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

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.

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。

本课题通过研究牛蛙消化道内植酸酶活性的时空变化和植酸酶基因的多样性，并分离鉴定牛蛙消化道内产植酸酶微生物，来探索牛蛙高效利用豆粕中植酸磷的原理。首先通过解剖观察和测量的方法确定牛蛙消化道包括口咽腔、食道、胃、十二指肠、空肠、回肠、直肠和肛门。然后配制全鱼粉饲料、豆粕蛋白替代鱼粉蛋白50%的饲料和全豆粕饲料，投喂牛蛙30天后，检测牛蛙空肠和回肠粘膜植酸酶活性为22.36～54.23nmol/mgprot/min，回肠的植酸酶活性高于空肠；饱食状态植酸酶活性高于饥饿状态；豆粕替代组植酸酶活性均高于鱼粉组。抗生素灌服实验发现，抗生素会显著降低鱼粉组牛蛙回肠及豆粕组空肠和回肠的植酸酶活性，而不影响鱼粉组牛蛙空肠的植酸酶活性，由此可推测牛蛙自身能够分泌植酸酶。从牛蛙消化道分离产植酸酶的微生物10株。枯草芽孢杆菌分泌的β折叠植酸酶（BPP）是动物消化道内常见的植酸酶，依据芽孢杆菌属的植酸酶序列，设计简并引物，以牛蛙消化道粘膜DNA为模板，扩增出6条植酸酶基因序列；以蛙源芽孢杆菌和蛙源阴沟肠杆菌的DNA为模板分别扩增出10条和11条植酸酶基因序列。而有关其他类型植酸酶基因的多样性还有待于进一步研究。同时，产植酸酶有益微生物的豆粕发酵及饲料添加的实验结果发现，蛙源酵母发酵豆粕后替代普通豆粕提高饲料磷的利用率，降低豆粕对肠道组织造成的损伤。豆粕饲料中添加适量蛙源产植酸酶枯草芽孢杆菌（10^7cfu/g）和阴沟肠杆菌（10^9 cfu/g）提高饲料磷的表观消化率，提高饲料效率和蛋白质效率，促进全体和血清钙磷的沉积，改善肠道健康状况，并能提高牛蛙的抗氧化能力。综上所述，牛蛙肠道的植酸酶活性具有时空变化规律，牛蛙消化道内BPP植酸酶基因具有种间多样性和种内多态性。牛蛙能够高效利用植酸磷的主要原因是其消化道内具有大量产植酸酶微生物，这些微生物在豆粕饲料的诱导下产植酸酶效率提高。牛蛙自身是否能够分泌植酸酶有待于进一步研究。