Cloning And Priliminary Functional Evaluation Of A New Promoter From Maize

Gene expression and regulation is one of the central content of plant gene engineering research. The promoters and its cis-elements are important for transcription level and have become the hot topics in the study of molecular biology. In present, constitutive promoters are used widely in plant genetic engineering. But constitutive promoters drive foreign gene highly expressed in all tissues and organs. Overexpression of unused foreign gene such as selective marker gene, do not have space and time on the specificity. This will consume a large number of nutrients and energy, which will inhibit other physiological activities and affect normal growth and development of plant. This will increase the adaptability of plants. Therefore, in order to overcome these shortcomings of constitutive promoters, we need to drive foreign genes in transgenic plants at a specific time and site expression, and the specificity of the transcription activity of moderate promoter.In PPR (Pentatricopeptide repeats) gene family from Maize do genome-wide analysis, we foud that the member of GRMZM2G129783 gene has a moderate leaf-specific expression. So we suggest the promoter of PPRGRMZM2G129783 gene is a moderate leaf-specific promoter. Therefore, based on the analysis of this research in bioinformatics, cloning PPRGRMZM2129783 gene full-length promoter pPPR1830 and its deletion promoters, build expression vectors for driving GUS reporter gene. By GUS histochemical staining analysis and GUS enzyme activity measurement of transgenic tobaccos, the function of the preliminary verify its specific promoter. The results were as follows:(1) The results of fluorescence quantitative PCR showed that PPRGRMZM2G129783 gene are expressed only in the seedling stem leaf, almost no expression in the root. So we conclude that the promoter of PPRGRMZM2G129783 gene is a green-tissue specific promoter.(2) Through sequence analyses in PLACE and PlantCARE, except for the core promoter elements such as TATA box and Inr, there also include seven light responsive elements:3-AF1 binding site, GT-1 motif, G-box, I-box, CATT motif,-10 promoter element, ATCT motif and five inducible responsive elements:MBS, LTRE, ABRE, CGTCA motif, ARF, also root-specific element:ROOTMOTIFTAPOX1.(3) A 1830 bp fragment of the 5’-upstream region of PPRGRMZM2G129783 gene promoter was isolated from the maize inbred line B73 (Zea mays), named pPPR1830. According to the results of sequence analysis, four 5’truncated segments are got by PCR, named respectively pPPRl387, pPPR437, pPPR146 and pPPR128, with whose respective lengths of 1387,437,146 and 128 bp. Five expression vectors were constructed by placing the 35S promoter of pRI201-;AN-35S-GUS, named pRI201-AN-pPPR1830-GUS, pRI201-AN-pPPR1387-GUS, pRI201-AN-pPPR437-GUS, pRI201-AN-pPPR146-GUS and pRI201-AN-pPPR128-GUS.(4) Injection of tobacco leaves, each in the above five expression vector, and with pRI201-AN-35S-GUS as the positive control, by GUS histochemical staining, detection of transient expression of GUS gene transcription activity of different length of promoter sequences for qualitative identification.The results show that besides full-length pPPR1830 promoter, other promoters can drive GUS gene express in leaf of tobacco.(5) All of expression vectors are transferred into tobaccos by Agrobacterium mediated transformation. And we got 25 positive tobaccos, respectively five strains of pPPR1830, three strains of pPPR1387, four strains of pPPR437, four strains of pPPR146, three strains of pPPR128, and six strains of positive control.(6) GUS histochemical staining analysis and quantitative measurement of GUS activity showed that the full length of pPPR1830 sequence, and pPPR1387, pPPR437 promoter sequences can drive GUS genes express in vegetative organs, such as roots, stems, leaves of transgenic tobaccos, but its activity all are lower than the control 35S. Whereas, pPPR146 and pPPR128 sequences have activities in stems and leaves only, and has not activity in roots. In addition, the activities of full-length pPPR1830 in roots, stems and leaves are lower than pPPR1387, less than pPPR437 in stems and leaves. It follows that in the upstream sequences of pPPR1830 there may be a negative regulatory elements.So we conclude that pPPR1830 may be a moderate tissue-specific promoter in monocotyledon, and there are some root-specific elements ROOTMOTIFTAPOX1 in the promoter sequence -397 to-106 bp, in the upstream sequences of pPPR1830 there may be a negative regulatory elements.The sequence of the promoter activity of moderate in between -1346 to -1799 bp, with further validation or modification is expected to be used in monocot transgenic engineer.