Cl Oning And Functional Studies Of Oryza Selfish Genes ORF2 And ORF3 Homolog In Wheat

In recent years,with the accumulation of wheat genome,transcriptome,and proteome data,the research on gamete-killing chromosomes has gradually become clear.Whether the wheat gene homologous to the rice selfish gene is involved in the phenomenon of gamete-killing chromosomes in wheat is more worthy of our in-depth study..Since the mechanism of action of the wheat gametomic chromosome and the rice selfish gene system seem to be transmitted by a"killer-protection"mechanism.Therefore,this study combined the rice selfish gene and the wheat gametomic chromosome phenomenon to explore the possible molecular mechanism of the gametolytic phenomenon,and to analyze the relationship between the two.TaORF2 and TaORF3 genes homologous to rice ORF2 and ORF3 were cloned from wheat.The two cloned genes were transferred into rice plants by Agrobacterium-mediated method,and positive transgenic rice plants were obtained by PCR identification,and the characteristics and functions of TaORF2 and TaORF3 genes were further analyzed.The main results are as follows:1.Cloning and sequence Analysis of TaORF2、TaORF3 geneIn this study,wheat TaORF2 and TaORF3 genes were cloned.TaORF2 gene contains 1554 nucleotides,encoding 517 amino acids;TaORF3 gene contains 1443nucleotides,encoding 480 amino acids.The TaORF2 protein sequence contains an AAA domain between amino acids 68-144 and a RIP domain between amino acids 296-430,with a molecular weight of 58.44614 k Da,a theoretical isoelectric point of 5.62,and an average hydrophilic coefficient.-0.426,is a non-secreted protein,TaORF3 protein,no conserved domain,molecular weight 53.14949k Da,theoretical isoelectric point 5.54,average hydrophilic coefficient-0.276,belongs to non-secreted protein.Evolutionary relationship analysis showed that TaORF2（KAF7064671.1）was highly homologous to emmer（XP＿037440434.1）and was relatively far from other species,and TaORF3（KAF7064673.1）protein is closely related to Os ORF3（AWW05364.1）on emmer and another branch.2.Construction of pCAMBIA1300-TaORF2,pCAMBIA1300-TaORF3,pCAM BIA1300-TaORF23 expression vectors and genetic transformation of riceThe cloned TaORF2 and TaORF3 genes were connected with the expression vector pCAMBIA1300 to construct pCAMBIA1300-TaORF2,pCAMBIA1300-TaORF3,pCAMBIA-TaORF23 recombinant vectors,and the TaORF2 and TaORF3 g enes were transferred into rice,screened by hygromycin resistance,and then subj ected to PCR 10 transgenic positive plants for TaORF2,TaORF3 and TaORF23were identified and obtained.3.Analysis of pollen fertility of TaORF23 transgenic plantsThe pollen fertility analysis of TaORF23 transgenic plants and wild-type plants by I₂-KI staining showed that wild-type pollen was more than 95%fertile,while transgenic plants were semi-sterile.It indicated that the wheat TaORF2 and TaORF3 genes,which are homologous to the rice selfish gene,may indeed play a"killer-protection"role in the wheat gametocisis.4.Physiological index analysis of TaORF2,TaORF3,TaORF23 transgenic plantsAfter measuring the physiological indicators of transgenic rice plants and wild-type rice plants,it was found that the proline content of the three transgenic rice plants was significantly higher than that of the wild-type rice Nipponbare variety at the same period,but the soluble sugar,soluble protein,MDA,H₂O₂,O₂^-was not significantly different from wild-type rice.