned,and their gene and protein characteristics were analyzed bioinformatically.At the same time,the cis-acting elements of their promoters were predicted.We revealed their organ-spcificity and their differential expression between more-branched rape and normal-branched rape by q RT-PCR.The overexpression vectors of Bn MAX1 and Bn LBO1 were constructed,and were genetically transformed into Arabidopsis thaliana.The main results are as follows:1.Nucleic acid and protein characteristics of MAX1 family and D53 family in B.napus and its parent speciesThe c DNAs of the MAX1 family in B.napus,B.rapa and B.oleracea are1776～1780 bp in length,all of which contain 4 introns.Their CDSs are 1593～1596 bp in length and encode polypeptides of 530～531 amino acids,which are basic proteins and all have typical structural features of MAX1-type cytochrome P450 s.The CDSs of SMXL6,SMXL7 and SMXL8 families in B.napus,B.rapa and B.oleracea are 2634～2877 bp in length,all containing 2 introns.Their CDSs encode proteins of 877～958 amino acids.They share predicted features with the class I Clp ATPase proteins,and most of them are basic.Bn SMXL6-2,Bn SMXL6-3,Br SMXL6-2,Bn SMXL8-2 and Bo SMXL8-1 have alternative splicing,which leads to early termination of translation,encoding truncated proteins with severely incomplete structures and no functions in theoretical prediction.It probably is a post-transcription negative regulation mechanism to achieve precise regulation of branching trait.2.Gene origin and evolution features of MAX1 and D53 in plant kingdomVascular plants,such as dicotyledon,monocotyledon,gymnosperms and ferns(broad concept),all have MAX1,while non-vascular plants such as mosses and algaes do not,indicating that MAX1 originated with the birth of vascular plants.The CYP711A1 of the vascular plant evolved from the CYP743A1 of green algae and the directed evolution of enzyme activity occurred.The direct ancestor gene of CYP711 has been lost in Physcomitrella patens and Marchantia polymorpha.CYP711A1 has selective loss events in ferns and gymnosperms.There is only one CYP711A1 gene in the common ancestor of coniferous gymnosperms(Picea glauca,Pinus taeda,etc.),basic angiosperm Amborella trichopoda and dicotyledonous plants.Many dicotyledon species(such as Cinnamomum micranthum,Medicago truncatula,Camellia sinensis and Citrus sinensis)have two CYP711A1 genes.The reason is the occurrence of a recent doubling event within the order or the family.CYP711A1 has been doubled in early common ancestor of monocotyledons,resulting in two or more CYP711A1 genes in every current monocotyledonous plant species,e.g.five paralogues in rice.There is one D53 gene in the basic angiosperm A.trichopoda(annotated as SMXL7),but there is no D53 in gymnosperms,ferns,bryophytes and green algaes,indicating that D53 originated along with angiosperms which are flowering plants.Doubling event of D53 gene did not occur in the common ancestor of monocotyledonous plants,but many doubling events of D53 within family or genera occurred in many monocotyledonous plants,resulting in two or more D53 genes within the same species,such as Musa acuminata,Elaeis guineensis and Oryza sativa.In the early common ancestor of dicotyledons after separation with camphor trees,D53 gene doubling event has occurred.In addition,there were recent doubling events of SMXL6 or SMXL8 at order-level or family-level in Brassicaceae,Medicago truncatula,Camellia sinensis and Gossypium arboreum.3.Organ specificity of MAX1 family and D53 family and their differential expression among branching-trait cultivarsq RT-PCR results indicate that Bn MAX1 is highly expressed in roots and flowers,and Bn MAX1-1 is also highly expressed in seeds.Br MAX1 is highly expressed in flowers,seeds and pericarps,and Bo MAX1 is highly expressed in leaves,pericarps,flowers and seeds.SMXL7 is highly expressed in roots and stems of B.napus,B.rapa and B.oleracea,and SMXL7 is also highly expressed in leaves of B.oleracea.In B.napus and B.rapa,SMXL8 is mainly expressed in roots and stems,while in B.oleracea it is mainly expressed in roots,stems and leaves.SMXL6 is expressed in various organs of the three species,and it is widely involved in plant growth and development.The expression patterns of Bn MAX1-1 and Bn MAX1-2 between two more-branching rapeseed lines 393 and 139 and normal-branching rapeseed cultivar ZS11 are similar.In the lower stem and the lateral branch,the expression levels of Bn MAX1 in more-branching lines are lower than those of normal-branching line ZS11,indicating that Bn MAX1 plays a certain role in branching diversity of B.napus.SMXL6,SMXL7 and SMXL8,which are homologous genes of D53,have different expressions between more-branching and normal-branching lines.Compared with the expressions of the members in Bn SMXL6 and Bn SMXL8,the expression characteristics of the members in Bn SMXL7 are more consistent.In the three organ parts,the expression levels of Bn SMXL7 in more-branching lines are lower than in ZS11,indicating that Bn SMXL7 may participate in regulating the branching trait diversity of B.napus.4.The progress of the transformation of upstream key-enzyme gene Bn MAX1 and downstream key-enzyme gene Bn LBO1 into A.thalianaThe cloned coding regions of Bn MAX1-1 and Bn LBO1-3 were ligated to the vector p FGC5941 M respectively,and transformed into Agrobacterium tumefasciens GV3101 to obtain overexpression vector engineering strains carrying the genes of interest.The constructed overexpression vectors were genetically transformed into A.thaliana,and then their progeny plants were screened to obtain transgenic plants.By preliminary observation,compared with the wild type,the branching number of Bn MAX1 overexpressing plants was reduced,indicating that MAX1 from B.napus is functionally involved in regulating branching trait.