Regulation Analysis And Utilization Of The Promoter Of Small Peptide Gene BnaC.SP6 In Brassica Napus

Cytoplasmic male sterility(CMS)and genic male sterility(GMS)are two common ways for heterosis utilization in Brassica napus.With the superiority of GMS,expecially the recessive GMS,new methods for heterosis utilization have been established with gene translation in maize,rice and other crops in recent years.The key for those new methods is finding pollen-specific promoters.To achieve this target,a pollen predominant expressing gene was identified from Brassica napus in this research.The functional regions in its promoter and a trans-factor for it were identified using different assays.Its pollen-specific promoter region was used to establish the transgenic maintainer line of recessive GMS line S45 A in Brassica napus.The major results are as follows: 1.Identification of small peptide gene Bna.SP6 from Brassica napusTwo ATSP6 homologs,Bn SP6_C08(611 bp)and Bn SP6_A08(475 bp),were cloned from B.napus ?ZS11‘.Their coding sequences(246 bp)were also cloned from the opening anthers of ?ZS11‘ and designated as BnaC.SP6 and Bna A.SP6.They encoded small peptides(81-a.a.)containing putative signal cleavage sites between residues 25 and 26,and had not any discernable motif.RT-PCR and GUS staining indicated that BnaC.SP6 was expressed in mature anthers and silique septum.2.Promoter deletion analysis of pBnaC.SP6 in Arabidopsis thalianaMotif search using Plant CARE revealed that the pollen-specific activation-related element POLLEN1LELAT52,LAT enhancer element,and GTGA MOTIF are located in pBnaC.SP6,which might help drive the expression of BnaC.SP6,predominantly in pollen of mature anthers.To identify functional regions in pBnaC.SP6(p1167),5?-deletions(p643,p447,p375,p306,p210,and p135)and 3?-deletions(p254 and p158)cassettes were introduced into Col.Histochemical GUS assay suggested that the-447 to-375 and-210 to-135 regions were crucial for the silique septum and pollen expression of BnaC.SP6,respectively.Furthermore,semi-thin anther sections and DAPI staining showed that the minimal promoter region of p158(-210 to-52)was sufficient for driving BnaC.SP6 expression specifically in pollen from early bicellular pollen stage to mature pollen.Comparative sequencing indicated that pollen-specific activity of p158 highly conserved in Brassica species.3.Expression pattern of Bna A.b ZIP1 and its function as a transcription factorqRT-PCR and GUS staining suggested that Bna A.b ZIP1 was dynamically expressed in floral organs and coexpressed with BnaC.SP6 in anthers.Therefore,the coding sequence(420 bp)of Bna A.b ZIP1 were cloned from opening anthers.It encoded a 139-a.a.protein with a basic leucine zipper(b ZIP)DNA-binding and dimerization domains localized between residues 17 to 68.Bna A.b ZIP1 was fused to the green fluorescent protein(GFP)to determine its subcellular localization.The GFP signal coincided with the cyan fluorescent protein signal of a previously characterized nuclear marker CCT domain protein(Os GHD7),indicating that Bna A.b ZIP1 is a nuclear-localized protein.X-Gal staining and the dual-luciferase reporter assay(DLR)results suggested that Bna A.b ZIP1 exhibited transcriptional activity and the C terminus of Bna A.b ZIP1 was necessary for its transcription activation activity.4.Transcriptional regulation of BnaC.SP6Sequence analysis revealed that p158 region contained a C-box(CACGTC)and an ABRE motif(TACGTG).Yeast one-hybrid assay demonstrated that Bna A.b ZIP1 specifically binds to the C-box but not to the ABRE motif in p158 region in yeast.In addition,the electrophoretic mobility shift and DLR assay results strongly supported that Bna A.b ZIP1 interacted directly with p158 by binding to the CACGTC(C-box)element.In vivo,Bna A.b ZIP1 could suppress the pollen activity of p158 in dexamethasoneinduced Bna A.b ZIP1/p158 plants.Thus,Bna A.b ZIP1 functions as a transcriptional repressor of BnaC.SP6.These results provide first insight into the transcriptional regulation of BnaC.SP6 in pollen activity.5.Utilization of p158The pollen-specific promoter region p158 of BnaC.SP6 and Bax gene were combined with Bn MS1 and a herbicide-resistant gene to generate a vector for establishing the transgenic maintainer line of S45 A,which was transformed into S45 AB.One potential maintainer line of S45 A was identified by test-crossing,which need to be tested again in the next generation.