Cloning And Evolutionary Analysis Of Homologous Sequences Of Clavata1 Gene In Brassica

In Brassicaceae,the Brassica plants have great economic value,abundant genetic variations and many cultivars,including a variety of important agricultural and horticultural crops.The CLV1 mutations may cause increased flower organs,enlarged siliques in Arabidopsis thaliana,with a concomitant increase in the number of seeds.Such structural changes in flower organs and siliques may represent the important yield traits in breeding.Thus,in this study we have cloned the homologous of CLV1 gene from different experimental materials of the Brassica diploid and tetraploid species and Arabidopsis thaliana by homologous cloning method.We obtained the sequences of all cloned homologous of CLV1 gene from different materials by sequencing.Then the molecular evolutionary analysis of the homologous ORF sequences and protein sequences of CLV1 was performed by bioinformatics methods.Based on their structural differences,we have analyzed their functional differences,and the main conclusions are as follows:(1)By the method of homologous cloning,the Blast in genome database and sequence similarity analysis,we found only one homologous site of CLV1 gene exists in B.rapa or B.oleracea or B.nigra,and two homologous sites exist in B napus or B juncea or B.carinata,which are located in two different subgenome.The homologous site of CLV1 gene in A and C genome genomes is located in chromosome A7 and C6,respectively.The homologous site of CLV1 gene in B genome may be located in chromosome B4 or B7,but more likely located in chromosome B7.(2)The microscopic collinearity analysis of the genomic region of the CLV1 loci showed that the order within AT1G75580 to AT1G75980 segments totaling 37 annotated genes is consistent homologous gene of which in the A,C genome segments.Most of the genes in the corresponding genomic regions have 1-2 a copy,four genes lost in the A and C genomes,only three genes exist three copies,indicating that gene loss phenomenon in Brassica genome triplication process.From the perspective of gene loss frequency,the LF subgenomic region collinearity in better,the loss of MF1 and MF2 genes is serious.(3)Based on the evolutionary analysis of CLV1 homologous ORF sequences revealed that the evolutionary tree can commendably separate CLV1 homologous copy from Arabidopsis,A,C and B genome.Each clade contain CLV1 orthologous gene and the CLV1 homologous copies of three allotetraploid complex genome are derived from two subgenome.Wherein,the homologous copies in A and C genome are clustered into a large branch,while the homologous copy in B genome as a separate taxon is assigned to another branch,indicating that the genetic relationship of the CLV1 homologous genes in A and C genome is closer than that of B genome.From the perspective of the entire evolutionary,the evolutionary pattern of the gene is in line with the evolution of the entire species,indicating that the gene in the whole process of the evolution of species is very conservative,and the evolutionary tree based on the CLV1 homologous ORF sequences can commendably and objectively reflect the phylogenetic relationship of Brassica species,suggesting that this gene plays a significant role in the growth and development.(4)The phylogenetic tree based on the homologous sequences alignment of the CLV1 protein showed that the CLV1 homologous protein of the Cruciferae plants which sequence similarity reach over 84%are owned by a large branch.The nearby branch which sequence similarity more than 60%contains TcaCLV1,GarCLV1A,GarCLV1B,GmaCLV1A,GmaCLV1B,also includes OsaFON1 and ZmaTD1,which sequence similarity is 57.41%and 55.73%,respectively.All of the above proteins constitute a big group named as Group I in the phylogenetic tree.The Arabidopsis AtBAM1 AtBAM2 and AtBAM3,soybean GmaRLK1,GmaRLK2 and GmaRLK3,as well as rice OsaRLK and OsaRLK1 constitute another big group named as Group ? in the entire evolutionary tree.According to this evolutionary relationship,all proteins of Group I may be CLV1 orthologous proteins,while those of Group ? may be CLV1 paralogous proteins.(5)Analyzing the specific structural domains and functional domain of the CLV1 homologous proteins separately,the length relationship of three domain of the CLV1 protein displayed the extracellular domain is the longest region,the intracellular(kinase domain)is longer,and the transmembrane domain is the shortest.Nevertheless,the sequence conservation relationship showed that the most conserved region is the intracellular domain(kinase domain),the extracellular domain comes second,and the transmembrane domain the least.The analysis of the extracellular domain showed that the sequences of LRR regions remained the same in Cruciferae plants,and highly conserved in other species.Among of the 21 LRRs,the LRR3,LRR4,LRR6,LRR8,LRR9,LRR 10,LRR14,LRR 19,LRR21 are more conserved.The amino-terminal and carboxy-terminal of LRR regions respectively have a highly conserved cysteine pair,indicating that the cysteine pairs play an important role in maintaining the structure and function of proteins.