Identification Of MADS-box Gene Family In Brassica Napus L. And Preliminary Analysis Of Regulation Network Of Pod Dehiscence

Brassica napus(AACC)is one of the major oil crops in our country,which is an allotetraploid species formed by the hybridization of B.rapa(AA)and B.oleracea(CC).As an ancient and big gene family,MADS-box genes play an important role in plant development,including regulation of flowering,flower and fruit development.In this study,the MADS-box genes of Brassica napus,B.rapa and B.oleracea were systematically analyzed,and the regulation mechanism of pod dehiscence in which MADS-box genes of Brassica napus involved was preliminarily investigated.The results are as follows:1.In this study,293,157 and 149 MADS-box genes were identified in Brassica napus,B.rapa and B.oleracea genome.MADS-box genes were classified into type Ⅰ(Mα,Mβ,Mγ)and type Ⅱ(MIKC~c and MIKC*)based on genes and proteins sequence characteristics and evolutionary analysis and MIKC~c was further divided into 13 sub-categories.Development of flower-organ ABCDE model and regulation of flowering time were the two most type of genes and it indicated the complexity of flower-organ development and flowering time regulation mechanism.By comparing the genome of Arabidopsis thaliana,B.rapa,B.oleracea and the corresponding An and Cn sub-genome of B.rapa,conclusions show that there may be mass loss or variation of genes during the whole genome triplication(WGT).2.The MADS-box orthologous gene pairs in the three rapeseeds and Arabidopsis thaliana were compared.Only 17 of the 107 Arabidopsis MADS-box genes has three orthologous genes in B.rapa or B.oleracea,indicating mass loss or variation of genes during the whole genome triplication(WGT).Meanwhile 101 of 133 B.rapa and B.oleracea orthologous gene pairs were found to be genetically retained in the An and Cn subgroups of Brassica napus.Type I phylogenetic tree has 122"sister pairs"consist of 49 An-Ar and 41Cn-Co orthologous pairs.Type Ⅱ phylogenetic tree has 130"sister pairs"consist of 64 An-Ar and 49 Cn-Co orthologous pairs.It indicates that the genetic relationship between Brassica napus and corresponding ancestor is closer than the homologous genes of Brassica napus.3.Through gene structure analysis,it found that the number and length of introns of the type Ⅱ gene of Brassica napus were larger than that of type Ⅰ,resulting in the overall length of type Ⅱ gene being larger than type Ⅰ.Through protein motif analysis,it found that in addition to the constituent motifs of the MADS-box and K-box domains,other motifs with unknown functions showed specific distribution in different subtypes of type Ⅰ and type Ⅱ and the gene s and proteins structure of the Brassica napus homologous genes in the same phylogenetic tree branch were very similar.4.The ful and ind mutants are constructed by CRISPR/Cas9.Interaction of Brassica napus homologous genes of Arabidopsis were analyzed to reveal possible regulation mechanism of pod dehiscence based on ful and ind mutants.Semi-quantitative results indicated that FUL negatively regulates the expression of SHP gene.Quantitative PCR results indicated that IND positively regulates the gene expression of NST1C04 and ADPG1A07downstream regulation network of pod dehiscence,which promotes the pod dehiscence.Meanwhile,the regulation of IND on the key enzyme of gibberellin synthesis,GA3ox1A06,was analyzed from the perspective of hormone-regulating pod dehiscence.It indicates that IND positively regulates the expression of GA3ox1A06 gene.The results above indicate the great similarity of the regulation mechanism of pod dehiscence between Brassica napus and Arabidopsis thaliana.