Genetic Engineering Study Of Antimicrobial Protein From Ginkgo Biloba Seeds And Evaluation Of A Seed-specific Promoter In Wheat

Gnk2-1, an antimicrobial protein was isolated from the seeds of Ginkgo biloba, which has the higher activity to fungus in vitro and the stable characterization. MALDI-TOF-MS identification showed higher homology(P<0.05) with a novel antifungal protein Gnk2(Ginkbilobin-2). Degenerate primers were designed to amplify a fragment of the antimicrobial protein gene, according to the ESI-MS/MS amino acid sequence results of internal peptide. The blast results showed the amplified fragment also had 99% similarity with the Gnk2 gene. Further, the homological primers were designed according to the Gnk2 gene sequence for cloning the gene cDNA of the antimicrobial protein by RT-PCR. The sequence analysis indicated that the structure of this antimicrobial protein was different from the others have known antifungal proteins, this antimicrobial protein has the extracellular domain of plant cysteine-rich receptor-like kinases(CRKs).The prokaryotic expression vector pGEX-GK2-1 and pET-32-GK2-1 were constructed by the gene recombination technique and then transfected the Escherichia coli BL21(DE3) for the further study to the antibiotic mechanism of this antimicrobial protein in vitro. The analysis of SDS-PAGE and Western blot showed that the GST recombinant protein was highly expressed with inclusion body in Escherichia coli. The pET-32-GK2-1 and pAcycDuet1 plasmid containing DsbC gene to be used for cotransformation the Escherichia coli cells. The analysis of SDS-PAGE and Western blot indicated that the recombinant protein was partly soluble in the supernate of thallus. In the meantime, a constitutive plant expression vector was constructed and transformed into cucumber (Cucumis sativus L.) cultivar nongcheng 3 via Agrobacterium-mediated transfer. The analysis of PCR,RT-PCR and Western blotting revealed that the target gene has been integrateed in the genome of cucumber and could be stably heredity. The results of disease resistance analysis is the first report of the ginkbilobin gene used to prove the application potential in the disease resistance reformation.The exploiting of new antimicrobial protein gene resource supply the basic material for the obtaining of transgenic disease resistance germplasm resources and the using of protein antibacterial antiseptic, biopesticide, new antibiotics. When using the cereal seeds as bioreactor to produce the special proteins, except that in need of these gene resources with applying value, but aslo require some high efficient endosperm-specific promoter. To develop the high efficient endosperm-specific expression promoter, not only is the base for improving cereal quality via genetic engineering technique, but also is the premise for producing particular proteins using cereal seeds as bioreactor.Glu-1Bx14 gene from wheat could specially high efficient expression during the development of endosperm. In this study, the 5'upstream regulated sequence of this gene 2.6kb was cloned using wheat variety Xiaoyan 6 as material via TAIL-PCR genome walking. The results of GUS transient expression revealed this promoter fragment have endosperm tissue-specific initiate activity. Sequence analysis showed the regulated sequence included the core transcription element, many potential transcription elements of endosperm-specific expression and distal nuclei matrix attachment regions(MARs).This article have defined the transcriptional start site by RLM-RACE method and studied the functional region of promoter via 5'deletion mutation and transient expression analysis of reporter gene. To select the region comprising necessary enhancer elements for tandem-repeat construct, It displayed 2 times the tandem-repeat reporter activity observed with the single copy construct and retained the endosperm tissue-specific characteristic. This article aim is to find a suitable length of promoter that included all necessary enhancer elements, and to reform and use. It was for obtaining a more higher efficient cereal endosperm-specific promoter and applying to the cereal gene engineering improvement and the research of endosperm bioreactor.