Isolation Of The Specific Promoter Of Efficiency Expression Of Protein From Alfalfa Or Soybean And Its Function Analysis In Transgenic Plant

Expression of recombinant proteins in transgenic plants is a platform technology for production of pharmaceutical proteins, orally delivered vaccines, antibodies and industrial enzymes. Transgenic plants provide an inexpensive and convenient system for the large-scale production of valuable recombinant proteins. Over the past ten years, several efficient plant-based expression systems have emerged, and>100recombinant proteins have now been produced in a range of different species. However, several constraints that hinder the widespread use of plants as bioreactors remain to be addressed. Important factors include the yield, quality and homogeneity of the final product. Achieving high levels of expression is crucial, therefore the studies focused on promoters. In this study, three special proteins, named alfalfa light-a (AL-a), alfalfa light-b (AL-b) and soybean no light-p (SN-p), were isolated from the2D-PAGE, which expressed abundantly in sprout of alfalfa and soybean under the growth condition of7days. Seven amino acids in N-terminal region were determined by BIOSUMS limited company for one of the proteins AL-a, with the sequence as VEVLLGA (Val-Glu-Val-Leu-Leu-Gly-Ala). The deduced amino acid sequence of the total protein was aligned in NCBI and Swiss-Prot Database. The BLAST results showed that the protein AL-a has high similarity to plastocyanin, a protein of pea. To identified the protein AL-a, we isolated the DNA fragment encoding the protein AL-a from genomic DNA of alfalfa and determined its nucleotides sequence by an automated DNA sequencer. The AL-a was highly homologous (90%) to the plastocyanin by multiple alignments for nucleic acid and protein sequences. So the AL-a protein was named alfalfa-plastocyanin (AP) and the deduced protein precursor was predicted to be chloroplast-specific via the ChloroP computer program (http://www.cbs.dtu.dk/services/ChloroP/). Meanwhile, the promoter of alfalfa-plastocyanin (P-AP) was isolated from light-alfalfa by PCR. The goal of this work was to perform efficient expression of heterologous gene of the replacement alfalfa-plastocyanin gene driven by the promoter in transgenic alfalfa. This may be a feasible method to increase the level of expression of recombinant proteins.The expression of plant-protein will be determined by its promoter. So we analyzed expression of alfalfa-plastocyanin by RT-PCR. The result suggested that alfalfa-plastocyanin gene express abundantly only in green leaf of alfalfa under the growth condition of7days light. In this study, we designed several primers of the promoter of alfalfa-plastocyanin according to published the published plastocyanin gene promoter region of Medicago truncatula (GenBank:CU013531.5) in NCBI genebank. We used PCR to amplify alfalfa plastocyanin gene promoter (AP-P) from genomic alfalfa DNA and obtained a269bp promoter. PlantCARE analysis of the promoter sequence indicated that both a typical TATA box and a CAAT box were located in the promoter sequence, and some of the cis-elements that are responsible for light responsiveness including G-box, GT1-motif, LAMP-element, Pc-CMA2a, Pc-CMA2b and Pc-CMA2c that are sufficient to regulate organ-specific and light-inducible transcription were also identified within this promoter region. A plant expression vector (pBI121-P-AP) containing a P-AP-GUS fusion construct was constructed and transferred into Agrobacterium tumefaciens EHA105. Agrobacterium tumefaciens EHA105containing pBI121-P-AP was transferred into alfalfa by seed soaking method. Histochemical staining suggested that GUS gene express in green leaf and GUS gene were amplified in transgenic alfalfa by PCR method. These data identified the tissue-specific expression on the alfalfa-plastocyanin gene promoter in alfalfa. Our results may make contributions in further research for edible anticancer vaccine, such as the high level HER-2production in transgenic alfalfa.