【作者】 崔杰；

【导师】 张树义；

【作者基本信息】 华东师范大学 ， 生态学， 2011， 博士

【摘要】 维生素C是脊椎动物体一种不可或缺的营养成分,它能够保护机体免除氧化损伤,缺乏维生素C能够导致机体坏血病。大部分的动物能够以葡萄糖为原料经由一系列的酶催化反应合成自身所需的维生素C。在爬行动物和低等鸟类中,维生素C的合成是在肾脏中进行的；而在高等的鸟类和哺乳动物中,维生素C的合成发生在肝脏中。动物丧失维生素C合成能力的现象是很罕见的。灵长类的类人猿亚目,如人类和大猩猩,是不能够合成维生素C的,而较原始的原猴亚目,如懒猴和狐猴,仍然保留这种合成能力；豚鼠同样也不能合成维生素C,然而其它啮齿目动物如大鼠、小鼠等式能够正常合成的；另外鸟类中比较高等的雀形目也出现了维生素C合成能力丧失的现象。这些物种之所以不能合成维生素C,是因为它们的体内都缺乏一种负责维生素C合成最后一步的关键酶——古洛糖酸内酯氧化酶(GULO)。在人和豚鼠中,编码该酶的基因——GULO积累了很多有害突变,发生了假基因化。前人的研究表明,所有蝙蝠,无论是食虫的还是食果的,由于体内缺乏GULO,无法自身合成维生素C。这些文章的研究对象主要针对新大陆蝙蝠物种,并且缺乏相关基因信息的研究。因此我们设计了一系列的包括从分子克隆、蛋白质表达、酶活性鉴定以及GULO基因进化分析的实验来系统的研究蝙蝠该基因的丧失情况。研究对象包括旧大陆蝙蝠中的两种食果蝙蝠(棕果蝠和犬蝠)以及两种食虫蝙蝠(大蹄蝠和马铁菊头蝠)。结果确出乎我们的意料。我们克隆到了棕果蝠和大蹄蝠全长GULO编码基因,并且该基因在两个物种中都有正常的mRNA表达。另随后进行的蛋白质表达检测发现棕果蝠和大蹄蝠能够正常表达GULO蛋白,而犬蝠和马铁菊头蝠同样也有蛋白水平表达,但是表达量较弱。(GULO活性检测实验表明棕果蝠和大蹄蝠表达的GULO蛋白具有酶活性,而犬蝠和马铁菊头蝠却丧失了酶活性。基因进化分析显示,棕果蝠和大蹄蝠的GULO基因处于强烈的纯化选择,与其基因仍然保留功能相一致,而对于丧失GULO活性的马来大狐蝠,该基因却处于松弛选择的状态,符合其基因丧失功能的特性。接下来的大规模的蝙蝠GULO基因测序以及进化分析显示,该基因的功能丧失在蝙蝠中是以一种阶段性的加速进化方式进行的。蝙蝠的总体基因选择压力高于劳亚大陆祖先12至54倍之多。狐蝠属的基因选择压力最高,接近于中性进化,表明其基因丧失功能开始与较早的时间(可能在3百万年左右的祖先状态就已经开始了),其它蝙蝠的基因选择压力相对较低(纯化选择),表明其基因功能丧失是最近才发生的。综上所述,我们的研究结果推翻了50年来人们对于蝙蝠不能合成维生素C的错误认识,更重要的是,我们揭示了一种基因(GULO)及其功能丧失的过程,并在此基础上首次提出了一个新的概念——假基因的过程化,并且揭示了蝙蝠GULO基因的功能丧失模式——阶段性的加速进化。因此,我们的研究结果无论对于研究维生素C生物化学途径的科研工作者,还是对于研究假基因化的进化学家而言都具有重大意义。更多还原

【Abstract】 Vitamin C (Vc) (or L-ascorbic acid) is an essential nutrient for all vertebrates, which protects the body against oxidative stress. Lack of vitamin causes the disease scurvy in organism. The vast majority of animals are able to synthesize their own Vc de novo, through a sequence of enzyme-driven steps, which convert glucose to Vc. In reptiles and birds in lower orders the biosynthesis is carried out in the kidneys; in birds in higher orders and mammals it is synthesized in the livers.Loss of Vc synthesis ability is rare in animals. Among the primates that have lost the ability to synthesize Vc are humans and chimpanzees etc., which together make up one of two major primate suborders, the anthropoidea, also called haplorrhini, and the other more primitive primates (strepsirrhini) i.e. lorises and lemurs have maintained the ability to make Vc. Synthesis also does not occur in guinea pigs in the rodent family caviidae, but occurs in other rodents (rats and mice, for example). And a number of species of passerine birds also do not synthesize, but not all of the birds. For the species that do not synthesize Vc de novo, they all lack the L-gulonolactone oxidase (GULO) enzyme, which is required in the last step of Vc synthesis. A non-functional GULO gene which accumulates many harmful mutations leading to pseudogenization is present in the genomes of the guinea pigs and humans.Previous researches suggested that all bats including insect and fruit-eating species have been reported to lose Vc synthesis for lack of GULO in their bodies. However, these studies were focused on New World species and there is a lack of GULO gene information in bats. To verify the loss-of-function of GULO in bats, we designed a series of experiments covering molecular, protein, enzyme and evolutionary rate levels. Our experimental targets include four Old World species:two frugivorous bats [the Leschenault’s Rousette(Rousettus leschenaultii) and the Greater Short-nosed Fruit bat(Cynopterus sphinx)] and two insectivorous bats [the Great Roundleaf bat(Hipposideros armiger) and the Greater Horseshoe bat(Rhinolophus ferrumequinum)]. To our surprise, the GULO genes of R. leschenaultii and H. armiger are intact and have regular mRNA expressions. Western blotting ascertains that all four bat species have GULO expression, in which R. leschenaultii and H. armiger have normal expressions however C. sphinx and R. ferrumequinum have weak expressions. Interestingly, Vc activity assay verifies that R. leschenaultii and H. armiger have normal GULO activities but C. sphinx and R. ferrumequinum have no functions. Our evolutionary analysis suggests that GULO genes of R. leschenaultii and H. armiger are under strong purifying selection, correlarating with their gene function, but for the Large Flying Fox(Pteropus vampyrus) (which loses Vc synthesis ability) its gene has been subjected to relaxed selection, which correlates with its loss-of-function.The following large-scale sequencing of bat GULO genes and the evolutionary analyses suggest that loss-of-function of GULO in bats follows the pattern of step-wise accelerated evolution. The whole evolutionary rates of bats are 12 to 54-fold higher than the ancestor of Laurasiatheria species. The GULO of Pteropus bats have the highest evolutionar rate and is close to be neutral evolution, which suggests an early time for their loss-of-function (probably started at around 3 million years ago). Other bat genes exhibit relative low evolutionary rates, suggesting their loss occurred recently.In summary, our results change a 50 year long-hold concept that bats cannot synthesize Vc and most importantly uncover the process of gene pseudogenization and come up with an new concept in evolutionary biology-progressive pseudogenization and uncover the pattern of loss-of-function-step-wise accelerated evolution, which will be of interest not only to scientists working on the biochemical pathways associated with Vc synthesis but also to evolutionary biologist in general who are interested in how adaptations may be lost over time.更多还原