饲喂高豆粕饲料牛蛙的消化道微生物植酸酶基因多样性和时空表达规律的研究

植物性蛋白质是替代鱼粉的重要原料，但其植酸（肌醇六磷酸）含量往往较高。由于单胃动物缺少内源型植酸酶，不能有效利用植酸，且植酸中存在的大量磷也因不能被充分利用而造成环境污染。如何提高水产动物对植酸磷的利用率成为水产饲料行业亟需解决的重要问题。前期研究发现，牛蛙饲料中使用豆粕将鱼粉由300g/kg替代至60g/kg不会影响牛蛙的生长，牛蛙对豆粕中磷的消化率高达81.6%，并已从牛蛙肠道内容物中分离得到能够分泌植酸酶的微生物。本项目拟用宏转录组技术筛选牛蛙消化道内微生物植酸酶基因，采用RT-PCR的方法研究微生物植酸酶基因转录水平上的时空变化，应用高通量测序技术分析牛蛙消化道内具有表达优势的微生物植酸酶基因多样性，结合植酸酶活力的测定，分析牛蛙消化道含有表达优势的植酸酶基因的微生物的多样性及作用规律，进一步解析牛蛙高效利用植酸磷的机制，为解决水产动物对植物蛋白原料中磷的利用问题提供思路。

In recent years, with rising fishmeal prices and reduced availability, replacement of fishmeal in aquafeeds has received more attention from academic institutions to industry. Vegetable protein is the main protein source for replacement of fishmeal in aquafeed. However, there are plenty of antinutrientional factors in vegetable ingredients including phytate, which is unavailable for all monogastric animals because of generate little phytase by themselves and is discharged as a source of environmental pollution. Our previous study shows that bullfrogs (Lithobates catesbeianus) fed the diet replacing fishmeal from 300g/kg to 60g/kg with soybean meal have similar growth performance with control group; the phosphorus apparent digestibility coefficients of soybean meal for bullfrog is 81.6%; also, some of microorganisms producing phytase have been isolated from the intestinal chyme of bullfrogs. These results indicate that the reason of bullfrog can make good use of phytic acid phosphorus is due to those microorganisms. More than 95% of the microorganisms in animal digestive tract cannot be cultivated, and the traditional method to understand the intestinal microbe is limited. In this project, phytase gene in digestive tract of bullfrogs fed the diet with high soybean meal level will be studied. Metatranscriptomics technology will be used to explore the diversity of phytase gene. RT-PCR technology will be used to study the space-time expression of phytase gene. High-throughput sequencing technology will be used to analyze dominant phytase gene. Phytase activity will be detected with GB/T 18634-2009. Finally, the diversity and the acting regularity of phytase gene of dominant microorganism in bullfrog intestine will be revealed, and the mechanism of efficient utilizing phytate phosphorus of bullfrogs will be analyzed. This will provide solutions for aquatic animal to utilize phosphorus in plant protein.