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生物科學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 SevenGeneThe 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-32PdCTP 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 copies 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 molecLar 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 resLts 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 accumLation 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 INTRODUCTIONCarotenoids 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 horticLtural 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 derivedcarotenoid, 15-cis-phytoene. Phytoene undergoes four desaturation reactions catalyzed by two enzymes, phytoene desaturase (PDS) and -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 -carotene (, -carotene) and the other to-carotene (, -carotene). Lycopene -cyclase (LCY-b) then converts lycopene into-carotene in two steps, whereas the formation of -carotene requires the action of two enzymes, lycopene - cyclase (LCY-e) and lycopene -cyclase (LCY-b). - carotene is converted into lutein by hydroxylations catalyzed by -carotene hydroxylase (HY-e) and-carotene hydroxylase (HY-b). Other xanthophylls are produced from-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 regLation 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, particLarly on growing 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 accumLated high levels of -cryptoxanthin and violaxanthin, respectively, whereas mature lemon accumLated extremely low levels of carotenoids. Goodner et al. demonstrated that mandarins, oranges, and their hybrids coLd be clearly distinguished by their -cryptoxanthin contents. Juices of red grapefruit contained two major carotenoids: lycopene and -carotene. More recently, we conducted a broad study on the organization of the variability of carotenoid contents in different cLtivated 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; (3) 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 -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 molecLar 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 cLtivated 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 resLting from the history of cLtivated Citrus. Thus from numerical taxonomy based on morphological traits or from analysis of molecLar markers , all authors agreed on the existence of three basic taxa (C. reticLata, mandarins; C. medica, citrons; and C. maxima, pummelos) whose differentiation was the resLt of allopatric evolution. All other cLtivated Citr us species (C. sinensis, sweet oranges; C. aurantium, sour oranges; C. paradisi, grapefruit; and C. limon, lemons) resLted from hybridization events within this basic pool except for C. aurantifolia, which may be a hybrid between C. medica and C. micrantha .Our previous resLts 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 resLted in the creation of secondary speci es. Such molecLar 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 biosynthetic 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 mLtilocus, (b) is the polymorphism revealed by RFLP and SSR markers in agreement with the general history of cLtivated 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.RESLTS AND DISCUSSIONGlobal 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 enzymes cut the cDNA probe sequence except HindIII for the Lcy-e gene. Intronic sequences and restriction sites on genomic sequences werescreened with PCR amplification using genomic DNA as template and with digestion of PCR products. The resLts 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 limited sample of the three basic taxa, only eight bands out of 58 coLd not be observed. In the basic taxa, the mean number of bands per genotype observed was 24.7, 24.7, and 17 for C. reticLata, 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 resLt 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 mandarins/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 molecLar markers such as genomic SSR as well as the organization obtained with qualitative carotenoid compositions. All these resLts 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. One of these bands was present in all individuals. There was no restriction site in the probe sequence. These resLts 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 accumLates 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 notPsy2 transcripts in endosperm correlated with endosperm ca rotenoid accumLation. These resLts underline the role of gene duplication and the importance of tissue-specific phytoene synthase in the regLation of carotenoid accumLation.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. reticLata, 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 pummelo 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. ResLts 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 gene 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 for 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 resLts from a complex hybrid combination with C. aurantium as one parent. It will benecessary to extend the analysis of the basic taxa to conclude whether these specific bands are present in the diversity of these taxa or resLt 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 individual, and seven profiles were differentiated among the 25 genotypes. These resLts provide evidence that Lcy-b is present at a single locus in the haploid Citrus genome. Two lycopene -cyclases encoded by two genes have been identified in tomato. The B gene encoded a novel type of lycopene -cyclase whose sequence was similar to capsanthin-capsorubin synthase. The B gene expressed at a high level in mutants was responsible for strong accumLation of-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 molecLar polymorphism was found within C. Paradisi, C. Sinensis, and C. Aurantium.Taken together, the information obtained 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:本文的首要目標是分析類胡蘿卜素生物合成相關等位基因在發(fā)生變異柑橘屬類胡蘿卜素組分種間差異的潛在作用;第二個目標是確定這些基因的拷貝數(shù)。本實驗應用限制性片段長度多態(tài)性(RFLP)和簡單序列重復(SSR)標記法對類胡蘿卜素生物合成途徑中的七個基因進行了分析。用R-32PdCTP標記PSY,PDS,ZDS, LCY-b,LCY-e,HY-b和ZEP cDNA片段作為作探針,使用若干限制性內(nèi)切酶對來自25種柑桔基因型基因組DNA的限制性片段長度差異進行了分析。而對于SSR標記,設計兩對引物分別擴增LCY-b和HY-b基因的表達序列標簽(ESTs)。在這7個基因中, LCY-b只有1個拷貝,而ZDS存在3個拷貝。利用RFLP和SSR分析發(fā)現(xiàn)基因的遺傳多樣性與核心分子標記一致。RFLP和SSR對PSY1,PDS1,LCY-b和LCY-e14個基因的分析結果足以解釋這幾個主要的商業(yè)栽培種的系統(tǒng)樹起源。此外,我們的分析結果表明,不同種類柑橘中類胡蘿卜素積累的番茄紅素環(huán)化酶LCY-b和LCY-e1等位基因存在種間差異。前人報道PSY,HY-b和ZEP基因與種間類胡蘿卜素含量差異密切相關,但本實驗發(fā)現(xiàn)這些等位基因并不起關鍵作用。關鍵詞:柑桔;類胡蘿卜素;生物合成基因;基因變異;系統(tǒng)發(fā)育前言類胡蘿卜素是植物光合組織中普遍存在的一類色素。在色素蛋白復合體中,它們作為光敏元件進行光合作用,并且防止過強光照強度引起的灼傷,并在園藝作物果實,根,或塊莖色澤和營養(yǎng)品質(zhì)上起著十分重要的作用。事實上,其中一些微量營養(yǎng)素是維生素A的前體,是人類和動物的飲食必不可少的組成部分。由于具有抗氧化性,類胡蘿卜素在預防慢性疾病也發(fā)揮著重要的作用。類胡蘿卜素生物合成途徑現(xiàn)在已經(jīng)明確。類胡蘿卜素通過核酸編碼的蛋白酶在質(zhì)體中合成。其直接前體是牻牛兒基牻牛兒基焦磷酸(GGPP,該前體同時也是赤霉素,質(zhì)體醌,葉綠素,維生素K,維生素E的前體)。在光合植物中,GGPP主要來源于2-C-甲基-D-赤藻糖醇-4-磷酸(MEP)途徑,兩分子的GGPP經(jīng)八氫番茄紅素合成酶(PSY)催化縮合形成一個八氫番茄紅素15-順式-八氫番茄紅素。八氫番茄紅素經(jīng)八氫番茄紅素脫氫酶(PDS)和-胡蘿卜素脫氫酶(ZDS)催化八氫番茄紅素轉換成紅色的聚- 順式-番茄紅素。最近,Isaacson等和Park等分別從番茄和擬南芥中分離編碼類胡蘿卜素異構(CRTISO)基因,該基因催化異構化聚順式-胡蘿卜素進入全反式類胡蘿卜素。CRTISO作用于番茄紅素前體環(huán)化反應形成一種全反式番茄紅素。植物番茄紅素的環(huán)化有兩條途徑:一個分支合成-胡蘿卜素,另一個分支合成-胡蘿卜素。番茄紅素環(huán)化酶(LCY-b)通過兩個步驟轉換成-胡蘿卜素,而形成的-胡蘿卜素的過程需要兩種酶,番茄紅素-環(huán)化酶(LCY-e)和番茄紅素環(huán)化酶(LCY-b)。-胡蘿卜素經(jīng)-胡蘿卜素羥化酶(HY-e)和-胡蘿卜素羥化酶(HY-b)的羧基化催化作用轉化為葉黃素。-胡蘿卜素經(jīng)HY-b的羥化反應催化和玉米黃質(zhì)環(huán)氧化酶(ZEP)環(huán)氧化催化作用合成其他葉黃素。到目前為止,柑橘中大多數(shù)的類胡蘿卜素生物合成的基因已被克隆和