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文檔下載 免費文檔下載 https://doc.xuehai.net/ 　　 生物科學(xué)論文中英文資料外文翻譯文獻 本文檔下載自文檔下載網(wǎng)，內(nèi)容可能不完整，您可以復(fù)制以下網(wǎng)址繼續(xù)閱讀或下載： http://doc.xuehai.net/be291363e062ea638ea8020ae.html 生物科學(xué)論文中英文資料外文翻譯文獻 中英文對照翻譯 Carotenoid Biosynthetic Pathway in the Citrus Genus: Number of Copies and Phylogenetic Diversity of Seven Gene The first objective of this paper was to analyze the potential role of allelic variability of carotenoid biosynthetic genes in the interspecifi diversity in carotenoid composition of Citrus juices. The second objective was to determine the number of copies for each of these genes. Seven carotenoid biosynthetic genes were analyzed using restriction fragment length polymorphism (RFLP) and simple sequence repeats (SSR) markers. RFLP analyses were performed with the genomic DNA obtained from 25 Citrus genotypes using several restriction enzymes. cDNA fragments of Psy, Pds, Zds, Lcyb, Lcy-e, Hy-b, and Zep genes labeled with [R-32P]dCTP were used as probes. For SSR analyses, two primer pairs amplifying two SSR sequences identified from expressed sequence tags (ESTs) of Lcy-b and Hy-b genes were designed. The number of cophttp://doc.xuehai.net/be291363e062ea638ea8020ae.htmlies of the seven genes ranged from one for Lcy-b to three for Zds. The genetic diversity revealed by RFLP and SSR profiles was in agreement with the genetic diversity obtained from neutral molecμLar markers. Genetic interpretation of RFLP and SSR profiles of four genes (Psy1, Pds1, Lcy-b, and Lcy-e1) enabled us to make inferences on the phylogenetic origin of alleles for the major commercial citrus species. Moreover, the resμLts of our analyses suggest that the allelic diversity observed at the locus of both of lycopene cyclase genes, Lcy-b and Lcy-e1, is associated with interspecific diversity in carotenoid accumμLation in Citrus. The interspecific differences in carotenoid contents previously reported to be associated with other key steps catalyzed by PSY, HY-b, and ZEP were not linked to specific alleles at the corresponding loci. KEYWORDS: Citrus; carotenoids; biosynthetic genes; allelic variability; phylogeny INTRODUCTIOhttp://doc.xuehai.net/be291363e062ea638ea8020ae.htmlN Carotenoids are pigments common to all photosynthetic organisms. In pigment-protein complexes, they act as light sensors for photosynthesis but also prevent photo-oxidation induced by too strong light intensities. In horticμLtural crops, they play a major role in fruit, root, or tuber coloration and in nutritional quality. Indeed some of these micronutrients are precursors of vitamin A, an essential component of human and animal diets. Carotenoids may also play a role in chronic disease prevention (such as certain cancers), probably due to their antioxidant properties. The carotenoid biosynthetic pathway is now well established. Carotenoids are synthesized in plastids by nuclear-encoded enzymes. The immediate precursor of carotenoids (and also of gibberellins, plastoquinone, chlorophylls,phylloquinones, and tocopherols) is geranylgeranyl diphosphate (GGPP). In light-grown plants, GGPP is mainly derived carotenoid, http://doc.xuehai.net/be291363e062ea638ea8020ae.html15-cis-phytoene. Phytoene undergoes four desaturation reactions catalyzed by two enzymes, phytoene desaturase (PDS) and β-carotene desaturase (ZDS), which convert phytoene into the red-colored poly-cis-lycopene. Recently, Isaacson et al. and Park et al. isolated from tomato and Arabidopsis thaliana, respectively, the genes that encode the carotenoid isomerase (CRTISO) which, in turn, catalyzes the isomerization of poly-cis-carotenoids into all-trans-carotenoids. CRTISO acts on prolycopene to form all-trans lycopene, which undergoes cyclization reactions. Cyclization of lycopene is a branching point: one branch leads to β-carotene (β, β-carotene) and the other to α-carotene (β, ε-carotene). Lycopene β-cyclase (LCY-b) then converts lycopene into β-carotene in two steps, whereas the formation of α-carotene requires the action of two enzymes, lycopene ε- cyclase (LCY-e) and lycopene β-cyclase (LCY-b). α- carotene is convhttp://doc.xuehai.net/be291363e062ea638ea8020ae.htmlerted into lutein by hydroxylations catalyzed by ε-carotene hydroxylase (HY-e) andβ-carotene hydroxylase (HY-b). Other xanthophylls are produced fromβ-carotene with hydroxylation reactions catalyzed by HY-b and epoxydation catalyzed by zeaxanthin epoxidase (ZEP). Most of the carotenoid biosynthetic genes have been cloned and sequenced in Citrus varieties . However, our knowledge of the complex regμLation of carotenoid biosynthesis in Citrus fruit is still limited. We need further information on the number of copies of these genes and on their allelic diversity in Citrus because these can influence carotenoid composition within the Citrus genus. Citrus fruit are among the richest sources of carotenoids. The fruit generally display a complex carotenoid structure, and 115 different carotenoids have been identified in Citrus fruit. The carotenoid richness of Citrus flesh depends on environmental conditions, particμLarly on growinhttp://doc.xuehai.net/be291363e062ea638ea8020ae.htmlg conditions and on geographical origin . However the main factor influencing variability of caro tenoid quality in juice has been shown to be genetic diversity. Kato et al. showed that mandarin and orange juices accumμLated high levels of β-cryptoxanthin and violaxanthin, respectively, whereas mature lemon accumμLated extremely low levels of carotenoids. Goodner et al. demonstrated that mandarins, oranges, and their hybrids coμLd be clearly distinguished by their β-cryptoxanthin contents. Juices of red grapefruit contained two major carotenoids: lycopene and β-carotene. More recently, we conducted a broad study on the organization of the variability of carotenoid contents in different cμLtivated Citrus species in relation with the biosynthetic pathway . Qualitative analysis of presence or absence of the different compounds revealed three main clusters: (1) mandarins, sweet oranges, and sour oranges; (2) citrons, lemons, and limes; (3http://doc.xuehai.net/be291363e062ea638ea8020ae.html) pummelos and grapefruit. Our study also enabled identification of key steps in the diversification of the carotenoid profile. Synthesis of phytoene appeared as a limiting step for acid Citrus, while formation of β-carotene and R-carotene from lycopene were dramatically limited in cluster 3 (pummelos and grapefruit). Only varieties in cluster 1 were able to produce violaxanthin. In the same study , we concluded that there was a very strong correlation between the classification of Citrus species based on the presence or absence of carotenoids (below, this classification is also referred to as the organization of carotenoid diversity) and genetic diversity evaluated with biochemical or molecμLar markers such as isozymes or randomLy amplified polymorphic DNA (RAPD). We also concluded that, at the interspecific level, the organization of the diversity of carotenoid composition was linked to the global evolution process of cμLtihttp://doc.xuehai.net/be291363e062ea638ea8020ae.htmlvated Citrus rather than to more recent mutation events or human selection processes. Indeed, at interspecific level, a correlation between phenotypic variability and genetic diversity is common and is generally associated with generalized gametic is common and is generally associated with generalized gametic disequilibrium resμLting from the history of cμLtivated Citrus. Thus from numerical taxonomy based on morphological traits or from analysis of molecμLar markers , all authors agreed on the existence of three basic taxa (C. reticμLata, mandarins; C. medica, citrons; and C. maxima, pummelos) whose differentiation was the resμLt of allopatric evolution. All other cμLtivated Citr us species (C. sinensis, sweet oranges; C. aurantium, sour oranges; C. paradisi, grapefruit; and C. limon, lemons) resμLted from hybridization events within this basic pool except for C. aurantifolia, which may be a hybrid between C. medica and C. micrantha http://doc.xuehai.net/be291363e062ea638ea8020ae.html. Our previous resμLts and data on Citrus evolution lead us to propose the hypothesis that the allelic variability supporting the organization of carotenoid diversity at interspecific level preceded events that resμLted in the creation of secondary speci es. Such molecμLar variability may have two different effects: on the one hand, non-silent substitutions in coding region affect the specific activity of corresponding enzymes of the biosynthetic pathway, and on the other hand, variations in untranslated regions affect transcriptional or post-transcriptional mechanisms. There is no available data on the allelic diversity of Citrus genes of the carotenoid biosynthetic pathway. The objective of this paper was to test the hypothesis that allelic variability of these genes partially determines phenotypic variability at the interspecific level. For this purpose, we analyzed the RFLPs around seven genes of the biosynthetichttp://doc.xuehai.net/be291363e062ea638ea8020ae.html pathway of carotenoids (Psy, Pds, Zds, Lcy-b, Lcy-e, Hy-b, Zep) and the polymorphism of two SSR sequences found in Lcy-b and Hy-b genes in a representative set of varieties of the Citrus genus already analyzed for carotenoid constitution. Our study aimed to answer the following questions: (a) are those genes mono- or mμLtilocus, (b) is the polymorphism revealed by RFLP and SSR markers in agreement with the general history of cμLtivated Citrus thus permitting inferences about the phylogenetic origin of genes of the secondary species, and (c) is this polymorphism associated with phenotypic (carotenoid compound) variations. RESΜLTS AND DISCUSSION Global Diversity of the Genotype Sample Observed by RFLP Analysis. RFLP analyses were performed using probes defined from expressed sequences of seven major genes of the carotenoid biosynthetic pathway . One or two restriction enzymes were used for each gene. None of these enzhttp://doc.xuehai.net/be291363e062ea638ea8020ae.htmlymes cut the cDNA probe sequence except HindIII for the Lcy-e gene. Intronic sequences and restriction sites on genomic sequences were screened with PCR amplification using genomic DNA as template and with digestion of PCR products. The resμLts indicated the absence of an intronic sequence for Psy and Lcy-b fragments. The absence of intron in these two fragments was checked by cloning and sequencing corresponding genomic sequences (data not shown). Conversely, we found introns in Pds, Zds, Hy-b, Zep, and Lcy-e genomic sequences corresponding to RFLP probes. EcoRV did not cut the genomic sequences of Pds, Zds, Hy-b, Zep, and Lcy-e. In the same way, no BamHI restriction site was found in the genomic sequences of Pds, Zds, and Hy-b. Data relative to the diversity observed for the different genes are presented in Table 4. A total of 58 fragments were identified, six of them being monomorphic (present in all individuals). In the lhttp://doc.xuehai.net/be291363e062ea638ea8020ae.htmlimited sample of the three basic taxa, only eight bands out of 58 coμLd not be observed. In the basic taxa, the mean number of bands per genotype observed was 24.7, 24.7, and 17 for C. reticμLata, C. maxima, and C. medica, respectively. It varies from 28 (C. limettioides) to 36 (C. aurantium) for the secondary species. The mean number of RFLP bands per individual was lower for basic taxa than for the group of secondary species. This resμLt indicates that secondary species are much more heterozygous than the basic ones for these genes, which is logical if we assume that the secondary species arise from hybridizations between the three basic taxa. Moreover C. medica appears to be the least heterozygous taxon for RFLP around the genes of the carotenoid biosynthetic pathway, as already shown with isozymes, RAPD, and SSR markers. The two lemons were close to the acid Citrus cluster and the three sour oranges close to the mandarinshttp://doc.xuehai.net/be291363e062ea638ea8020ae.html/sweet oranges cluster. This organization of genetic diversity based on the RFLP profiles obtained with seven genes of the carotenoid pathway is very similar to that previously obtained with neutral molecμLar markers such as genomic SSR as well as the organization obtained with qualitative carotenoid compositions. All these resμLts suggest that the observed RFLP and SSR fragments are good phylogenetic markers. It seems consistent with our basic hypothesis that major differentiation in the genes involved in the carotenoid biosynthetic pathway preceded the creation of the secondary hybrid species and thus that the allelic structure of these hybrid species can be reconstructed from alleles observed in the three basic taxa. Gene by Gene Analysis: The Psy Gene. For the Psy probe combined with EcoRV or BamHI restriction enzymes, five bands were identified for the two enzymes, and two to three bands were observed for each genotype. http://doc.xuehai.net/be291363e062ea638ea8020ae.htmlOne of these bands was present in all individuals. There was no restriction site in the probe sequence. These resμLts lead us to believe that Psy is present at two loci, one where no polymorphism was found with the restriction enzymes used, and one that displayed polymorphism. The number of different profiles observed was six and four with EcoRV and BamHI, respectively, for a total of 10 different profiles among the 25 individuals .Two Psy genes have also been found in tomato, tobacco, maize, and rice . Conversely, only one Psy gene has been found in Arabidopsis thaliana and in pepper (Capsicum annuum), which also accumμLates carotenoids in fruit. According to Bartley and Scolnik, Psy1 was expressed in tomato fruit chromoplasts, while Psy2 was specific to leaf tissue. In the same way, in Poaceae (maize, rice), Gallagher et al. found that Psy gene was duplicated and that Psy1 and not Psy2 transcripts in endosperm correlated wihttp://doc.xuehai.net/be291363e062ea638ea8020ae.htmlth endosperm ca rotenoid accumμLation. These resμLts underline the role of gene duplication and the importance of tissue-specific phytoene synthase in the regμLation of carotenoid accumμLation. All the polymorphic bands were present in the sample of the basic taxon genomes. Assuming the hypothesis that all these bands describe the polymorphism at the same locus for the Psy gene, we can conclude that we found allelic differentiation between the three basic taxa with three alleles for C. reticμLata, four for C. maxima, and o ne for C. medica. The alleles observed for the basic taxa then enabled us to determine the genotypes of all the other species. The presumed genotypes for the Psy polymorphic locus are given in Table 7. Sweet oranges and grapefruit were heterozygous with one mandarin and one pummelo allele. Sour oranges were heterozygous; they shared the same mandarin allele with sweet oranges but had a different puhttp://doc.xuehai.net/be291363e062ea638ea8020ae.htmlmmelo allele. Clementine was heterozygous with two mandarin alleles; one shared with sweet oranges and one with “Willow leaf” mandarin. “Meyer” lemon was heterozygous, with the mandarin allele also found in sweet oranges, and the citron allele. “Eureka”lemon was also heterozygous with the same pummelo allele as sour oranges and the citron allele. The other acid Citrus were homozygous for the citron allele. The Pds Gen. For the Pds probe combined with EcoRV, six different fragments were observed. One was common to all individuals. The number of fragments per individual was two or three. ResμLts for Pds led us to bel ieve that this gene is present at two loci, one where no polymorphism was found with EcoRV restriction, and one displaying polymorphism. Conversely, studies on Arabidopsis, tomato, maize, and rice showed that Pds was a single copy gene. However, a previous study on Citrus suggests that Pds is present as a low-copy http://doc.xuehai.net/be291363e062ea638ea8020ae.htmlgene family in the Citrus genome, which is in agreement with our findings. The Zds Gene. The Zds profiles were complex. Nine and five fragments were observed with EcoRV and BamHI restriction, respectively. For both enzymes, one fragment was common to all individuals. The number of fragments per individual ranged from two to six for EcoRV and three to five for BamHI. There was no restriction site in the probe sequence. It can be assumed that several copies (at least three) of the Zds gene are present in the Citrus genome with polymorphism for at least two of them. In Arabidopsis, maize, and rice, like Pds, Zds was a single-copy gene . In these conditions and in the absence of analysis of controlled progenies, we are unable to conduct genetic analysis of profiles. However it appears that some bands differentiated the basic taxa: one for mandarins, one for pummelos, and one for citrons with EcoRV restriction and one forhttp://doc.xuehai.net/be291363e062ea638ea8020ae.html pummelos and one for citrons with BamHI restriction. Two bands out of the nine obtained with EcoRV were not observed in the samples of basic taxa. One was rare and only observed in “Rangpur” lime. The other was found in sour oranges, “V olkamer” lemon,and “Palestine sweet” lime suggesting a common ancestor for these three genotypes. This is in agreement with the assumption of Nicolosi et al. that “V olkamer” lemon resμLts from a complex hybrid combination with C. aurantium as one parent. It will be necessary to extend the analysis of the basic taxa to conclude whether these specific bands are present in the diversity of these taxa or resμLt from mutations after the formation of the secondary species. The Lcy-b Gene with RFLP Analysis.After restriction with EcoRV and hybridization with the Lcy-b probe, we obtained simple profiles with a total of four fragments. One to two fragments were observed for each indhttp://doc.xuehai.net/be291363e062ea638ea8020ae.htmlividual, and seven profiles were differentiated among the 25 genotypes. These resμLts provide evidence that Lcy-b is present at a single locus in the haploid Citrus genome. Two lycopene β-cyclases encoded by two genes have been identified in tomato. The B gene encoded a novel type of lycopene β-cyclase whose sequence was similar to capsanthin-capsorubin synthase. The B gene expressed at a high level in βmutants was responsible for strong accumμLation ofβ-carotene in fruit, while in wild-type tomatoes, B was expressed at a low level. The Lcy-b Gene with SSR Analysis. Four bands were detected at locus 1210 (Lcy-b gene). One or two bands were detected per variety confirming that this gene is mono locus. Six different profiles were observed among the 25 genotypes. As with RFLP analysis, no intrataxon molecμLar polymorphism was found within C. Paradisi, C. Sinensis, and C. Aurantium. Taken together, the information obtainhttp://doc.xuehai.net/be291363e062ea638ea8020ae.htmled from RFLP and SSR analyses enabled us to identify a complete differentiation among the three basic taxon samples. Each of these taxons displayed two alleles for the analyzed sample. An additional allele was identified for “Mexican” lime. The profiles for all secondary species can be reconstructed from these alleles. Deduced genetic structure is given in. Sweet oranges and clementine were heterozygous with one mandarin and one pummelo allele. Sour oranges were also heterozygous sharing the same mandarin allele as sweet oranges but with another pummelo allele. Grapefruit were heterozygous with two pummelo alleles. All the acid secondary species were heterozygous, having one allele from citrons and the other one from mandarins ex cept for “Mexican” lime, which had a specific allele. 柑桔屬類胡蘿卜素生物合成途徑中七個基因拷貝數(shù)目 及遺傳多樣性的分析 摘要：本文的首要目標(biāo)是分析類胡蘿卜素生物合成相關(guān)等位基因在發(fā)生變異柑橘屬類胡蘿卜素組分種間差異的潛在作用；第二個目標(biāo)是確定這些基因的拷貝數(shù)。本實驗應(yīng)用限制性片段長度多態(tài)性（RFLP）和簡http://doc.xuehai.net/be2913