| Due to the accumulation of specific carotenoids, citrus fruits display a wide range of colorations and are appreciated by consumers with significant economic value. Citrus fruits are also of high scientific value owing to the diversity of carotenoid composition and content among varieties. So far, citrus carotenoids have been well characterized, however, carotenoid metabolism is a complex process with many genes participating in and coordinating interaction. Research on the molecular mechanism of the major carotenoid biosynthetic genes is far from knowing enough, further investigations to understand the regulatory mechanism of carotenoid metabolism is needed. The mechanism has been conducted in this research through the research on the promoter of the gene (Psy) which encoding the rate-limiting enzyme of carotenoid biosynthesis pathway, to find the regulatory pattern and to acquire the information about the upstream regulatory gene. The result was presented as two parts:Two similar but non-identical sequences (CsPsyla and CsPsy1b) were isolated from sweet orange. A functional analysis based on heterologous expression in E. coli indicated that CsPsyla was a more efficient converter of geranylgeranyl diphosphate to phytoene, to understand the mechanism of citrus color trait; The promoters of the sweet orange genes (CsPsy1a and CsPsy1b) were isolated, then fused to the β-glucuronidase (GUS) reporter gene and introduced as a transgene into Arabidopsis thaliana, to investigate the promoter activity based on GUS activity. The main results were as follows:1Isolation and functional characterization of phytoene synthase genes from sweet orange(1) Two CsPsyl sequences were isolated from sweet orange cv. Anliu, and named as CsPsy1a and CsPsy1b. The two sequences were98.6%identical to one another, one being of length1,311bp and the other1,317bp. They encoded436and438amino acids, respectively. The sequences differed from one another at18nucleotide positions, resulting in nine predicted peptide differences. Seven of which were generated by SNP and the other two by different number of AAT repeat units within a microsatellite.(2) Real time RT-PCR was used to study the expression of the two genes during fruit development. Transcription profiling showed that CsPsyla abundance increased throughout fruit development in both the peel and pulp, while that of CsPsy1b increased early in fruit development and then stabilized. The abundance of the two transcripts was similar in the peel, but that of CsPsy1a present was higher in the pulp.(3) A full length Psy cDNA was isolated from pummelo cv. Gaophuang. Its sequence proved to be identical to that of CsPsy1b. HPLC was used to compare the composition and content of carotenoids from these two cultivars, the result displayed a pronounced difference in their carotenoid content. The transcription in cv. Gaophuang of main carotenoid biosynthetic genes (except for Hyb) was higher than in cv. Anliu (yellow fleshed fruit), these results showed that the transcription of other major carotenoid synthesis genes could not explain the large difference in carotenoid content between the two species.(4) Two CpPsyl sequences were isolated from grapefruit cv. Marsh, and named as CpPsy1a and CpPsy1b. The peptide sequence of CpPsyla was identical to that of CsPsy1a, and CpPsy1b was99%identical to CsPsy1b. The number of residues encoded by these three genes (CsPsy1a, CsPsy1b and CpPsy1b) varies slightly (respectively,437,438and439, due to polymorphism in the number of AAT triplet present. Ten predicted peptide differences were found among the three genes, eight of them were generated by SNP and the other two by different number of AAT repeat units within a microsatellite.(5) The full-length coding region of three genes (CsPsy1a, CsPsy1b and CpPsy1b) were amplified using PCR and further inserted into pET-28a (+), resulting in the prokaryotic expression vector pET-CsPsy1a, pET-CsPsy1b and pET-CpPsy1b, respectively. After transformation of E. coli strain BL21which produces the PSY substrate GGPP, the HPLC profile of both types of transgenic cell included a peak with a retention time of19.0-21.5min which was not detectable in the empty vector control. Interesting, there was a demonstrable negative correlation between the number of microsatellite repeat units present and PSY activity.(6) An E. coli expression platform was used to test the effect of site-directed mutagenesis in CsPSYla on enzymatic activity, particularly that of the number of microsatellite repeat units presented. Four different variants were generated (pET-CsPSY1a-1, pET-CsPSY1a-2, pET-CsPSYla-3and pET-CsPSYla-4). After transformation of E. coli strain BL21which produces the PSY substrate GGPP, functional analysis suggested that there was a clear correlation between the number of microsatellite repeat units present and PSY activity.2Identification and functional characterization of the CsPsyla and CsPsylb promoters from sweet orange (1) The promoter of the sweet orange gene CsPsy1a of sweet orange was isolated using chromosome walking. Its sequence included a number of regulatory elements predicted to be responsive to light, hormone and other stress cues. The promoter’s transcription start site was determined using5’RACE based on RNA extracted from the pulp of sweet oranges cv. Anliu. Two sites were identified, the first a cytidine (C)186bp upstream of the translation initiation codon and an adenine (A)518bp upstream of translation initiation codon. PLACE and PlantCARE analyses suggested a potential TATA box551bp from the5’end of the ATG and33bp upstream of the potential TSS (’+1’) that was further from the translation initiation codon. In addition, a possible CAAT box was located at-83bp.(2) The926-bp region upstream of CsPsyla was fused to the β-glucuronidase (GUS) reporter gene, and introduced as a transgene into Arabidopsis thaliana. The pattern of GUS expression in the transgenic plants showed that the CsPsy1a promoter drove expression in the young seedlings, the leaves, the roots and in parts of the flower, in a developmentally regulated fashion. These results included that the CsPsy1a promoter drove expression in photosynthetic and non-photosynthetic tissue, and was induced by developmental cues.(3) Five5’-deletions expression vectors named V1～V5were constructed, and transformed into Arabidopsis. A promoter deletion analysis revealed that the region-479nt to-306nt positively regulated expression, the region-306nt to-93nt was associated with negative regulation of expression and the region-46nt to+21nt maintained basal promoter activity. The transgenic Arabidopsis seedlings were treated with different light quality during de-etiolation, A promoter deletion analysis revealed that Two highly repetitive sequences (Spl and A-box), located in the region from-479nt to-306nt, may act as light-responsive elements regulating CsPsy1a transcription during photomorphogenesis. The transgenic plants carrying V5::GUS were treated with various stresses and hormones treatment, of these, sucrose treatment showed a significantly different value from the control by one-sided t tests. The promoter deletion constructs were used to explore the sucrose induction of the CsPsy1a promoter, the results showed that the cis-acing element(s) responsive to sucrose must be located in the region between-479nt to-306nt.(4) The promoter of the sweet orange gene CsPsy1b of sweet orange was isolated using chromosome walking. Its sequence included a number of regulatory elements predicted to be responsive to light, hormone and other stress cues, as well as the CsPsy1a promoter sequence. There were many different cis-elements in the two sequences, for example, many light responsive element (G-box) were present in the CsPsy1b promoter sequence, but none was discovered in the CsPsyla promoter sequence. In a word, the bioinformatics analysis indicated that the two promoters was mainly regulated by light, and induced by various stresses.(5) The CsPsy1b promoter was fused to the P-glucuronidase (GUS) reporter gene, and introduced as a transgene into Arabidopsis thaliana. The pattern of GUS expression in the transgenic plants included that the CsPsy1b promoter drove expression in photosynthetic and non-photosynthetic tissue, and was induced by developmental cues, as well as the CsPsy1a promoter.(6) The transgenic Arabidopsis seedlings carrying CsPsy1ap::GUS and CsPsy1bp::GUS were treated with different light quality treatment (red, far red, blue and white), the results showed that two promoters were induced by different light quality treatment, however, there were some difference maybe due to the different light responsive elements in their promoters; The transgenic plants carrying CsPsy1ap::GUS and CsPsy1bp::GUS were treated with different light intensity treatment, the results showed that CsPsy1a promoter activity was enhanced by dim light, and the CsPsy1b promoter had no response to different light intensity; The transgenic plants carrying CsPsy1ap::GUS and CsPsy1bp::GUS were treated with different photoperiod treatment, CsPsy1a promoter activity was enhanced by long-day, and the CsPsy1b promoter had no response to different photoperiod. |
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