林麝化脓性疾病与细菌识别相关TLR基因表达及多态性的关系研究

Musk, a kind of precious Chinese traditional medicine, is too rare to meet the need. Forest musk deer is the original animal of musk. The high incidence of purulent diseases seriously hindered the increase of forest musk deer population and limited the yield of musk in production. Toll like receptors (TLRs) play an important role in pathogenic micro-organism recognition and the infection signal transduction. Thus, the project put forwards: the expression and polymorphisms of bacterial recognition related TLR genes play a key role in the occurrence of purulent diseases in forest musk deer.. We have mastered the key technology of artificial musk acquisition with forest musk deer alive. Additionally, we have completed the clinical symptom observation of purulent diseases, morbidity statistics, the pathological section making and sample collection. Therefore, the complete sequences, including exons and introns, of bacterial recognition related TLR genes in forest musk deer will be cloned. On this basis, the polymorphisms of target gene exon and intron sequences between the health and purulent disease susceptible forest musk deer will be detected and analyzed. Meanwhile, the effects of purulent diseases on the expression of these genes in main organs of forest musk deer will be detected using real-time PCR, to elucidate the relationship between the target genes and purulent diseases in the aspects of gene expression and polymorphisms. This project will provide a theoretical basis for forest musk deer breeding and improving the resistance to purulent diseases. It is very important in resource protecting of forest musk deer and increasing the yield of musk.

麝香为名贵中药，资源量少，供不应求。麝香重要基原动物林麝化脓性疾病的高发严重阻碍了林麝种群数量增长，极大限制了麝香产量的提高。Toll样受体（Toll-like receptors, TLRs）在病原微生物识别及感染信号传递中起重要作用，因此本项目提出：细菌识别相关TLR基因表达及多态性对林麝化脓性疾病的发生具有关键作用。.项目组掌握了“活麝取香”关键技术，已完成林麝化脓性疾病的临床症状观察、发病率统计、病理切片制作及样本采集等工作。本项目拟克隆林麝细菌识别相关TLR基因全序列，检测和分析同一种群中健康林麝和化脓性疾病易感林麝目的基因全序列的多态性，同时，研究化脓性疾病对林麝主要脏器中各目的基因表达量的影响，从表达及多态性两方面揭示目的基因与林麝化脓性疾病发生的关系。本项目为林麝的选种育种和提高林麝种群对化脓性疾病的抵抗力提供理论基础，对林麝资源的保护与名贵中药麝香产量的提高具有重要意义。

麝香为名贵中药，具有开窍醒神、活血通络、消肿止痛等功效，由于天然麝香产量极低，市场上供不应求，价格一直维持在黄金的3倍左右。林麝为天然麝香最重要的基原动物，但由于林麝化脓性疾病的高发严重阻碍了林麝种群数量增长，极大限制了麝香产量的提高。TLR在病原微生物识别及感染信号的传导中起重要作用，因此，探讨林麝TLR基因表达量及多态性，对林麝对化脓性疾病的抗性研究有重要作用。本研究通过RACE法和PCR法首次克隆了TLR1、TLR2、TLR4、TLR5、TLR6基因序列，并上传至GenBank。在此基础上，设计荧光定量PCR引物检测了化脓和非化脓林麝心肝脾等目的组织中相关TLR基因的表达量差异，结果表明，脾脏中化脓组林麝TLR1、TLR2基因表达量显著高于对照组（P<0.05）。这表明，罹患化脓性疾病使得脾脏中TLR1基因和TLR2基因的表达量上调，推测TLR1基因和TLR2基因可能参与了对抗化脓性疾病的过程中。此外，筛选出了一种快速高效提取林麝毛发DNA的方法，并在此基础上采用PCR-SSCP法检测了化脓和非化脓林麝目的基因的多态性差异，结果表明，均未检测到显著的多态性差异。通过该研究，从基因表达和多态性两个方面揭示了目的基因与林麝化脓性疾病发生的关系，为林麝的选种育种和提高林麝对化脓性疾病的抗性提供了一定的理论基础。