| Jasmonic acid signaling is consisted of SCFCOI1 ubiquitin protein complex, JAZ repressor protein family and the MYC family of transcription factors. The F-box protein COI1, the scaffold protein SKP1, Cdc53/cullin and Rbxl were the main compontents of SCFCOI1 complex which assembling were regulated by Cand1, RUB, and CSN. We have proved that JA can induce laticifer differentiation, regulate natural rubber biosynthesis, and further cloned HbCOI1, HbJAZ1, and HbLMYCl genes. In order to characterize the components of SCFCOI1 ubiquitin-protein complex in laticifer cells, HbCull, HbSKP1 and HbRBXl genes were cloned and their expression patterns were analysesd by real-time PCR in the study. The prokaryotic expression vectors of HbCull, HbSKP1, HbRBX1, HbCOI1 and HbJAZ1 were constructed, and the fused proteins were expressed. In addition, yeast one-hybrid bait vector of PHbCOI1 full promoter and three core promoter repeat yeast one-hybrid bait vector were constructed for further screening transcription factors. The results are as follows:1, Full length cDNAs of HbCull, HbSKPl and HbRBXl were cloned by RT-PCR and RACE. The HbCull was 2762 bp in length, containing a 2235 bp ORF encoding 744 amino acid residues, a 374 bp 3’untranslation region and a 153 bp 5’untranslation region. The molecular mass of the putative protein is 87 kDa with its theoretical pi of 6.56. The deduced amino acid sequence had specific domain of CULLIN superfamily and Cullin Nedd8, sharing 97%、97%、95%、82%、87% and 82% identity with Ricinus communis, Populus trichocarpa, rape (Vitis vinifera), Arabidopsis, Picea sitchensis and tobacco, respectively. The HbSKPl was 778 bp in length, containing a 543 bp ORF which encodes 180 amino acid residues, a 188 bp 3’untranslation region and a 47 bp 5’untranslation region. The molecular mass of the putative protein is 20.3 kDa with its theoretical pi of 4.38. The deduced amino acid sequence had specific domains of Skpl-POZ and Skpl, sharing 61%、64%、50%、48%、48% and 47%identity with Populus trichocarpa, Ricinus communis, Soybean, Arabidopsis, and rape(Vitis vinifera) Capsicum annuum, respectively; The RBX1 cDNA was 638 bp in length, named HbRBX1, containing a 351 bp ORF which encodes 116 amino acid residues, a 255 bp 3’untranslation region and a 32 bp 5’untranslation region. The molecular mass of the putative protein is 13 kDa with its theoretical pi of 6.2. The deduced amino acid sequence had the specific domains of RING superfamily, sharing 94%、93%、92%、92%、90%'90% identity with Soybean, Arachis hypogasa, Arabidopsis, Sitka spruce, Populus trichocarpa, and rape (Vitis vinifera), respectively.2, Real-time quantitative RT-PCR analysis showed that HbCull expression was up-regulated by tapping, JA, ethephon up while mechanical wounding had no obvious effect on HbCull expression. HbSKP1 expression was up-regulated by all of the factors, tapping, mechanical wounding, and ethephon, but not jasmonic acid, whereas the HbRBX1 expression was up-regulated by tapping, mechanical wounding, jasmonic acid and ethylene.3, The prokaryotic expression vector of HbCull, HbSKP1, HbRBX1, HbCOI1 and HbJAZ1 were constructed and expressed in E. coli BL21 (DE3) in the forms of HbCul1-His(6), HbSKP1-His(6), HbCOIl-His(6), and HbJAZ1-His(6).4, HbWDRl gene was screened from the yeast one-hybrid system by using HbCOI1 promotors. It was 4036 bp in length, containing a 3387 bp ORF which encodes 1128 amino acid residues, a 481 bp 3’untranslation region and a 168 bp 5’untranslation region. The molecular mass of the putative protein is 123.85 kDa with its theoretical pI of 6.77. The deduced amino acid sequence had the sepecific domains of LisH, CTLH, and WD40, sharing 89%、86%、85% and 73% identity with Ricinus communis, Populus trichocarpa, Vitis vinifera and Arabidopsis, respectively.5, This study is fundamental for identifying the compontents of JA signaling pathways in laticifer cells, and laid a good foundation for further research on the regulation of natural rubber biosynthesis by JA signaling pathway. |
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