Function Analysis Of Self-incompatibility-related Genes MLPK And ARC1 In Brassica Napus

Brassica plants have evolved self-incompatibility systems to prevent self-fertility,promote outcrossing and maintain genetic diversity.Self-incompatibility genes that have been discovered contains SCR,SRK,THL-1,THL-2,Exo70A1,GLO1,ARC1 and MLPK in Brassicaceae.ARC1 was interfered in Brassica napus"W1"and it partially broke self-incompatibility.MLPK can break self-incompatibility completely after mutating in self-incompatible Brassica rapa,but not in self-incompatible Arabidopsis Athalina Col-0?SRKb-SCRb?and that in Brassica napus was unknown.In this study,CRISPR and RNAi techniques were used to verify function of ARC1 and MLPK in Brassica napus?S-70?.The main results are as follows:1.BnMLPK has 4 copies in Brassica napusFour copies of BnMLPK were cloned in?S-70?and named BnA3MLPK,BnC3MLPK,BnA4MLPK,and BnC4MLPK.Amino acid sequence of BnA3MLPK and BnC3MLPK are similar to amino acid sequence of BrMLPK and BoMLPK,respectively;Amino acid sequence of BnA4MLPK and BnC4MLPK are similar to amino acid sequence of BrMLPKn1 and BoMLPKn1 respectively.BnA3MLPK has two transcripts,named BnA3MLPKf1 and BnA3MLPKf2.The others only had one respectively.There is hydrophobic region in BnA3MLPKf2 N terminal and N-myristoylation motif in proteins of other transcripts.All of them have serine/threonine protein kinase in C-terminal.2.Gene editing of BnMLPK successfully broke self-incompatibility.A CRISPR vector was constructed for BnMLPK.We got 6 transgene lines with BnMLPK being edited from 31 positive plants via genetic transformation.50 plants of120 T₁ generation plants had elongated pods and much seeds.Gene editing of BnMLPK successfully broke self-incompatibility and it was an positive regulator for SI response.3.BnMLPK influences the expression of self-incompatibility-related genesSC factor GLO1 was down-regulated after self-pollination in?S-70?,However it was not changed after self-pollination in Bnmlpk mutant.In addition,expression of SRK in Bnmlpk mutant is lower than that in wildtype?S-70?before pollination.So BnMLPK influenced the expression of SC factor GLO1 and was related to expression of SI factors.4.RNAi of BnARC1 partially break self-incompatibility response in?S-70?Two copies of BnARC1 were cloned in?S-70?and named BnARC1-1 and BnARC1-2.A CRISPR vector was constructed and an interference vector was constructed as a control.After genetic transformation,we got 8 transgene lines with BnMLPK being edited from 42 positive plants via genetic transformation.Pods of 180 T₁ generation plants of them had no seeds or very a little seeds.3 T₀ generation positive plant were obtained by RNAi vector,Part of pods from 1/4 of their T₁ generation plants had seeds.So RNAi of BnARC1 partially broke self-incompatibility response.