生物科學論文外文翻譯

1、浙江師范大學本科畢業(yè)設計(論文)外文翻譯譯文一：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journal of agricltural and food chemistry. 2007, 55(18): 74057417.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 cdn

2、a片段作為作探針，使用若干限制性內切酶對來自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

3、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)在已經明確。類胡蘿卜素通過核酸編碼的蛋白酶在質體中合成。其直接前體是牻牛兒基牻牛兒基焦磷酸（ggpp，該前體

4、同時也是赤霉素，質體醌，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

5、兩條途徑：一個分支合成b-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b-胡蘿卜素，而形成的b-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b環(huán)化酶（lcy-b）。-胡蘿卜素經-胡蘿卜素羥化酶（hy-e）和b-胡蘿卜素羥化酶（hy-b）的羧基化催化作用轉化為葉黃素。b-胡蘿卜素經hy-b的羥化反應催化和玉米黃質環(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測序，但對柑橘類水果類胡蘿卜素合成的復雜調控的認識仍然十分有限的，需要進一步了解柑橘中這些有差異的等位基因拷貝數(shù)，因為這

6、些基因會影響柑橘類水果中最來源豐富的類胡蘿卜素組成。果實類胡蘿卜素的結構較為復雜，在柑橘類水果中已查明有115種不同的類胡蘿卜素。柑橘果肉類胡蘿卜素豐富程度取決于環(huán)境條件，特別是生長條件和地理環(huán)境。但影響類胡蘿卜素質量變化的主要因素是遺傳的多樣性。kato等表明中國柑橘和橙汁積累高水平的-隱黃質和紫黃質，成熟的檸檬積累低水平的類胡蘿卜素。goodnr等發(fā)現(xiàn)紅西柚汁含有兩個主要類胡蘿卜素：番茄紅素和胡蘿卜素。最近，我們對栽培變異柑桔品種類胡蘿卜素組分含量不同從生物合成途徑上進行了廣泛的研究。根據(jù)是否含有不同的化合物將其分成三類：（1）中國（普通）柑桔，甜柑，酸橘；（2）蜜餞，檸檬，和酸橙；（3）

7、柚和葡萄柚。我們的研究也明確了致使類胡蘿卜素結構的多樣關鍵步驟。在酸柑橘中八氫番茄紅素合成是一個限制步驟；番茄紅素形成-胡蘿卜素和-胡蘿卜素是被酸式磷酸鹽限制在第三個分類中（柚和葡萄柚）；只有第1組品種能夠合成紫黃質。在同一研究中，以是否存在的類胡蘿卜素（以下這種分類也被稱為類胡蘿卜素組織多樣性）和遺傳多樣性評價為基礎，我們認為應通過生化或分子分子標記遺傳多樣行評估在種間一級組織中的多樣性，類胡蘿卜素的組成如同工酶或隨機擴增多態(tài)性dna（rapd）對大量的柑桔品種進行分類。此外，我們還得出結論認為，在種間水平，類胡蘿卜素結構多樣性和柑橘屬的進化過程有關系，而不是突變事件或人為的甄選過程。事實上

8、，在種間水平，在柑桔栽培歷史上，表型變異和遺傳多樣性的關系具有普遍性和一般性，這和不能適應環(huán)境的不平衡相關聯(lián)。因此，從數(shù)值分類的基礎上，或從形態(tài)性狀的分子標記分析，所有的研究者均認為：存在著三種基本分類（寬皮桔類；中國柑桔；枸櫞，蜜餞；文旦，柚)，其差異是由于異域的演變。其他種植柑桔的品種（甜橙類，甜桔；酸橙類，酸桔；柑橘屬葡萄柚，葡萄柚；檸檬類，檸檬）是雜交的結果，而酸橙類則可能是佛手柚和薇甘菊的雜交種。先前研究柑桔演變的結果和數(shù)據(jù)使我們提出這樣的假設：等位基因變異導致類胡蘿卜素水平的結構差異，原因在于次級代謝產物的產生。這種分子變異可能有兩種不同的影響：一方面，非沉默替換編碼區(qū)影響生物合成

9、途徑相應酶的作用；另一方面，非編碼區(qū)域的變化影響轉錄或轉錄后機制。到目前為止，沒有人研究過柑桔中類胡蘿卜素生物合成途徑的等位基因多樣性。本實驗的目的是為了研究基因變異是否部分決定種間水平表型變異性。為此，我們應用rflp分析了類胡蘿卜素生物合成途徑中的7個基因（psy，pds，zds，lcy-b，lcy-e，hy-b，zep），以及兩個ssr序列分析一批有代表性品種的lcy-b和hy-b基因，旨在解決下列問題：（a）這7個基因是單基因還是多基因位點；（b）rflp法和ssr標記法顯示的差異性與栽培柑桔的記錄一致，從而可以推論次級產物基因系統(tǒng)發(fā)生的起源。（c）多樣性與表型的（類胡蘿卜素化合物）變

10、化相關聯(lián)。結果與討論本實驗應用rflp分析法來觀察基因型樣本的整體差異。用類胡蘿卜素生物合成途徑中的7個主要基因的表達序列作為探針進行rflp分析，每一個基因用一個或兩個限制性內切酶，篩選內含序列及酶切位點的基因組序列，以基因組dna為模板pcr擴增和酶切pcr產物。結果表明沒有一個pds和lcy-b片段的內含子序列。在這兩個片段克隆和序列分析相應的基因組序列中沒有檢查出內含序列（數(shù)據(jù)未顯示）。相反，我們發(fā)現(xiàn)pds，zds，hy-b，zep和lcy-e基因組含有rflp探針序列。ecorv并沒切斷pds，zds，hy-b，zep和lcy-e基因組序列。以同樣的方式，沒有發(fā)現(xiàn)pds，zds和hy

11、-b的基因組序列的bamhi酶切位點。表4是對于不同基因多樣性觀察有關的數(shù)據(jù)。總共58個片段被確定，它們中的六個是單一同態(tài)的（存在于個體中）。三個基本分類單元的有限樣本中，58個之外只有8個條帶不能被觀察到。在基本分類單位，每個基因型遺傳距離的平均數(shù)分別是，寬皮橘類24.7，中國柑橘24.7，檸檬類17這與次級物種的28（酸橙類）到36（橙類）不同。每個基本的分類單元個體rflp條帶的平均數(shù)均低于次級物種類群。結果表明次級物種比基于三個基因分類的基本物種更加雜合。這是合理的如果我們假設次級物種起源于三個基本分類，此外經rflp圍繞類胡蘿卜素生物合成途徑的基因分析檸檬類好像是最雜合程度最小的分類

12、單元。如同功酶，rapd，ssr標記法所示。四種甜橘子的分析顯示所有的基因同樣的標記，三種酸橙類的代表和三種葡萄柚也是。在接下來的研究中，次級代謝產物僅有一個個體參加。基因多樣性的機體組成顯示相鄰系統(tǒng)樹以所有rflp標記波帶的有無的片段的不同指標為基礎。區(qū)分出八個不同的標記。主要的線束被識別；第一個組是中國柑橘和甜橘，第二文旦和葡萄柚，第三檸檬和酸的柑橘屬的多數(shù)。兩種檸檬接近酸柑橘屬的線束而三種酸橘子接近橘子或甜橙的線束。以rflp標記為基礎的遺傳多樣性的機體組成獲得類胡蘿卜素合成途徑的七個基因和通過不定性分子組成的時標獲得的是相似的，通過定性分子組分獲得的也是一樣。所有這些結果表明觀察rfl

13、p和ssr片段是完好的系統(tǒng)標記。這和我們的基礎假說相似，主要的區(qū)別在于涉及類胡蘿卜素生物合成途徑基因先于次級雜交物種的產生，因此在三類基礎分類中等位基因的構成源于等位基因的重組。psy基因分析 因為psy探針和ecorv或bamhi限制性內切酶結合，所以可以把五個染色體條帶看成是兩種酶，觀察兩或三個染色體條帶的基因型。這些條帶中的一個出現(xiàn)在所有個體中。沒有限制性酶切位點在探針序列中。這些結果使我們相信psy在兩個基因位點出現(xiàn)，一個用限制性內切酶發(fā)現(xiàn)沒有多態(tài)性，另一個有多態(tài)性。用ecorv和bamhi不同的標記觀察分別是六或四，總共10個不同的標記在25個個體中。兩種psy基因也在番茄，煙草，玉

14、米，水稻中被發(fā)現(xiàn)。相反地，在擬南芥和在胡椒中僅僅發(fā)現(xiàn)一個psy基因，在它的果實中也積累胡蘿卜素。根據(jù)bartley和scolnik的研究，psy1表達在番茄果實的色素細胞中，psy2特殊在它在葉片組織中表達。同樣的方法，在禾本科(玉米，水稻)中，gallagher等發(fā)現(xiàn)psy基因是被復制出來的，在胚乳中psy1而并非是psy2轉錄產物與胚乳類胡蘿卜素積累有關。這些結果強調基因復制的作用和特有組織的八氫番茄紅色合酶在類胡蘿卜素積累的調控中的重要性。所有的多態(tài)性條帶出現(xiàn)在基礎分類群的基因組。假定該假說，在相同的基因位點為psy基因所有的條帶描述多態(tài)性，我們能斷定我們發(fā)現(xiàn)等位基因的區(qū)別在三類基礎分類

15、，柑三個等位基因，中國柑橘四個等位基因，檸檬類一個等位基因。觀察三類基礎分類的所有等位基因，然后我們能確定所有物種基因型。表七中給出psy基因多態(tài)性位點推測的基因型。甜橘和葡萄柚是中國柑橘和甜橙的雜合。四種酸橙是雜合的；它們和中國柑橘共用相同的等位基因但和柚有一個不同的等位基因?？巳R門氏小柑橘是兩種中國柑橘（柑橘和酸柑）等位基因的雜合；一個與甜橙共用，而一個與柳橙共用?！癿eyer”檸檬是雜合的，中國柑橘的等位基因也在甜橙和檸檬中被發(fā)現(xiàn)，“eureka”檸檬也是柚四種酸橙等位基因和檸檬等位基因的雜合。其它的酸柑橘屬是純合的。 pds基因 將ecorv與pds探針結合，觀察到六個不同的片段。一個

16、為所有個體所共有。每個個體的片段數(shù)量是兩或三個。這些結果使我們相psy在兩個基因位點出現(xiàn)，用限制性內切酶發(fā)現(xiàn)一個沒有多態(tài)性，另一個有多態(tài)性。相反地，對擬南芥，番茄，玉米和水稻的研究顯示pds是一個單獨的拷貝基因。然而，前人對柑橘屬的研究表明pds基因作為一個低拷貝的基因家族在柑橘屬基因組中，這與我們的發(fā)現(xiàn)相矛盾。zds基因 zds標記是復合體。通過ecorv和bamhi限制可以分別觀察到九和五個片段。這兩種酶中的一個片段是所有個體都有。對于ecorv每個單獨個體基因片段的數(shù)量從二變到三，對于bamhi從三變到五。沒有限制性酶切位點在探針序列。假定zds基因的幾個拷貝（至少三個）出現(xiàn)于柑橘屬基因

17、組中，至少它們中的兩個有多態(tài)性。在擬南芥，玉米和水稻中，pds，zds是單拷貝的基因。在這些條件下和缺乏可控后代分析的情況下，我們不能處理基因遺傳分析的標記。然而它看起來好像一些條帶區(qū)分三類基礎分類：一條是中國柑橘的，一條是柚的，一條是通過ecorv限制性內切的柚和bamhi限制酶切的檸檬。經ecorv基礎分類的樣品中九個之外又兩個沒有被觀察到。僅僅在“rangpur”酸橙中才能觀察到一條比較薄的。其他的被發(fā)現(xiàn)酸橙，“volkamer”檸檬，巴基斯坦甜橙暗示這三種基因型有同一個祖先。這與nicolosi等的設想相符?！皏olkamer”檸檬起源于以橙作為親本的雜種結合。加大對三種基礎分類的分析

18、是必需的，總之這些特殊的條帶出現(xiàn)在分類中或者源于次級物種形成后的突變。 rflp法分析lcy-b基因 在ecorv限制之后，和lcy-b探針雜交，我們用四個片段的全部獲得簡單的標記。每個個體觀察一到兩個片段，在25個基因型中觀察到7個條帶。這些結果為在柑橘屬單倍體基因組中l(wèi)cy-b出現(xiàn)在單一的位點提供依據(jù)。在番茄中已經識別兩種編碼番茄紅素柚b-環(huán)化酶的基因。b基因編碼一個新形式的番茄紅素b-環(huán)化酶它的序列和辣椒玉紅素合酶的相似。在果實中b基因表達高水平的突變體對于強大的b-胡蘿卜素積累而在野生型番茄中b表達水平較低。ssr法分析lcy-b基因 通過引物1210（lcy-b基因）分析發(fā)現(xiàn)四個條帶

19、。每個品種發(fā)現(xiàn)一或兩個條帶這證實基因是單一位點。在25個基因型中有六個標記。與rflp一樣，在酸橘類，甜橘類，和酸橙類中沒有發(fā)現(xiàn)內部分類群分子的多態(tài)性?？傊ㄟ^rflp和ssr法的分析獲得的信息使我們確定在三類基礎分類樣品中存在完全的變異。分析樣本每一個類群顯示兩個基因位點。一個額外的為墨西哥酸橙。所有次級物種的標記可以改造于其他等位基因。推動遺傳結構的得出。甜橙和克萊門氏小柑橘是中國柑橘和柚等位基因的雜合。酸橙也是中國柑橘和甜橘類雜合的，但是和另一種柚的等位基因。葡萄柚是兩種柚等位基因的雜合。所有酸的次級物種都是雜合的，一個等位基因來自檸檬另外一個來自中國柑橘除了墨西哥柚，它有一個特殊的基

20、因位點。原文一：carotenoid biosynthetic pathway in the citrus genus: number of copies and phylogenetic diversity of seven genejournal of agricltural and food chemistry.2007, 55(18)：74057417the first objective of this paper was to analyze the potential role of allelic variability of carotenoid biosynthetic g

21、enes 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)

22、 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 sequenc

23、es 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 molec

24、lar 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

25、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 linke

26、d to specific alleles at the corresponding loci.keywords: citrus; carotenoids; biosynthetic genes; allelic variability; phylogenyintroductioncarotenoids are pigments common to all photosynthetic organisms. in pigment-protein complexes, they act as light sensors for photosynthesis but also prevent ph

27、oto-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

28、 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 gib

29、berellins, plastoquinone, chlorophylls,phylloquinones, and tocopherols) is geranylgeranyl diphosphate (ggpp). in light-grown plants, ggpp is mainly derived from the methylerythritol phosphate (mep) pathway). the condensation of two molecles of ggpp catalyzed by phytoene synthase (psy) leads to the f

30、irst colorless carotenoid, 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

31、 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 o

32、f 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

33、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

34、(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 d

35、iversity 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 ric

36、hness 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 accumla

37、ted 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 conta

38、ined 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 c

39、ompounds 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 f

40、or 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 classificat

41、ion 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 al

42、so 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 phe

43、notypic 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 ana

44、lysis 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 citrus species (c. sinensis, sweet oranges; c. aurantium, sour o

45、ranges; 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 va

46、riability supporting the organization of carotenoid diversity at interspecific level preceded events that reslted in the creation of secondary species. such moleclar variability may have two different effects: on the one hand, non-silent substitutions in coding region affect the specific activity of

47、 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 pa

48、per 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 polymo

49、rphism 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

50、 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 genotyp

51、e 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 th

52、e 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 reslts indicated the absence of an intronic sequence for psy and lcy-b fragments. the absence of intron in these two

53、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,

54、 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

55、 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 specie

56、s. 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 hybridizat

57、ions 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 clo

58、se 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 qua

59、litative 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.g