The Functional Analysis Of Brassica Rapa BrAtg6 Gene In Resisting The Infection Of Plasmodiophorales Brassica

Chinese cabbage clubroot occurs in many regions of China,which has caused a sharp decline in Chinese cabbage production in some areas.In our previous research,a protein G17,which was significantly up-regulated expressed in the root of inoculated ‘742’ Chinese cabbage,was screened by two-dimensional electrophoresis(2-DE)-mass spectra(MS)technique.In this study,the full-length cDNA sequence of the protein was obtained by homologous cloning,and the expression pattern of the corresponding gene was analyzed by quantificational real-time polymerase chain reaction(qRT-PCR)and in situ hybridization,and the resistance of Arabidopsis mutants was identified.The following results were obtained:1.The protein was identified as Bra003466 by mass spectrometry.NCBI blastn analysis of Bra003466 cDNA sequence showed that there was a about 600 bp redundant sequence at its5’ end.The primer was designed according to the cDNA sequence of the highest homology gene XM-013793011.2 of Brassica napus,and the sequence was obtained by PCR amplification in 742 strain of Chinese cabbage.2.The analysis of open reading frame and protein structure of target gene showed that the expressed product was composed of 521 amino acid residues with molecular weight of58.90 KDa and isoelectric point of 6.21.It is predicted that the protein does not have signal peptide sequence and transmembrane domain.So that the protein may be located in the cytoplasmic matrix or organelle matrix,and the protein has only one APG6 superfamily conserved domain,which belongs to the autophagy protein family.Therefore,the gene was named as BrAtg6.3.qRT-PCR analysis of BrAtg6 in root,stem and leaf of Chinese cabbage inoculated by Plasmodiophorales bacteria(uninoculated plants used as control)showed that BrAtg6 was only expressed in inoculated root,but the expression is difficultly found in other organ or control roots.Compared to control root,BrAtg6 has a higher expression in the infected and diseased roots,and its expression level is higher in the diseased roots than in infected roots.In conclusion,BrAtg6 may be involved in the interaction between Chinese cabbage and Plasmodiophorales bacteria.4.The results of In situ hybridization showed no hybridization signal was found in uninoculated roots,uninoculated/inoculated stems and uninoculated/inoculated leaves by Plasmodiophorales bacteria,but it is only showed on inoculated roots.Furthermore,the hybridization signal was stronger in the diseased root than in the infected roots.The results indicated that the expression of BrAtg6 was related to the infection degree ofPlasmodiophorales bacteria,and BrAtg6 might play a role in Chinese cabbage resistance to the infection of Plasmodiophorales bacteria.5.The Arabidopsis mutant atg6 was obtained on the TAIR site.The mutant atg6 was screened by "three primer" method.The disease resistance was identified in mutant atg6 and wild type(WT)at 12 h,24h,36 h,48h,60 H and 72 h after inoculation by Plasmodiophorales bacteria.The results showed that infection speed is different between the mutant and the wild type.The root hairs of mutant atg6 can be infected from 36 h after inoculation,while the wild type WT can be infected at 72 h after inoculation.It is concluded that BrAtg6 gene may be associated with resistance to clubroot.