MiR-205b靶向HSD11B1在单色光影响鸽产蛋中的作用机制

Pigeon is an altrices, with long breeding cycle and low egg production efficiency, monochromatic lights affect the laying performance of poultry. Our previously study showed the red light raised the laying rate, while the blue light had opposite effect. High-throughput sequencing and bioinformatics analysis obtained differentially expressed miR-205b and HSD11B1 gene, preliminarily validated miR-205b targeting HSD11B1. However, the mechanism of miR-205b regulates monochromatic lights affecting egg production of pigeon by targeting HSD11B1 is still unclear. In this study, luciferase reporter gene assay identifies the targeting regulatory relations between miR-205b and HSD11B1. RT-qPCR is used to detect the expressions of miR-205b and HSD11B1 in ovaries at different reproductive period of pigeons under monochromatic lights. The follicular granular cells from ovaries are isolated and cultured in vitro, the miR-205b will be over-expressed and knocked down, the expressions of HSD11B1 and key genes of steroid hormone biosynthesis signaling pathway will be detected by RT-qPCR and Western Blot and the amounts of steroid hormones will be measured by ELISA. This results will confirm the effect of miR-205b on synthesis of steroid hormones and related biosynthesis signaling pathway by targeting HSD11B1. Furthermore, observing and recording follicular development of pigeon during the egg production interval under different monochromatic lights. The amounts of steroid hormones in serum and the expressions of miR-205b, HSD11B1 and key genes of steroid hormone biosynthesis signaling pathway in the ovaries and follicles will be detected. Analyzing the expressions of genes and correlating with the hormone levels and production data. The results of this study will systematic illustrate the mechanism of miR-205b regulates monochromatic lights affecting egg production of pigeon by targeting HSD11B1, which will provide theoretical basis for the application of monochromatic lights in pigeon breeding.

鸽属晚成鸟，繁殖周期长、产蛋效率低；单色光影响禽类产蛋性能。前期研究发现，红光提高鸽产蛋率，蓝光则相反；测序和分析获得差异表达miR-205b和HSD11B1，初步验证两者的靶向关系，但两者在单色光影响鸽产蛋中的作用机制尚不清楚。本研究通过双荧光素酶报告系统确证两者靶向作用；RT-qPCR分析两者在单光色下不同繁殖期鸽卵巢中表达特征；检测过表达和敲低miR-205b的鸽卵泡颗粒细胞中类固醇激素合成量及其合成通路关键基因和HSD11B表达，确定miR-205b靶向HSD11B1对类固醇激素合成量及其合成通路的影响；观察记录单色光下产蛋间隔期鸽卵泡发育情况，检测类固醇激素合成量、卵巢和卵泡中miR-205b、HSD11B1及信号通路基因表达，分析各基因表达关系，与激素水平和生产数据关联。系统性阐明miR-205b靶向HSD11B1在单色光影响鸽产蛋中的作用机制，为养鸽业单色光应用提供理论基础。

随着畜牧业结构调整，肉鸽养殖业已成为特禽养殖业龙头产业，而鸽繁殖周期长、产蛋率低使其不能满足市场需求，严重制约了肉鸽养殖业的发展。前期发现红光能提高肉鸽产蛋率而蓝光则是抑制，利用高通量测序技术和生物信息学分析发现差异cli-miR-205b和HSD11B1基因，初步验证两者间存在靶向关系。本项目利用双荧光素酶报告系统检测发现cli-miR-205b通过结合HSD11B1的3'UTR下调其表达。首次成功培养出鸽颗粒细胞，构建了cli-miR-205b重组腺病毒，RT-qPCR检测鸽颗粒细胞，rAd-cli-miR-205b（OE）表达水平显著高于miR-205b野生型（WT）组，rAd-cli-miR-205b-sh（SH）显著低于WT组，重组病毒可在HEK293A细胞中复制，且效价可到达1011 PFU/mL。RT-qPCR检测感染的鸽颗粒细胞，发现HSD11B1和类固醇激素生物合成通路关键基因（CYP3A5、CYP19A1和CYP11A1）在OE组的表达水平显著低于其阴性对照组，SH组显著高于其阴性对照组。WB法检测发现，与WT组相比，HSD11B1的蛋白表达水平在OE组表达下降，而SH组升高。CCK-8实验表明，cli-miR-205b的过表达显著抑制了肉鸽颗粒细胞的增殖，而cli-miR-205b的干扰显著诱导了肉鸽颗粒细胞的增殖。综上所述，cli-miR-205b在肉鸽颗粒细胞中被下调，其过表达通过触发细胞凋亡来抑制颗粒细胞的增殖。cli-miR-205b通过靶向HSD11B1调控鸽卵巢颗粒细胞类固醇激素生物合成，介导肉鸽产蛋。此外，红光可提高母鸽卵巢中E2的浓度和ERβ的表达，增加了公鸽垂体和睾丸中AR的表达，红光通过影响类固醇激素的合成影响肉鸽的产蛋量。本项目不仅系统性阐明miR-205b靶向HSD11B1在单色光影响鸽产蛋中的作用机制，还从个体水平验证了光色通过影响肉鸽类固醇激素水平影响其产蛋性能，为养鸽业单色光应用提供理论基础。