Evolutionary Analysis Of The PGIPs Gene Family And Preliminary Study On The Function Of BrPGIP4 And BrPGIP12 In Brassica Rapa

Plant PGIPs(polygalacturonase-inhibiting proteins)is a specific binding protein located in the plant cell wall,which belongs to the leucine rich repeat(LRR)family,all of the plant PGIP proteins contain up to 10 LRR motifs,as well as occur to the variation in number of LRRs frequently.Each LRR consists of 24 amino acid residues[LxxLxxLxLxxNxLt/sgxIPxxLGx(L= l/L/V/F;N = N/T/S/C;C = C/S;x =any amino acid)],and these conserved LRRs structure constitute functional region of proteins interactions with other proteins,thus they can specifically inhibit endo polygalacturonase(endo-PG)secretion of pathogens,then prevent pathogens of host plants and improve plant resistance to pathogens.Based on the previous studies,also the completion of the genome sequencing,making it possible to identificate the PGIPs genes of Brassica crops,and explore the role of PGIPs gene in disease resistance,including of clubroot.In this study,we identified the PGIPs gene family members of major Brassica crops,and analyzed the gene structure,chromosome location,physiological and biochemical functions,conserved LRR domain,evalutionary and so on.Meanwhile,the expression patterns in different tissues of B.rapa has been carried out.At last,we constructed the over-expression vector and amiRNA expression vector of BrPGIP12 and BrPGIP4,and some transformants have been obtained so far.The main results show as follows:(1)The known protein amino acid sequence of Arabidopsis PGIPs gene has been imported in B.rapa gene database and NCBI database and to search B.rapa PGIP genes,and to make the last identification with conservative structure analysis of LRRs.Finally,we successfully identified 12B.rapa PGIP genes and named these genes.These 12 genes locate in A01 A10,A03,A05 chromosome of B.rapa,some genes cluster of adjacent arrays,their open reading frame length are between 618bp and 1044bp,coded 205aa～347 aa amino acid polypeptide,molecular weight between 22.8 kD and 38.6 kD.The theoretical value of pI are between 5.82～9.46.In addition,there are 1～3 glycosylation sites in these B.rapa PGIP genes.Except of the three genes,the rest of the subcellular location have been predicted in the secretion pathway.All of the 12 B.rapa PGIP proteins contain LRRs domain structure,and the number of LRR domain vary from four to nine,the average is 7.1.The analysis results indicate that the gene structure,protein domain,evolutionary character are particularly conservative among these PGIP genes.In accordance with the same methods,the identification and bioinformatics analysis of PGIPs genes from B.oleracea,B.napus,B.juncea and B.nigra have been carried out,respectively.(2)The PGIPs gene in root,stem,leaf,flower and silique of B.rapa in expression using real-time fluorescence quantitative PCR technology have been analyzed.Results show that BrPGIP2,BrPGIP10 in roots,stems are very low expression levels,BrPGIP12,BrPGIP11,BrPGIP9and BrPGIP1 are highly expressed in root and stem.Meanwhile,the expression of BrPGIP2,BrPGIP10,BrPGIP8,BrPGIP5 in the leaves maintain very low level,BrPGIP1 expression is particularly high in the leaves.In addition,the expression of BrPGIP8 and BrPGIP5 in flowers and siliques in quantity is very low,while expression of BrPGIP12 and BrPGIP7 was relatively high in siliques.(3)The overexpression vector and amiRNA expression vector of BrPG1P12 and BrPGIP4 have been constructed,and the genetic transformation of flowering Chinese cabbage ’Youqing 49 ’for these 4 vectors and empty vector pBI 121 have been carried out subsequently.So far,we obtained 2 transformants of amiRNA923 8,4 transformants of amiRNA5918-8 transformants of OE9238-3 transformants of OE5918 and 3 transformants of empty vector of pBI 121.Moreover,the expression characteristic of BrPGIPl 2 and BrPGIP4 by real-time fluorescent quantitative PCR technique in the transformants of 5 vectors have been carried out.Results indicated that the expression level of BrPGIP12 and BrPGIP4 in transformants increased,most notably the OE9-5 and OE9-6 lines.The expression level of BrPGIP12 was 5 times higher than that in CK,the expression of BrPGIP4 in the transformants increased slightly.The expression level of BrPGIP4 has decreased in the amiRNA transformants,most obviously,the expression level of BrPGIP4 decreased about half in is am5-2 transformant.The resistance of clubroot for transformants is being done now.These results in this study will provide research foundation and theoretical basis for thorough research of biology function of members of of Brassica plants PGIP genes family in the near future.