Cloning And Expression Analysis Of A Novel Polygalacturonase Inhibitory Protein Gene BcMF19 In Brassica Campestris Ssp. Chinensis

The male sterile line is a valuable system for its hybrid seed production.Although there are many ways in the utilization of hererosis,the application of male sterility becomes an important way in heterosis breeding to reduce the production cost and simplify the seed production procedures.Brassica crop is one of the most universal crop in utilizing of heterosis with rich variety resources and wide distribution.It is very useful for producing F1 hybrid seeds to collecting and creating the male sterility line.The isolation and functional analysis of the genes related to pollen development were carried out,so as to further understand the molecular mechanism of pollen development in Brassica.In this study,a gene coding polygalacturonase inhibitory protein(PGIP) was successfully cloned from the fertile B line of the Chinese cabbage-pak-choi(Brassica campestris L.ssp.chinensis Makino) genie male sterile AB line(Bcajh97-O1A/B) based on an eDNA-amplified fragment length polymorphism(cDNA-AFLP) diverse fragment with rapid amplification of eDNA ends(RACE) and homologous gene amplification method.Analyze its sequence characteristics and predict protein structure and function.Real time polymerase chain reaction(RT-PCR) and in situ hybridization was performed to investigate the expression pattern of the gene during different stages of plant development and forecast the gene's function.Based on the results above,we the gene,and understand the systematic regulation and molecular mechanism of pollen development in Brassica.(1) We have obtained an expression different fragment from the fertile B line of the B.campestris ssp. chinensis genie male sterile AB line(Bcajh97-01A/B) based on cDNA-AFLP.Then we cloned an cDNA of polygalacturonase inhibitory protein gene differentially expressed in pollen using rapid amplification of the cDNA ends(RACE) and homologous expansion named Brassica Campestris Male Fertility 19(BcMF19) (GQ902048).Based on the cDNA of BcMF19,we designed primers to amplify DNA of BcMF19.The open reading frame and deduced of BcMF19 was analyzed by GENETYX software.The amino acid sequence of BcMF19 possesses the basic feature of PGIPs,containing an N-terminal signal peptide,several potential N-glycosylation sites,two disulfide bridges flanking both N-and C-terminal regions,and 10 leucine-rich repeat(LRR) consensus sequences.The tertiary structure of BcMF19 compose aβ-sheet layers (LxxLxLxxN/CxL) and anα-helix connected by loop ring formation.The adjacent LRRs are usually arranged around a common axis parallelly to form a curved horseshoe-shaped structure.(2) We isolated the homologue of BcMF19 from 17 cultivars from genera Brassica and Raphanus of family Cruciferae by homology cloning,then compared these sequences with BcMF19 by multiple alignment. The similarity of DNA from BcMF19 homologous genes is from 97.3%～100%,which indicated that homologues of BcMF19 from family Cruciferae were highly conserved with low variation in DNA sequence. The similarities between B.napus var.napobrassica cv.Datou 1 and B.campestris ssp.chinensis var. communis cv.Aijiaohuang,B.juncea var.multiceps cv.Xuelihong and B.nigra,B.campestris ssp.chinensis cv.Aiiiaohuang and B.campestris ssp.chinensis var.multicep cv.Maertou achieve 99.9%.The lowest similarity is 97.3%between B.campestris ssp.pekinensis cv.Huangya 14 and B.Carinata,however 100% similarity between B.nigra and R.sativus cv.SaKura Shimada Kon.The comparison between BcMF19 and 48 PGIPs from other species showed that there wre extensive differences in N-terminal except Cysteine residues.The LRRs of conservative sequences from BcMF19 and other PGIP from Cruciferae were consistent conserved,however there was no similarity in Non-conservative sequences.Evolutionary tree constructed by NJ showed that BcMF19 was not clustered together with PGIP from Cruciferae.So we presumed that BcMF19 is in another evolution branch apart from other PGIP genes.(3) Real-time quantitative polymerase chain reaction(RT-PCR) and in situ hybridization was performed to investigate the expression pattern of BcMF19.Gene transcripts expressed in fertile plants was higher than in sterile plants,especially during pollen meiosis stage,tetrad period and the stage of mature pollen grain. However,it should be pointed out that BcMF19 is not pollen or anther specific,due to its expression in other sporophytic tissues.It indicates that BcMF19 is also involved in other developmental events.(4) In situ hybridization results showed that the gene was located in the tapetum and microspore in Bcajh97-01B.Maybe the gene impacts the development of tapetum and affects the formation of pollen wall.It is also worthy to note that the expression of BcMF19 in the ovules of flower buds in later stages of development was found from the in situ hybridization analysis,but we presumed that BcMF19 may not be expressed in the ovule but rather,in the embryo.We presumed that BcMF19 appeared again in the embryo for its essential roles in development events after the zygotic stage,or the phenomenon could be due to the paternal genomic imprinting in the plant,and further investigation is required to conclusively explain this phenomenon.