| Isoprenoids not only play important roles in plant growth and development, but also have significant commercial value. Recently, terpenoid metabolic engineering has attracted widespread attention. To obtain valuable isoprenoids by using metabolic engineering, it is significant to understand the regulation mechanism of isoprenoid biosynthesis pathway.HMGR is known as a rate-limiting enzyme of the MVA pathway in catalyzing the conversion of 3-hydroxy-3-methylglutaryl coenzyme A(HMG-CoA) to mevalonate(MVA)in plants. Plant HMGR is encoded by a small multigene family. Plant HMGR has a critical regulatory role in the MVA pathway, not only for normal plant development, but also for responses to various physiological and environmental stimuli. In this study, we first investigated the expression patterns of MdHMGR1, MdHMGR2 and MdHMGR3 genes in different tissues and after treatment with hormones. The MdHMGR2 promoter was isolated,sequenced, and analyzed through bioinformatics tools. The GUS spatial and temporal expression patterns driven by the MdHMGR2 promoter and its deletion fragments were identified in transgenic Arabidopsis. To elucidate the regulation mechanisms of MdHMGR2 expression and apple MVA metabolic pathway, Structural and functional analysis of the MdHMGR2 promoter was identified in transgenic Arabidopsis. The main results were as follows:(1) Although the MdHMGR1, MdHMGR2, and MdHMGR3 genes were all expressed in tender leaves, old leaves, stems, fruits and roots, the expression patterns of the three genes varied significantly among different tissues. MdHMGR1 gene was expressed at high levels in roots and fruits, and the expression levels in different tissues were not very different. The highest expression level of the MdHMGR2 gene was exhibited in roots, followed by fruits and stems, with the lowest expression level in old leaves. MdHMGR3 gene was expressed at high levels in fruits and roots, and the expression levels in different tissues were not very different.(2) The expression patterns of the MdHMGR1, MdHMGR2, and MdHMGR3 genes varied significantly treated with ABA, ETH, GA, IAA, MeJA, and SA. MdHMGR1 expression was slightly enhanced by these hormone treatments with a maximum level at 2 h or 4 h. Hormone treatments of ETH, IAA, MeJA, and SA significantly increased MdHMGR2 mRNA expression with a maximum level at 4 h. However, MdHMGR2 expression was only slightly enhanced by ABA and GA with a maximum level at 4 h. MdHMGR3 expression was slightly induced by hormone treatments with a maximum level at 2 h or 4 h except for ABA.(3) The primers pHMGR2 F and pHMGR2 R were designed based on the sequence upstream of the MdHMGR2 encoding region from the sequence MDC015298.78 in the apple genome database. A 1509 bp upstream DNA sequence including the putative promoter of the MdHMGR2 gene was obtained from “white winter pearmain” apple genomic DNA.(4) The transcription start site of the MdHMGR2 gene was identified by 5′-rapid amplification of cDNA ends(5′ RACE). The gene-specific primers HMGR2 GSP and HMGR2 NGSP were designed according to the MdHMGR2 cDNA sequence(GenBank:EF580921.1). The second round PCR products were cloned into the pMD-18 T vector and 15 positive clones were sequenced to determine the transcription start site. Finally, an adenine156 bp upstream of the translation initiation codon ATG was identified as the transcription start site.(5) The MdHMGR2 promoter sequence was analyzed for putative cis-acting regulatory elements using Plant CARE and PLACE databases. A putative TATA box(TATAAAA) was detected at positions-35 to-29, which is consistent with the previous data for a TATA box 32± 7 upstream from the transcription start site. A number of potential cis-acting elements associated with hormone-related responses and tissue-specific expression were identified in the MdHMGR2 promoter.(6) A series of promoter fragments of MdHMGR2 were generated by PCR amplification and then the amplified promoter fragments were cloned into the corresponding sites of plant binary vector pBI121-GUS, replacing the CaMV35 S promoter. The several recombinant plasmids were designated as MdHMGR2P-D1, MdHMGR2P-D2, MdHMGR2P-D3,MdHMGR2P-D4, MdHMGR2P-D5, and MdHMGR2P-D6. All constructs were verified by sequencing and were then transferred into the Agrobacterium tumefaciens strain GV3101 by using the freeze-thaw method. The Agrobacterium-mediated transformation of Arabidopsis was performed using the floral dip method. The T3 homozygous transformants of three independent lines for each construct was used for promoter analysis.(7) In order to investigate the expression pattern of the MdHMGR2 gene during the entire life cycle, GUS expression pattern driven by the full MdHMGR2 promoter was analyzed in MdHMGR2P-D1 transgenic Arabidopsis. In MdHMGR2P-D1 transgenic plants, strong GUS expression was detected in roots, hypocotyls, cotyledons and newly growing true leaves of the3-day-old, 5-day-old, 10-day-old, and 15-day-old seedlings. GUS activity was observed atlow levels in roots and newly growing true leaves of 30-day-old transgenic plants. In50-day-old Arabidopsis, GUS activity was located in newly growing true leaves, while no GUS staining was observed in mature leaf tissues. During the stage of inflorescence development, the GUS signal was found only in anthers and stigmas. Moreover, pollens and anther walls had GUS staining in the anthers. There was no GUS expression in petals, pistils,and siliques.(8) MdHMGR2P-D2 transgenic plants share similar temporal and spatial GUS expression profiles as MdHMGR2P-D1 transgenic plants. While in MdHMGR2P-D3 plants, faint GUS activity was located in cotyledons, newly growing true leaves and lower part of roots of3-day-old, 5-day-old, 10-day-old, and 15-day-old seedlings. In 30-day-old and 50-day-old transgenic Arabidopsis, newly growing true leaves had unique GUS staining in the vegetative organs. GUS expression was also detectable in anther walls, pollens, and stigmas of flowers.Fluorometric analysis revealed that MdHMGR2P-D1 plants showed the highest GUS activity while the GUS expression level in MdHMGR2P-D2 and MdHMGR2P-D3 plants decreased by 33% and 79% compared to that of MdHMGR2P-D1 plants, respectively. Loss of the region from-1050 to-827 induced a sharp decrease in GUS activity. In addition, GUS expression was undetectable in MdHMGR2P-D4, MdHMGR2P-D5 and MdHMGR2P-D6 transgenic plants in any tissues and at any developmental stages. Taken together, the results illustrated that the region from-1050 to-827 was critical for promoter quantitative activity.(9) Treatment with ETH, IAA, MeJA, and SA increased GUS activity in MdHMGR2P-D1 transgenic plants. There was no GUS signal in MdHMGR2P-D4,MdHMGR2P-D5, and MdHMGR2P-D6 transgenic plants with hormones as determined by histochemical staining. Further, fluorometric analyses were performed in MdHMGR2P-D1,MdHMGR2P-D2, and MdHMGR2P-D3 transgenic plants treated with hormones.MdHMGR2P-D1 plants produced 2.5-fold increases in GUS activity treated with ETH while no GUS activity induction was found in MdHMGR2P-D2 and MdHMGR2P-D3 plants treated with ETH. MdHMGR2P-D1, MdHMGR2P-D2, and MdHMGR2P-D3 plants were all responsive to IAA, MeJA, and SA treatment. These results suggested that the ethylene responsive elements were likely present in the MdHMGR2 promoter region from-1050 to-1005.(10) MdHMGR3-like gene was isolated which had 97.04% homology with MdHMGR3 gene encoding sequence. The expression level of the MdHMGR3-like gene in roots was much higher than in other tissues and was enhanced by ETH, MeJA, and SA treatments.MdHMGR3-like gene promoter was isolated and its homology with MdHMGR3 genepromoter was 49.21%. The transgenic Arabidopsis containing MdHMGR3 gene promoter showed a constitutive GUS expression pattern at high level, while GUS was expressed in a root-specific manner driven by MdHMGR3-like gene promoter in transgenic Arabidopsis. |
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