魁蚶幼体卵黄蛋白原表达动态规律及其与母源免疫的关系

Vitellegenin (Vg) and Vg-cleaved yolk proteins play important nutritional and immune defense roles in ovipara embryos and larvae. The ark shell, Scapharca broughtonii, is an important shellfish species in China; but because the gonads of parent S. broughtonii are coated in foots, it is difficult to identify their maturity, and dysplasia or increased mortality of embryo and larvae occurs often due to insufficient Vg. Maternal immunization is expected to increase the Vg content in early offspring through the mediation of Heat Shock Proteins 90 (HSP90) and estrogen receptor (ER)，thereby strengthening nutrition and immunity supply for embryos and larvae. This project will take half-sib families with the same father as research target, use inactivated Vibrio anguillarum to stimulate parent S. broughtonii and set it as the maternal immunization group. Molecular biological techniques and immunology methods will be used to obtain Vg, HSP90 and ER genes and proteins, then studies will be carried out on the dynamic spatio-temporal expression pattern of Vg during the early development stages of the S. broughtonii larvae, the influence of maternal immune on Vg expression, the conjugation effect of HSP90 and ER as well as the influence of protein inactivation on Vg expression, thus to reveal the effect of HSP90-ER during Vg synthesis. This study will identify the dynamic change of vitellogenin expression during early- stage larvae of S. broughtonii and its relationship with maternal immune, reveal the inherent molecular mechanism of HSP90-ER mediating Vg expression, and provide theoretical basis for increasing the survival rate of larval S. broughtonii in a scientific way.

卵黄蛋白原（Vg）及其降解产物对卵生动物胚胎和幼体具有重要的营养和免疫保护作用。魁蚶是一种大型蚶科贝类，性腺被斧足包被，成熟度难以鉴别，人工催产常造成母源Vg传递不足，导致幼体发育不良和死亡。母源免疫有望通过HSP90-ER介导增加幼体Vg含量，强化营养和免疫力供给，促进幼体生长发育。本项目以魁蚶同父异母半同胞家系为研究对象，采用分子生物学和免疫学方法，分离鉴定Vg、热休克蛋白90（HSP90）、雌激素受体（ER）基因和蛋白，研究Vg在幼体发育早期的时空动态表达规律，明确灭活鳗弧菌母源免疫对幼体Vg表达的影响，并分析HSP90与ER的结合作用及蛋白沉默对Vg合成的影响，从而阐明HSP90-ER在Vg合成中的作用。本研究，将明确魁蚶幼体Vg表达动态规律及其与母源免疫的关系，揭示HSP90-ER介导母源免疫影响Vg表达变化的分子基础，为科学提高魁蚶幼体成活率提供理论依据。

魁蚶（Scapharca broughtonii）是一种重要的大型经济蚶科贝类，其亲本卵黄蛋白原（Vg）的积累和母源传递对于苗种前期生长发育具有重要意义。基于Vg是一种免疫因子和热休克蛋白90-雌激素受体（HSP90-ER）介导合成Vg的相关研究报道，本项目开展了魁蚶Vg在子代早期发育过程中表达规律及母源免疫对其影响的研究。取得如下结果：（1）利用同源扩增和RACE技术克隆获得了魁蚶Vg、ER、HSP90和热休克蛋白70（HSP70）基因全长cDNA，并构建染色体水平的魁蚶基因组对基因进行验证和挖掘，同时制备了Vg、ER、HSP90抗体；（2）分离纯化到Vg天然蛋白，电泳检测表明其以同源二聚体形式存在，体外抑菌实验表明Vg蛋白对哈维氏弧菌生长有一定抑制作用；（3）qRT-PCR和ELISA结果显示Vg具有明显的母源传递特点，受精后Vg基因和蛋白含量均降低后升高，推测担轮幼虫期开始自身合成，且壳顶幼虫期以后合成能力显著加强。相关性分析表明，Vg与ER基因在子代幼虫发育早期表达具有显著相关性，但与HSP90、HSP70相关性不显著，CO-IP实验没能检测到卵细胞浆液中HSP90和ER的复合体；（4）催产前3天，对亲贝注射灭活鳗弧菌进行母源免疫，卵及子代Vg基因表达未显著提高，注射HSP90和ER抗体也没有显著降低Vg的表达；投喂β-葡聚糖15天能显著提高卵内Vg的含量，且其后代早期发育也具有显著生长优势；（5）基于上述结果，研制了提高海洋双壳贝类性腺成熟度及免疫力的复合制剂，在魁蚶和栉孔扇贝中效果良好，其机制有待进一步研究。综上所述，本项目研究了魁蚶Vg在子代幼虫发育早期的表达变化规律，探讨了母源免疫对其表达的影响，对于认识Vg合成调控积累了数据，为从免疫角度提高亲贝和幼虫Vg含量提供了思路和依据。