【作者】 李中媛；

【导师】 姚斌；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2013， 博士

【摘要】 碳元素和磷元素是生物体维持正常生命活动的必需元素，植食性动物体内的这些元素主要从植物中摄取。植物中的碳和磷主要是以木质纤维素和植酸的形式存在，但是它们很难被单胃动物直接利用，而反刍动物则可以有效利用植物中的木质纤维素和植酸。这主要是因为反刍动物的瘤胃是一个天然的高效生物反应器，它富含的大量真菌、细菌和原虫等微生物可以分泌多种降解酶。目前人们采用宏基因组测序等方法对瘤胃环境微生物及其降解酶进行了很多研究，已经发现大量的糖苷水解酶和其他有价值的酶类，但是目前对瘤胃环境中发挥作用降解酶的研究还很少。本文旨在研究饲喂周期内反刍动物瘤胃环境GH10木聚糖酶和CP植酸酶的转录水平基因多样性及其转录变化情况，发掘出具有潜在应用价值的GH10木聚糖酶和CP植酸酶，并为进一步开发瘤胃环境基因资源提供有利的理论依据，本文以绵羊瘤胃为研究对象，对饲喂周期内瘤胃环境的pH、细菌数量和相关酶活性的变化进行监测，结果表明瘤胃环境的pH和木聚糖酶活性的变化趋势大体一致，饲喂后开始下降，4小时降至最低点，然后开始缓慢上升，饲喂后24小时达到饲喂前水平；微生物数量的变化趋势稍有不同，呈现在饲喂后12小时达到最高，然后开始缓慢下降，饲喂后24小时达到饲喂前水平的变化趋势，这些参数的变化说明了瘤胃环境在饲喂周期内具有一定的动态性。根据瘤胃环境的变化趋势，构建糖苷水解酶第十家族（GH10）木聚糖酶在4个（饲喂后0、4、9和16小时）cDNA和1个（饲喂后16小时）DNA基因片段克隆库，共获得44条不同的GH10木聚糖酶基因片段，它们与已知的GH10木聚糖酶序列一致性在56–90%之间。通过比较饲喂后16小时cDNA和DNA基因片段克隆库的木聚糖酶基因，结果发现只有9个基因是两个克隆库的共有基因，表明基因水平和转录水平的GH10木聚糖酶基因组成存在很大差异，说明转录水平的研究方法更能有效的发掘出真正发挥功能的GH10木聚糖酶基因。然后通过比较饲喂后不同时间点cDNA克隆库的木聚糖酶基因，结果发现只有6个基因是4个cDNA克隆库的共有基因，说明在饲喂周期内随着时间的推移，得到转录的木聚糖酶基因组成也存在非常显著的变化；进一步采用qPCR定量方法对这6个共有木聚糖酶基因进行绝对定量分析，结果表明在基因水平上它们的变化趋势基本一致，但是在转录水平上这些共有基因则呈现多样的变化趋势，从而反映了瘤胃环境木聚糖酶的转录具有复杂的动态性；此外，相关性分析表明木聚糖酶的转录与环境因素（pH、细菌数量和木聚糖酶活性）呈显著相关性。构建瘤胃环境半胱氨酸植酸酶（CP）的4个（饲喂后0、4、9和16小时）cDNA和1个（饲喂后9小时）的DNA基因片段克隆库，共获得46条不同的CP植酸酶序列，这些序列与已知CP植酸酶序列的一致性在40–96%之间。通过比较饲喂后9小时cDNA克隆库和DNA克隆库的CP植酸酶基因，结果发现两个克隆库的基因组成有很大差异，说明采用cDNA克隆库的方法可以更好地发掘功能性基因。同时，对4个时间点cDNA克隆库的植酸酶基因进行比较，结果表明同GH10木聚糖酶一样，饲喂周期内CP植酸酶的基因组成差异很大，并且不同基因的转录趋势呈现多样性。根据已获得的大量GH10木聚糖酶和CP植酸酶基因片段，从瘤胃环境中直接克隆得到10个新颖的GH10木聚糖酶基因和4个CP植酸酶基因，其中木聚糖酶XynB和XynC在大肠杆菌中进行表达纯化后，具有明显的木聚糖酶活性，以榉木木聚糖为底物的比活力分别为73.9U/mg和142.3U/mg。二者的酶学性质相近，其最适pH都是6.0；具有很好的pH稳定性，在pH5.0–8.0和39°C条件下处理1个小时后剩余80%的相对酶活；最适温度都是40°C，在20°C还有60%的相对酶活；在40°C和最适pH条件下处理1个小时后可以分别剩余90%和86%的酶活。这两个酶的基本酶学性质跟瘤胃的生理环境（39°C，pH5.0–7.0）非常相近，推测瘤胃环境来源的木聚糖酶与瘤胃环境存在一种相互适应相互选择的关系。木聚糖酶XynA是转录水平上的高丰度基因，由407个氨基酸组成，包括一个信号肽（24aa），一个GH10木聚糖酶结构域和其C端的一段富含脯氨酸的序列（60aa）。序列比对分析表明该C端序列没有同源序列。为了探索该C端序列的功能，将XynA和XynA-Tr （C端序列截短酶）分别在大肠杆菌中进行表达，结果表明该C端序列可以提高酶的最适温度（从45°C升至50°C），和扩宽pH的作用范围（从pH5.5–6.5扩展至pH5.0–7.5）；最特别的是，它可以显著提高木聚糖酶的比活(1135U mg^–1v.s.97U mg^–1)和催化效率(133.5ml s^–1mg^–1v.s.7.3ml s^–1mg^–1)。圆二色谱仪比较二者结构差异的结果表明XynA的二级结构具有更多的α螺旋。等温滴定量微热仪测定二者与低聚寡糖亲和力的结果表明XynA与低聚寡糖有更高的亲和力，从而揭示了该C端序列提高催化效率的原因。此外，将该C端序列与另一个木聚糖酶XynB进行融合表达，结果发现它也可以提高木聚糖酶XynB的最适温度和催化效率，说明这段序列具有通用性，可以为今后改造其他木聚糖酶提供了思路。本论文通过对瘤胃环境GH10木聚糖酶和CP植酸酶的转录水平基因多样性的研究，获得了大量新颖的功能性基因资源。通过对饲喂周期内不同时间点GH10木聚糖酶和CP植酸酶基因组成的比较，表明了瘤胃环境GH10木聚糖酶和CP植酸酶基因呈现多样的转录变化趋势，从分子水平揭示了瘤胃环境降解酶的动态性和复杂性，为瘤胃环境微生物资源的开发提供有利依据。通过对代表性基因的结构与功能的研究，一方面证实了基因的功能性，另一方面为进一步改造木聚糖酶的相关性能提供了新的思路。更多还原

【Abstract】 Carbon and phosphorus are essential elements to living organisms. Herbivorous animals obtainthese elements from plant-based feed, of which lingocellulose and phytase represent the main storedforms of carbon and phosphorus, although these can not be digested by monogastric animals. Ruminantanimals harbor varieties of microorganisms in the rumen that secrete different enzymes and have theability to easily utilize these substrates. Currently, metagenomic sequencing method has been used tostudy ruminal micro-organisms leading to the identification of a great variety of glycoside hydrolasesand other enzymes. To date, there is scant information on ruminal enzymes at the transcriptional level.This study analyzes the genetic composition and expression characteristics of ruminal xylanase andphytases during the feeding cycles of ruminants in order to identify the functional genes and to providemore information on the ruminal environment.The rumen of Small Tail Han sheep was selected as the excellent microorganism source to studythe ruminal xylanases of glycoside hydrolase （GH） family10and cysteine phytases. DNA and cDNAlibraries of xylanase and phytase during a feeding cycle were constructed, and their diversity at thetranscriptional level was studied. Analysis of the rumen pH, bacterial population, and enzymatic activityduring a feeding cycle indicated that rumen pH and xylanase activity showed similar trends. The valuesdecreased with feeding for4h, then increased up to the initial level. The trend of bacterial populationwas different, showing the highest at12h and reduced then to the initial level. The results reveal thatthe rumen environment is dynamic with regular changes during feeding cycles.According to the changing rule of rumen environment, four cDNA libraries at0h,4h,9h and16hand one DNA library at16h of GH10xylanases were constructed, and44distinct gene fragments withidentities of <95%were identified. Genes in the DNA and cDNA libraries at16h showed significantdivergence with only nine genes identical. The result indicated that evaluating functional genes at thetranscription level is a more reliable indicator for understanding fluctuations than that of genomic level.Comparison of the relative abundance of xylanase genes indicated that six xylanase genes were detectedat all time points of the feeding cycle and showed a complex trend of gene expression over24h. Furtherquantitative analysis with qPCR indicated that these six representative genes showed similar trends atthe genomic level but varied at transcriptional level. These results reveal that xylanases have complexdynamics of expression in the rumen. Correlation analysis indicated that the rumen is a dynamicecosystem where the transcript profiles of xylanase genes are closely related to ruminal conditions,especially rumen pH and bacterial population.Using the same strategy, four cDNA libraries at0h,4h,9h and16h and one DNA library at9hof cysteine phytases were constructed, and a total of46unique fragments （<95%identities） of cysteinephytase genes were retrieved at both genomic and transcriptional levels. Most of these fragments hadlow identities （30–70%） with known sequences. Comparison of the cDNA and DNA libraries at9hindicated that the constitution and abundance of cysteine phytase genes were divergent, and morefunctional genes were identified at the transcriptional level. Furthermore, comparative analysis of thecysteine phytase during the feeding cycle shows the complex dynamic expression of phytase in therumen.Of the large number of novel gene fragments retrieved from the sheep rumen, ten full-length xylanase genes and four phytase genes were cloned, and three of them were chosen for further studies.Recombinant XynB and XynC were produced in Escherichia coli and had similar enzymatic properties.With beechwood xylan as the substrate, XynB and XynC showed the specific activities of73.9U/mgand142.3U/mg, respectively. Both enzymes showed the maximal activity at pH6.0. The enzymes werestable between pH5.0and8.0, retaining more than80%of their initial activity after incubation at39°Cfor1h. The optimal temperature for enzymatic activity was40°C, and the enzymes exhibitedapproximately60%relative activity at20°C. Recombinant XynB and XynC were thermostable at40°C,retaining90%and86%of their relative activities after incubation for1h, respectively. These enzymecharacteristics are similar to the physiological conditions of rumen （39°C and pH5.0–7.0）, revealingthat ruminal xylanases and rumen may adapt to each other in a mutual selection style.XynA was the most predominant gene at the transcriptional level and encoded a407-residuepolypeptide. It consisted of a signal peptide of24amino acids, a catalytic motif of GH10, and aproline-rich C-terminal sequence of60amino acids without homolog. To determine its function, matureXynA and its C terminus-truncated mutant derivative XynA-Tr were both expressed in E. coli. TheC-terminal oligopeptide had significant effects on the function and structure of XynA. Its presenceincreased the temperature optimum （from45C to50C） and broadened the ranges of temperature andpH optima （from40–50C to40–60C and from pH5.5–6.5to pH5.0–7.5）. It also improved thespecific activity (1135U mg^–1v.s.97U mg^–1) and catalytic efficiency (133.5ml s^–1mg^–1v.s.7.3mls^–1mg^–1). Analysis of the secondary structures with circular dichroism spectroscopy indicated that XynAhad more α-helical components. Binding analysis with isothermal titration calorimetry showed thatXynA had greater binding capacity to xylooligosaccharides, which was essential to the improvement ofcatalytic efficiency. Similar results were obtained when fused the C-terminal sequence to anotherxylanase （XynB） of the same source and same family. This result suggested the C-terminal oligopeptideis versatile to other xylanases and reveals an engineering strategy to improve the enzyme catalyticperformance.In summary, this study has identified a large amount of functional genes in the rumen of Small TailHan sheep based on the transcriptional analysis of ruminal GH10xylanases and cysteine phytasesduring a feeding cycle. The changes in xylanase and phytase genes are revealed by comparing theconstitution and abundance of genes between the genomic and transcriptional levels. By analyzing thecomplexity and dynamics of ruminal enzymes at the molecular level, this study provides valuableinformation for the exploitation of the microbial source in the rumen. Characterization of therepresentative xylanase （XynA, XynB and XynC） not only verifies the functions of these genes, but alsoprovides a strategy for xylanase improvement based on the study of the relation between structure andfunction.更多还原