元转录组分析瘤胃中木质纤维素降解酶系的组成

Highly efficient degradation of lignocellulose in rumen results from the concerted action of a host of microbes and their fibrolytic enzymes. Metagenomic surveys have drastically increased our knowledge of lignocellulose degradation in rumen. Because of the huge difference in biomass among bacteria, fungi and protozoa in rumen, the majority of the sequences obtained through metagenomic approaches are of bacterial origin. In addition, the metagenomic information permits the understanding of the functional potential of rumen microbes and not of the process of the enzymatic degradation. This project aims to obtain lignocellulolytic genes that are highly expressed during lignocellulose degradation in yak rumen by metatranscriptomics and subsequent pyrosequencing, to analyze the expression profiles of these genes and to identify microbes that harbor these genes. Microarrays designed on the basis of the above analysis will be employed to determine the expression profiles of fibrolytic genes during the course of lignocelluloses degradation. In situ geneFISH will be used to verify the relationship between cellulolytic activities and microbes that produce them. This successful execution of this project will help understand cellulose degradation in rumen.

反刍动物瘤胃中木质纤维素的高效降解是多菌种/多基因/多酶系协同作用的结果。对瘤胃元基因组的分析极大地丰富了我们对瘤胃微生物降解木质纤维素的认识。由于瘤胃中细菌、真菌和原生动物生物量差别巨大，导致元基因组获得的序列绝大部分来自瘤胃细菌，并且基因组本身只反映细胞的功能潜力，从瘤胃微生物元基因组中获得的木质纤维素降解酶基因信息并不能代表纤维素降解过程中的实际情况。因此，本研究拟通过牦牛瘤胃微生物元转录组高通量测序，获得大量降解过程中高表达的降解酶基因，分析其序列多样性特征和对应微生物类群特征，据此设计合成瘤胃微生物木质纤维素降解功能基因芯片，结合芯片杂交分析降解酶基因在瘤胃木质纤维素降解过程中表达量的相对比例和变化，并通过基因荧光原位杂交进行功能基因与微生物类群对应性的验证，确定瘤胃木质纤维素降解的酶系组成和特点，并推测其降解模式，为瘤胃木质纤维素高效降解机制的研究奠定基础。

本项目开展1年以来，完善了瘤胃微生物元转录组RNA提取和纯化方法，对饲喂后1小时的2个瘤胃样品的元转录组RNA高通量测序，获得高质量reads约126万条（非核糖体RNA序列约111万条，平均长度471bp）。本项目对元转录组中编码木质纤维素（纤维素、半纤维素和寡糖）降解酶基因（序列）及其对应的微生物物种进行分析。结果表明瘤胃纤维素降解的主要贡献者是Ruminococcus，Fibrobacter和Epidinium的GH5，GH9，GH45和GH48家族蛋白；其中在元基因组中很少发现的GH48结构域降解酶在元转录组中有相对较高丰度的表达，推测它在瘤胃纤维素降解中具有重要作用并对其在瘤胃中的多样性进行了进一步的研究；瘤胃半纤维素降解的主要贡献者是Ruminococcus, Prevotella和Fibrobacter的GH10，GH11和GH26家族蛋白；瘤胃中寡糖的降解更多地通过Prevotella and Bacteroides的GH1，GH2，GH3，GH43家族蛋白完成。元转录组的研究结果表明，尽管基于16S rRNA基因序列多样性的分析和瘤胃元基因组的研究显示瘤胃中存在大量未获培养的微生物，但瘤胃中木质纤维素降解主要的贡献者为瘤胃研究早期所确定的R. albus, R. falvefaciens, F. succinogenes和P. ruminicola等。此外，dockerin和CBM10的较高表达也显示厌氧细菌和真菌的纤维小体在瘤胃木质纤维素降解中发挥作用。研究成果为进一步揭示瘤胃木质纤维素高效降解机制奠定基础。已发表论文1篇，完成整理并拟投稿论文1篇。