Expression analysis of the bidirectional promoter of the stilbene synthase gene from the Chinese wild Vitis quinquangularis

Abstract: 【Objective】To learn of the activities and differences of bidirectional stilbene synthase promot-ers from the Chinese wild V. quinquangularis‘Danfeng-2'. As the position of the 48 genes of the stilbenesynthase family from V. vinifera has been reported,we analyzed and found that the stilbene synthasegenes Vv STS31 and Vv STS32 space 1 737 bp,and started expressing in the opposite direction. We notedthat the sequence between these two genes is a bidirectional promoter. As China is one of the original ar-eas for the grape,it is filled with extensive resistance germplasm resources. Among them,V. quinquangularis‘Danfeng-2'not only has good resistance,but also a high content of resveratrol. Since the genomicDNA sequences of Chinese wild V. quinquangularis have a high degree of similarity with V. vinifera,pro-moters,cloning of bidirectional stilbene synthase from Chinese wild V. quinquangularis‘Danfeng-2'canprovide a novel resistance gene resource for the grapes.【Methods】Bidirectional stilbene synthase promot-ers from wild V. quinquangularis‘Danfeng-2'were cloned by genome walking. The sequences of bidirec-tional stilbene synthase promoters from‘Danfeng-2'were submitted to the promoter prediction website Plant CARE(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/),and the corresponding cis-act-ing elements were obtained and marked on the sequences. In order to analyze the promoter activity of bidi-rectional stilbene synthase promoters,they were fused with a Uid A gene(β-glucuronidase,GUS). Recom-binant plasmid Pvq DSTS31::GUS and Pvq DSTS32::GUS were transient transformed into grape leaves us-ing Agrobacterium-mediated vacuum infiltration. The expression patterns of GUS in grape leaves underdifferent stress treatments,including powdery mildew,high temperature,low temperature,Me JA,ABAand SA,were analyzed using histochemical staining and GUS quantification. Treatment of the biotic andabiotic stresses were conducted as follows:(1) Powdery mildew treatment: grape powdery mildew were in-oculated from infected leaves to test leaves which were infiltrated after 48 h using the friction extrusionmethod,and then placed in an illumination incubator for 24 h.(2) Salicylic acid(SA) treatment: grapeleaves in vitro infiltrated after 48 hours were sprayed with 1 mmol·L-1SA solution,and afterwards wereincubated in the same conditions as the powdery mildew treatment.(3) Methyl jasmonic(Me JA) acid treat-ment: 100 μmol·L-1Me JA solution prepared by 10 mmol·L-1MES solution using the culture method wasthe same as above.(4) Abscisic acid(ABA) treatment: the treatment concentration of ABA is 100 μmol·L-1.(5) Low temperature(or high temperature): the grape leaves in vitro infiltrated in Agrobacterium liquid af-ter 48 h at room temperature were transferred to a 4 ℃(or 40 ℃) incubator for 24 h. In order to under-stand the gene expression promoted by bidirectional stilbene synthase promoters,the fruit c DNA of differ-ent mature periods from V. quinquangularis‘Danfeng- 2'and V. vinifera‘Cabernet Sauvignon'wereused as templates. The expression of Vq STS12/Vv STS31 and Vq STS33/Vv STS32 were analyzed by usingthe quantitative real-time PCR method.【Results】In order to understand the homologous sequences of V.vinifera bidirectional stilbene synthase promoters in the transcription initiation of Vv STS31 and Vv STS32 in V. quinquangularis‘Danfeng-2',the sequences of Vv STS31 and Vv STS32 were blasted in NCBI. Theresults showed that the homologous sequence of Vv STS31 was Vq STS12(Genbank accession No.JQ868686) with a similarity rate of 98.47%; the homologous sequence of Vv STS32 was Vq STS33(Genbankaccession No. JQ JQ868665) with a similarity rate of 92.71%. Predictive analysis of the bidirectional stil-bene synthase promoters from‘Danfeng-2'was made on the promoter prediction website Plant CARE.From the cis-acting elements,promoters contained many regulatory elements associated with stress-in-ducible,wherein,Box-W1 is a fungal elicitor responsive element in Petroselinum crispum; TGACG-mo-tif is a cis-acting regulatory element involved in the Me JA-responsiveness of Hordeum vulgare; ABRE isa cis-acting element involved in the ABA responsiveness of Arabidopsis thaliana and Oryza sativa; MBS isa MYB binding site involved in drought-inducibility in A. thaliana,acting on the binding of MYB tran-scription factors and DNA; GCN4_motif and Skn-1_motif are required for endosperm expression; SORLIPresponds to light; and SITEIIATCYTC is involved in cell DNA polymerization. Predictive analysis resultsshowed that“Danfeng-2”stilbene synthase gene bidirectional promoter may have an important role in thedefensive reaction to various stresses. By comparing the bidirectional stilbene synthase promoter of three V. quinquangularis lines‘Danfeng-2'‘83-4-96'and‘Shang-24'as well as three V. vinifera varieties‘Cabernet Sauvignon'‘Chardonnay'and‘Riesling',we found that the bidirectional promoter sequenceof V. quinquangularis is 491 bp shorter than V. vinifera. By removing the missing 491 bp,the other se-quence similarity rate is 98.81%. At the six development periods of grape fruits,Vq STS12 and Vq STS33 started by bidirectional stilbene synthase promoter of‘Danfeng-2'had a higher expression level than Vv STS31 and Vv STS32 which were started by the bidirectional stilbene synthase promoter of‘CabernetSauvignon'. The basic expression trends of the four genes are: Before the turning-color period of the ber-ries,the expression of the four genes increased sustainedly until late veraison,after a dropping period,the gene expression reached a peak at the half-ripe stage with berries with inter mediate Brix values,then continued to decrease during the berry ripening period. The only exceptions were that the Vq STS12 of the‘Danfeng-2'didn't reduce at late veraison and the Vq STS33 of the‘Cabernet Sauvignon'didn'treduce during the berry ripening period. To study the promoter activity,the GUS gene was fused to the 3'end sequences of the bidirectional promoter. Histochemical staining and fluorescent quantitation analysisof the GUS gene were conducted. The results showed that Pvq DSTS12 was positively regulated by the pow-dery mildew incubation,Me JA,and heat and cold stress,but had no significant changes in response toSA and ABA. We found that Pvq DSTS33 was positively regulated by Me JA and negatively regulated byheat and cold stress,and was insensitive to powdery mildew incubation,SA and ABA.【Conclusion】Re-al-time PCR results showed that,Vq STS12 and Vq STS33 started by the bidirectional stilbene synthasepromoter of‘Danfeng-2'had higher expression levels than Vv STS31 and Vv STS32 started by the bidirec-tional stilbene synthase promoter of‘Cabernet Sauvignon'. We also noted that the Vq STS12/Vv STS31 and Vq STS33/Vv STS32 basic expression trends at six development periods increased sustainedly before theberries turned color until late veraison,After falling,the gene expression reached a peak,then contin-ued to decrease during the berry ripening period. However,the Vq STS12 of‘Danfeng-2'didn't reduceat late veraison. Pvq DSTS12 was positively regulated by powdery mildew incubation,Me JA,and heatand cold stress. We found that Pvq DSTS33 was positively regulated by Me JA and negatively regulated byheat and cold stress but not affected by powdery mildew incubation. Both promoters had no significantchanges in response to SA and ABA. The research on bidirectional stilbene synthase promoter activitysets the foundation for the use of a plant natural bidirectional promoter.