Dissection Of Structure And Expression Regulation Mechanism Of Castor Bean RcFAH12

Castor bean(Ricinus communis.L)seed oil is the commercial source of ricinoleate,a valuable raw material used in many industries.However,the output of castor production is low,the harvest is not mechanized,the demand for more labor,the enthusiasm of farmers to plant is low.As a result,Castor oil production cannot meet market demand due to the reduced planting enthusiasm and planting area.It is of great value to the castor oil industry to improve the yield characters and resistance characters of castor by biotechnology,or to synthesize hydroxylated fatty acids by other plant systems.Oleoyl-12-hydroxylase(RcFAH12)is a key enzyme in the biosynthesis of ricinoleate.In the present study,we cloned and analyzed the RcFAH12 and its promoter region and tested deletions in the promoter sequence for their effects on the expression level of a GUS reporter gene in transgenic Arabidopsis plants.Screened transcriptional regulators and revealed the gene expression regulation mechanism.The main results are as follows:(1)The structure of RcFAH12The full-length c DNA of the RcFAH12(Submitted to Gene Bank:MK913424.1)was cloned by RACE with Tongbi No.5 50DAP seeds.By comparison with the c DNA,the genomic form of RcFAH12(Submitted to Gene Bank:MK913425.1)consists of two exons and one intron with a 2438 bp 5′untranslated region(UTR),183 bp 3′UTR.The exons are 127 bp and 1373bp and are separated by one intron of 2285 bp.The intron conforms to the GT-AG rule.The poly-A signal(AATAAA)in the RcFAH12 c DNA was present at 100 bp upstream of the poly-A tail.The RcFAH12 protein-coding sequence is located in the second exon.The open reading frame is 1164bp long and encodes 387 amino acid residues with an expected molecular weight of 44.43 k D and a theoretical isoelectric point of 8.95.(2)Sequence characteristics and function of RcFAH12 promoterAccording to the results of RACE,the transcriptional start site of RcFAH12 was located.The sequence of the region-2506～+99bp was cloned and used as the full-length promoter.The35s promoter in front of the GUS gene in plant expression vector p CAMBIA1303 was replaced by 5’deletion,3’deletion and both ends deletion fragments of RcFAH12 promoter,respectively.The expression of the GUS gene driven by the RcFAH12 promoter in different tissues of Arabidopsis thaliana was studied.Among them,3’deletion and two terminal deletion fragments FP494,FP584,FP502 and FP613 respectively contain a transcriptional starting site predicted by TSSPlant,which is the same as the 5’deleted FP256,which can drive GUS gene expression.Indicating that the RcFAH12 gene have multi-transcriptional starting site.According to the analysis of the Plant CARE database,the region of-2506～+99bp contains 101TATA frames and54 CAAT boxes,abscisic acid response elements(ABRE),essential anaerobic induction elements(ARE),methyl jasmonate response elements(CGTCA,TGACG-motif),low temperature response elements(LTR),Zein metabolic regulatory elements(O2-site),participating salicylic acid response elements(TCA-element),etc.,indicating that the gene may be regulated by many hormones.The expression of the GUS gene driven by different deletion fragments of the promoter in Arabidopsis thaliana showed that FP1611 could drive GUS expression in all tissues except in anthers,while FP2605,FP1611,FP256,FP502 and FP613drive GUS expression power in seed and weaker in other tissues,FP2605 driven GUS were highly expressed in seeds and leaves.The expression of GUS driven by FP256,FP584 and FP502 was higher in seeds but lower in other tissues,which showed seed-specific expression.FP2159,FP1178,FP677 and FP584 showed specific expression in anthers.The results showed that there were seed-specific negative regulatory elements between-578～-158bp and-2060～-1513,and seed-specific positive regulatory elements between-2506～-2061,-1512～-1081bp and-1079～-579bp.G-box and ABRE in FP256 may be involved in driving GUS gene expression in seeds.(3)The transcription factor that interacts with the RcFAH12 promoterConstructs castor 50DAP seed homogenous c DNA library-p GADT7-Rc50DAP that can be used for yeast one-hybrid and yeast two-hybrid.The library capacity is 1.25×10~7 CFU,the average insert fragment is about 1200bp,and the positive rate is 100%.Multiple bait vectors were constructed for library screening,including bait vector p HIS2-MDPL with-1180～+125bp as bait sequence,GCN4＿motifs that required for endosperm expression as bait sequence,and Skn-1＿motif that required for endosperm expression as bait sequence.With the library screening and Blast,it was found that one of the gene Rc WRKY48 which belongs to the family of WRKY transcription factors was interact with Skn-1＿motif.The Rc WRKY48 was cloned and with different deletion fragments of the RcFAH12 promoter connected upstream of the GUS gene was transiently co-expressed in tobacco leaves.The interaction between Rc WRKY48 and RcFAH12 promoter was detected.The interaction between Rc WRKY48 and RcFAH12 promoter was confirmed.Furthermore,the expression vector containing Rc WRKY48 was transformed into transgenic Arabidopsis thaliana with FP2605 to initiate GUS gene expression by Agrobacterium tumefaciens,and the T3 generation was obtained.GUS staining analysis showed that the expression of GUS was enhanced,indicating that Rc WRKY48 can promote the expression of the RcFAH12 gene.