生物科學(xué)論文外文翻譯

1、浙江師范大學(xué)本科畢業(yè)設(shè)計(jì)(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(gè)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(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(gè)目標(biāo)是確定這些基因的拷貝數(shù)。本實(shí)驗(yàn)應(yīng)用限制性片段長(zhǎng)度多態(tài)性（rflp）和簡(jiǎn)單序列重復(fù)（ssr）標(biāo)記法對(duì)類胡蘿卜素生物合成途徑中的七個(gè)基因進(jìn)行了分析。用r-32pdctp標(biāo)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(nèi)切酶對(duì)來(lái)自25種柑桔基因型基因組dna的限制性片段長(zhǎng)度差異進(jìn)行了分析。而對(duì)于ssr標(biāo)記，設(shè)計(jì)兩對(duì)引物分別擴(kuò)增lcy-b和hy-b基因的表達(dá)序列標(biāo)簽（ests）。在這7個(gè)基因中，lcy-b只有1個(gè)拷貝，而zds存在3個(gè)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(biāo)記一致。rflp和ssr對(duì)psy1，pds1，lcy-b和lcy-e14個(gè)基因的分析結(jié)果足以解釋這幾個(gè)主要的商業(yè)栽培種的系統(tǒng)樹起源。此外，我們的分析結(jié)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(bào)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(guān)，但本實(shí)驗(yàn)發(fā)現(xiàn)這些等位基因并不起關(guān)鍵作用。關(guān)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(fù)合體中，它們作為光敏元件進(jìn)行光合作用，并且防止過(guò)強(qiáng)光照強(qiáng)度引起的灼傷，并在園藝作物果實(shí)，根，或塊莖色澤和營(yíng)養(yǎng)品質(zhì)上起著十分重要的作用。事實(shí)上，其中一些微量營(yíng)養(yǎng)素是維生素a的前體，是人類和動(dòng)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(yù)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(guò)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

4、同時(shí)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(lái)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(gè)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(zhuǎn)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(gòu)（crtiso）基因，該基因催化異構(gòu)化聚順式-胡蘿卜素進(jìn)入全反式類胡蘿卜素。crtiso作用于番茄紅素前體環(huán)化反應(yīng)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(gè)分支合成b-胡蘿卜素，另一個(gè)分支合成-胡蘿卜素。番茄紅素環(huán)化酶（lcy-b）通過(guò)兩個(gè)步驟轉(zhuǎn)換成b-胡蘿卜素，而形成的b-胡蘿卜素的過(guò)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(jīng)-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(zhuǎn)化為葉黃素。b-胡蘿卜素經(jīng)hy-b的羥化反應(yīng)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測(cè)序，但對(duì)柑橘類水果類胡蘿卜素合成的復(fù)雜調(diào)控的認(rèn)識(shí)仍然十分有限的，需要進(jìn)一步了解柑橘中這些有差異的等位基因拷貝數(shù)，因?yàn)檫@

6、些基因會(huì)影響柑橘類水果中最來(lái)源豐富的類胡蘿卜素組成。果實(shí)類胡蘿卜素的結(jié)構(gòu)較為復(fù)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(zhǎng)條件和地理環(huán)境。但影響類胡蘿卜素質(zhì)量變化的主要因素是遺傳的多樣性。kato等表明中國(guó)柑橘和橙汁積累高水平的-隱黃質(zhì)和紫黃質(zhì)，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(gè)主要類胡蘿卜素：番茄紅素和胡蘿卜素。最近，我們對(duì)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(jìn)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(guó)（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(jié)構(gòu)的多樣關(guān)鍵步驟。在酸柑橘中八氫番茄紅素合成是一個(gè)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(gè)分類中（柚和葡萄柚）；只有第1組品種能夠合成紫黃質(zhì)。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(píng)價(jià)為基礎(chǔ)，我們認(rèn)為應(yīng)通過(guò)生化或分子分子標(biāo)記遺傳多樣行評(píng)估在種間一級(jí)組織中的多樣性，類胡蘿卜素的組成如同工酶或隨機(jī)擴(kuò)增多態(tài)性dna（rapd）對(duì)大量的柑桔品種進(jìn)行分類。此外，我們還得出結(jié)論認(rèn)為，在種間水平，類胡蘿卜素結(jié)構(gòu)多樣性和柑橘屬的進(jìn)化過(guò)程有關(guān)系，而不是突變事件或人為的甄選過(guò)程。事實(shí)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(guān)系具有普遍性和一般性，這和不能適應(yīng)環(huán)境的不平衡相關(guān)聯(lián)。因此，從數(shù)值分類的基礎(chǔ)上，或從形態(tài)性狀的分子標(biāo)記分析，所有的研究者均認(rèn)為：存在著三種基本分類（寬皮桔類；中國(guó)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(jié)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(jié)果和數(shù)據(jù)使我們提出這樣的假設(shè)：等位基因變異導(dǎo)致類胡蘿卜素水平的結(jié)構(gòu)差異，原因在于次級(jí)代謝產(chǎn)物的產(chǎn)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(yīng)酶的作用；另一方面，非編碼區(qū)域的變化影響轉(zhuǎn)錄或轉(zhuǎn)錄后機(jī)制。到目前為止，沒(méi)有人研究過(guò)柑桔中類胡蘿卜素生物合成途徑的等位基因多樣性。本實(shí)驗(yàn)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(yīng)用rflp分析了類胡蘿卜素生物合成途徑中的7個(gè)基因（psy，pds，zds，lcy-b，lcy-e，hy-b，zep），以及兩個(gè)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(wèn)題：（a）這7個(gè)基因是單基因還是多基因位點(diǎn)；（b）rflp法和ssr標(biāo)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(jí)產(chǎn)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(guān)聯(lián)。結(jié)果與討論本實(shí)驗(yàn)應(yīng)用rflp分析法來(lái)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(gè)主要基因的表達(dá)序列作為探針進(jìn)行rflp分析，每一個(gè)基因用一個(gè)或兩個(gè)限制性內(nèi)切酶，篩選內(nèi)含序列及酶切位點(diǎn)的基因組序列，以基因組dna為模板pcr擴(kuò)增和酶切pcr產(chǎn)物。結(jié)果表明沒(méi)有一個(gè)pds和lcy-b片段的內(nèi)含子序列。在這兩個(gè)片段克隆和序列分析相應(yīng)的基因組序列中沒(méi)有檢查出內(nèi)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(méi)切斷pds，zds，hy-b，zep和lcy-e基因組序列。以同樣的方式，沒(méi)有發(fā)現(xiàn)pds，zds和hy

11、-b的基因組序列的bamhi酶切位點(diǎn)。表4是對(duì)于不同基因多樣性觀察有關(guān)的數(shù)據(jù)?？偣?8個(gè)片段被確定，它們中的六個(gè)是單一同態(tài)的（存在于個(gè)體中）。三個(gè)基本分類單元的有限樣本中，58個(gè)之外只有8個(gè)條帶不能被觀察到。在基本分類單位，每個(gè)基因型遺傳距離的平均數(shù)分別是，寬皮橘類24.7，中國(guó)柑橘24.7，檸檬類17這與次級(jí)物種的28（酸橙類）到36（橙類）不同。每個(gè)基本的分類單元個(gè)體rflp條帶的平均數(shù)均低于次級(jí)物種類群。結(jié)果表明次級(jí)物種比基于三個(gè)基因分類的基本物種更加雜合。這是合理的如果我們假設(shè)次級(jí)物種起源于三個(gè)基本分類，此外經(jīng)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(biāo)記法所示。四種甜橘子的分析顯示所有的基因同樣的標(biāo)記，三種酸橙類的代表和三種葡萄柚也是。在接下來(lái)的研究中，次級(jí)代謝產(chǎn)物僅有一個(gè)個(gè)體參加?；蚨鄻有缘臋C(jī)體組成顯示相鄰系統(tǒng)樹以所有rflp標(biāo)記波帶的有無(wú)的片段的不同指標(biāo)為基礎(chǔ)。區(qū)分出八個(gè)不同的標(biāo)記。主要的線束被識(shí)別；第一個(gè)組是中國(guó)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(biāo)記為基礎(chǔ)的遺傳多樣性的機(jī)體組成獲得類胡蘿卜素合成途徑的七個(gè)基因和通過(guò)不定性分子組成的時(shí)標(biāo)獲得的是相似的，通過(guò)定性分子組分獲得的也是一樣。所有這些結(jié)果表明觀察rfl

13、p和ssr片段是完好的系統(tǒng)標(biāo)記。這和我們的基礎(chǔ)假說(shuō)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(jí)雜交物種的產(chǎn)生，因此在三類基礎(chǔ)分類中等位基因的構(gòu)成源于等位基因的重組。psy基因分析 因?yàn)閜sy探針和ecorv或bamhi限制性內(nèi)切酶結(jié)合，所以可以把五個(gè)染色體條帶看成是兩種酶，觀察兩或三個(gè)染色體條帶的基因型。這些條帶中的一個(gè)出現(xiàn)在所有個(gè)體中。沒(méi)有限制性酶切位點(diǎn)在探針序列中。這些結(jié)果使我們相信psy在兩個(gè)基因位點(diǎn)出現(xiàn)，一個(gè)用限制性內(nèi)切酶發(fā)現(xiàn)沒(méi)有多態(tài)性，另一個(gè)有多態(tài)性。用ecorv和bamhi不同的標(biāo)記觀察分別是六或四，總共10個(gè)不同的標(biāo)記在25個(gè)個(gè)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(gè)psy基因，在它的果實(shí)中也積累胡蘿卜素。根據(jù)bartley和scolnik的研究，psy1表達(dá)在番茄果實(shí)的色素細(xì)胞中，psy2特殊在它在葉片組織中表達(dá)。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(fù)制出來(lái)的，在胚乳中psy1而并非是psy2轉(zhuǎn)錄產(chǎn)物與胚乳類胡蘿卜素積累有關(guān)。這些結(jié)果強(qiáng)調(diào)基因復(fù)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(diào)控中的重要性。所有的多態(tài)性條帶出現(xiàn)在基礎(chǔ)分類群的基因組。假定該假說(shuō)，在相同的基因位點(diǎn)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(chǔ)分類

15、，柑三個(gè)等位基因，中國(guó)柑橘四個(gè)等位基因，檸檬類一個(gè)等位基因。觀察三類基礎(chǔ)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(diǎn)推測(cè)的基因型。甜橘和葡萄柚是中國(guó)柑橘和甜橙的雜合。四種酸橙是雜合的；它們和中國(guó)柑橘共用相同的等位基因但和柚有一個(gè)不同的等位基因?？巳R門氏小柑橘是兩種中國(guó)柑橘（柑橘和酸柑）等位基因的雜合；一個(gè)與甜橙共用，而一個(gè)與柳橙共用?！癿eyer”檸檬是雜合的，中國(guó)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(jié)合，觀察到六個(gè)不同的片段。一個(gè)

16、為所有個(gè)體所共有。每個(gè)個(gè)體的片段數(shù)量是兩或三個(gè)。這些結(jié)果使我們相psy在兩個(gè)基因位點(diǎn)出現(xiàn)，用限制性內(nèi)切酶發(fā)現(xiàn)一個(gè)沒(méi)有多態(tài)性，另一個(gè)有多態(tài)性。相反地，對(duì)擬南芥，番茄，玉米和水稻的研究顯示pds是一個(gè)單獨(dú)的拷貝基因。然而，前人對(duì)柑橘屬的研究表明pds基因作為一個(gè)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(biāo)記是復(fù)合體。通過(guò)ecorv和bamhi限制可以分別觀察到九和五個(gè)片段。這兩種酶中的一個(gè)片段是所有個(gè)體都有。對(duì)于ecorv每個(gè)單獨(dú)個(gè)體基因片段的數(shù)量從二變到三，對(duì)于bamhi從三變到五。沒(méi)有限制性酶切位點(diǎn)在探針序列。假定zds基因的幾個(gè)拷貝（至少三個(gè)）出現(xiàn)于柑橘屬基因

17、組中，至少它們中的兩個(gè)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(biāo)記。然而它看起來(lái)好像一些條帶區(qū)分三類基礎(chǔ)分類：一條是中國(guó)柑橘的，一條是柚的，一條是通過(guò)ecorv限制性內(nèi)切的柚和bamhi限制酶切的檸檬。經(jīng)ecorv基礎(chǔ)分類的樣品中九個(gè)之外又兩個(gè)沒(méi)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(gè)祖先。這與nicolosi等的設(shè)想相符?！皏olkamer”檸檬起源于以橙作為親本的雜種結(jié)合。加大對(duì)三種基礎(chǔ)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(jí)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(gè)片段的全部獲得簡(jiǎn)單的標(biāo)記。每個(gè)個(gè)體觀察一到兩個(gè)片段，在25個(gè)基因型中觀察到7個(gè)條帶。這些結(jié)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(diǎn)提供依據(jù)。在番茄中已經(jīng)識(shí)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(gè)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(shí)中b基因表達(dá)高水平的突變體對(duì)于強(qiáng)大的b-胡蘿卜素積累而在野生型番茄中b表達(dá)水平較低。ssr法分析lcy-b基因 通過(guò)引物1210（lcy-b基因）分析發(fā)現(xiàn)四個(gè)條帶

19、。每個(gè)品種發(fā)現(xiàn)一或兩個(gè)條帶這證實(shí)基因是單一位點(diǎn)。在25個(gè)基因型中有六個(gè)標(biāo)記。與rflp一樣，在酸橘類，甜橘類，和酸橙類中沒(méi)有發(fā)現(xiàn)內(nèi)部分類群分子的多態(tài)性。總之，通過(guò)rflp和ssr法的分析獲得的信息使我們確定在三類基礎(chǔ)分類樣品中存在完全的變異。分析樣本每一個(gè)類群顯示兩個(gè)基因位點(diǎn)。一個(gè)額外的為墨西哥酸橙。所有次級(jí)物種的標(biāo)記可以改造于其他等位基因。推動(dòng)遺傳結(jié)構(gòu)的得出。甜橙和克萊門氏小柑橘是中國(guó)柑橘和柚等位基因的雜合。酸橙也是中國(guó)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(jí)物種都是雜合的，一個(gè)等位基因來(lái)自檸檬另外一個(gè)來(lái)自中國(guó)柑橘除了墨西哥柚，它有一個(gè)特殊的基

20、因位點(diǎn)。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