1、生物科学论文中英文资料外文翻译文献中英文对照翻译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
2、 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
3、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
4、) 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
5、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
6、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.
7、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 inten
8、sities. 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 (suc
9、h 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,phylloqu
10、inones, 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
11、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
12、 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
13、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 hyd
14、roxylation 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.
15、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
16、 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 s
17、hown 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 b
18、e 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 t
19、he 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 divers
20、ification 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 sa
21、me 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 o
22、r 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
23、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 c
24、Ltivated 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 evolutio
25、n. 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 r
26、esLts 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: o
27、n 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 d
28、iversity 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 bi
29、osynthetic 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:
30、 (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 phenoty
31、pic (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 us
32、ed 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
33、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. Eco
34、RV 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 identifi
35、ed, 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, respect
36、ively. 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 the
37、se 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 mar
38、kers.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 ne
39、utral 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 g
40、enes 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 wi
41、th 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 a
42、t 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 als
43、o 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 speci
44、fic 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-sp
45、ecific 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 differ
46、entiation 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
47、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
48、 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 cit
49、ron 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
