基于瘤胃转录组探究双峰驼沙漠适应性分子机制

旨在比较胚胎期和成年期双峰驼瘤胃在组织形态、编码基因表达上的变化，挖掘影响双峰驼瘤胃发育的关键基因和通路，从消化系统探究双峰驼的沙漠适应性机制。本研究选取3峰10~12岁健康状况良好的成年期阿拉善双峰驼，以及3峰9~10月龄健康状况良好的胚胎期阿拉善双峰驼，采集瘤胃组织样品，制作石蜡组织切片，观察成年期与胚胎期双峰驼瘤胃，比较其组织结构之间的差异。分别提取成年期和胚胎期双峰驼的瘤胃组织总RNA，利用Illumination Hiseq 2000测序平台分别进行转录组测序，对转录组数据进行质控、比对、差异基因筛选、GO、KEGG富集分析。随机选择6个差异表达基因进行荧光定量试验，关联转录组数据与荧光定量试验的表达趋势。结果表明，胚胎期瘤胃组织中上皮细胞和肌纤维细胞清晰可见，分布密集，在成年期的瘤胃组织中，可见明显的肌纤维，肌纤维直径较宽，肌纤维间的空隙较大。转录组测序结果显示，每个样本获得至少10G的数据量，各样本的质控率都在90%以上，Q30数据都在88%以上。对测序数据进行分析，以胚胎期为对照组，成年期为试验组，筛选到1 207个差异表达基因，其中上调基因456个，下调基因751个；对差异表达基因进行层次聚类分析，结果显示，成年期的3个个体（M1、M2、M3）表达模式接近，胚胎期的3个个体（T1、T2、T3）表达模式接近。对差异表达基因进行GO和KEGG富集分析，结果显示，上调差异表达基因显著富集到62条显著的GO条目，下调差异表达基因显著富集到366条显著的GO条目，73条显著的KEGG通路。差异表达基因主要富集在代谢过程的负调控、RNA生物合成过程的负调控、基因表达的负调控等GO条目中，KEGG主要富集到MAPK信号通路、PI3K-Akt信号通路、糖尿病并发症中的AGE-RAGE信号、胰岛素信号通路、醛固酮的合成与分泌等通路中。同时筛选到MAPK12、MAPK13、FABP5、PPARγ、CaMK1等与双峰驼沙漠适应性相关的基因。荧光定量结果显示，差异基因在成年期和胚胎期瘤胃组织中的表达模式与RNA-Seq的结果一致。上述结果表明，双峰驼的瘤胃组织可能与脂肪细胞分化有关。在沙漠环境中，双峰驼可能降低瘤胃组织代谢效率、通过胰岛素抵抗作用提高血糖耐受性以及促进醛固酮的合成与分泌以调节血压平衡，以更好地在沙漠干燥缺水的环境中生存。

Exploring the Molecular Mechanism of Bactrian Camel's Desert Adaptation Based on Rumen Transcriptome

This study aimed to compare the changes in tissue morphology and the expression of coding genes in the rumen tissues of the Bactrian camel between embryonic and adult stages, to discover the key genes and pathways affecting the development of rumen of the Bactrian camel, and to explore the desert adaptive mechanism of the Bactrian camel from the digestive system. Three adult Alxa Bactrian camels aged 10-12 years old with good health and three embryonic Alxa Bactrian camels aged 9-10 months old with good health were selected to make paraffin tissue sections, observe the rumen of adult and embryonic Bactrian camels, and compare the differences in tissue structure. The total RNA was extracted from rumen tissue of adult and embryonic Bactrian camels, RNA-Seq analysis was performed by Illumination Hiseq 2000 platform, the RNA-Seq data were performed quality-controlled, alignment, differential genes screening, GO and KEGG analysis. Six differentially expressed genes were randomly selected for RT-qPCR, the results of RNA-Seq and RT-qPCR was compared. The results showed that epithelial cells and muscle fiber cells were clearly visible and densely distributed in the rumen of Bactrian camel at embryonic stage. In the rumen of Bactrian camel at adult stage, obvious muscle fibers could be observed, the diameter of the muscle fibers was wider and the space between the muscle fibers was larger. The RNA-Seq sequencing results showed that at least 10G data was obtained from each sample, the clean ratio were more than 90%, and Q30 data were more than 88%. The embryonic rumen was set as the control group and the adult rumen as the experimental group, 1 207 differentially expressed genes were screened, contained 456 up-regulated genes and 751 down-regulated genes. The hierarchical clustering analysis of differentially expressed genes showed that the similar expression pattern was detected among adult individuals(M1, M2, M3) and among embryonic individuals (T1, T2, T3). The GO enrichment analysis results showed that the up-regulated differentially expressed genes were significantly enriched in 62 GO terms, down-regulated differentially expressed genes were significantly enriched in 366 GO terms. GO terms were mainly enriched in negative regulation of metabolic process, negative regulation of RNA biosynthetic process, negative regulation of gene expression, etc. The KEGG pathway analysis of differentially expressed genes showed that 73 significant KEGG pathways were enriched in MAPK signaling pathway, PI3K-Akt signaling pathway, AGE-RAGE signaling pathway in diabetic complications, insulin signaling pathway, aldosterone synthesis and secretion, etc. At the same time, MAPK12, MAPK13, FABP5, PPARγ, CaMK1 and other genes related to desert adaptation of Bactrian camel were screened. The expression pattern of 6 differentially expressed genes detected by RT-qRCR was consistent with the results of RNA-Seq analysis. The above results indicate that the rumen of Bactrian camel may be related to adipocyte differentiation. And in a desert environment, the Bactrian camel might reduce the metabolic efficiency of rumen, improve blood glucose tolerance through insulin resistance, and promote the synthesis and secretion of aldosterone to regulate blood pressure balance to better survive in the dry and water-deficient environment of the desert.