| Keratinocyte growth factor-1(KGF-1)can induce keratinocyte proliferation,migration,and differentiation by specifically binding to a variety of human and animal epithelial cells,and has a healing effect on wounds without adverse inflammatory responses.Human keratinocyte growth factor-1(hKGF-1)can reduce the incidence and duration of mucositis caused by high-dose chemotherapy and radiation therapy in malignant tumors treatment.An efficient approach for hKGF-1 is urgently needed to address the shortage of hKGF-1 in market caused by the low production and poor stability of natural hKGF-1.Rice kernels are of complete eukaryotic expression system,which is beneficial to the expression,post-translationally processing,accumulation and stabling of hKGF-1,and they are easy for transportation.Hence,expression of hKGF-1 in rice kernels is cost efficient and it is easy to obtain high-expression,which can create conditions for downstream processing and clinical application of hKGF-1.In this study,a seed-specific promoter,pGBSSI,was cloned from maize,and its transcriptional activity was verified.The promoter was used to drive the expression of human keratinocyte growth factor(hKGF-1)gene in order to generate rice plants with high hKGF-1 expression.This study provides a new approach to obtain high expression and the natural security of KGF-1.Mainly results are as followed:(1)A seed-specific promoter,pGBSSI,which is 999bp in length,was cloned from maize.Sequence analysis showed that the promoter sequence contained a CpG island and multiple cis-acting elements such as light responses,stress response,hormone response,stress induction and development-related elements.A plant expression vector pCBG-Gus was constructed and transformed into maize immature embryos with Agrobacterium-mediated method.Resistant maize plants were obtained after screening and differentiation.Transgenic maize plant was obtained by Bar-gene-Aurum-labeled immuno-strip test combined with PCR detection.The qPCR analysis showed that the Gus gene had the highest transcription level in leaves.Gus histochemical staining analysis showed that the highest expression of Gus was found in mature grains,while it was not detectable in other tissues or were of low activity.This indicates that Gus protein gradually accumulated in the grain during grain maturation.The promoter pGBSSI can be utilized in expression foreign proteins in gramineous crops.(2)The DNA sequence of hKGF-1 gene was optimized according to the rice codon usage bias and artificially synthesized.A poly-histidine(His-Tag)sequence was added to the 5’-end for the purification of recombinant proteins.A seed-specific expression vector pCBG-hKGF-1 was constructed with the optimized gene.Agrobacterium-mediated transformation was performed to introduce the pCBG-hKGF1 was transformed into the callus of rice cultivar Nipponbare.Two hundred and thirty-three resistant regenerated plantlets were obtained after screening and regeneration.(3)Forty-six rice transformed events were obtained by PCR detection of hKGF-1 and aurum-labeled immune-strip assay on Bar protein.Twelve transformed events with single copy insertion of hKGF-1 were obtained by Southern Blotting.RT-PCR results suggested that hKGF-1 was transcribed in the 12 transformed events.Western Blotting results showed that hKGF-1 was expressed in 10 of the transformed e vents.ELISA results revealed that the expression of hKGF-1 protein could reach up to 10.8 μg/g of brown rice.Recombinant hKGF-1 was purified by Ni+affinity chromatography and the mitogenic activity was tested.The results showed that the recombinant hKGF-1 protein from rice species has the activity of stimulating the proliferation of mouse 3T3 fibroblast.The proliferation rate of the cells was linearly positive correlation the concentration of recombinant hKGF-1 around lng/ml to 75ng/ml.At the concentration of 5ng/ml,which located in the linear concentration scope,the rice-derived recombinant hKGF-1(OshKGF-1)exhibited the same mitogenic activity as commercial standard hKGF-1. |
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