解淀粉芽孢杆菌SJ06脂肽粗提物与吡唑醚菌酯协同对玉米小斑病菌的抑制效果

运用对峙培养法测定解淀粉芽孢杆菌SJ06菌株对玉米小斑病菌的拮抗活性;采用菌丝生长速率法测定发酵上清液、脂肽粗提物对玉米小斑病菌的抑菌活性,以及SJ06脂肽粗提物与吡唑醚菌酯复配的协同抑菌活性。结果表明:对峙培养SJ06对玉米小斑病菌的抑制率为71.43%;SJ06发酵上清液和脂肽粗提物对病菌的EC_(50)值分别为0.88μL/mL和0.23μL/mL,吡唑醚菌酯对病菌的EC_(50)值为1.10μg/m L;SJ06脂肽粗提物与吡唑醚菌酯复配对玉米小斑病菌的毒性比均大于1,说明复配对病菌的抑制效果均为增效作用,其中,复配体积比为4∶6时毒性比为1.21,增效作用最强。     

Effects of dietary Bacillus pumilus on growth performance,innate immunity and digestive enzymes of giant freshwater prawns (Macrobrachium rosenbergii)

The present study was conducted to investigate the effects of Bacillus pumilus on the growth performance, innate immunity and digestive enzymes of the giant freshwater prawn, Macrobrachium rosenbergii. Prawns (mean weight: 1.81 ± 0.01 g) were fed different levels of B. pumilus at doses of 0, 1 × 10⁷, 1 × 10⁸ and 1 × 10⁹ CFU/g to produce one control group and three experimental groups (G1, G2 and G3), respectively. After a 60‐day experimental feeding period, the results showed that the final weight, weight gain rate (WGR) and specific growth rate (SGR) were significantly higher in experimental groups than in the control (p < 0.05), whereas these improvements did not differ significantly (p > 0.05) among experimental groups. The improvements in phagocytic activity, respiratory burst (RBs) activity and the activity of catalase (CAT), nitric oxide synthase (NOS) and acid phosphatase (ACP) were significantly higher in G2 and G3 than in the control and G1 (p < 0.05). Further, the activity of CAT, NOS and ACP significantly increased with an increase in the dietary level of B. pumilus (p < 0.05). The activity of phenoloxidase (PO) in G3 was significantly higher than that in the control (p < 0.05). The activity of amylase was significantly higher in the groups G2 and G3 than that in the control group. G3 improved the activity of protease compared with the control (p < 0.05). In conclusion, the results of this study indicated that the potential use of B. pumilusat a dose of 1 × 10⁸ CFU/g feed could improve the growth, immunity and digestive enzymes of M. rosenbergii.     

一种高产L-赖氨酸枯草芽孢杆菌的构建研究

为了选育一种非抗性、高产出、遗传稳定性好、低生产成本的L-赖氨酸生产菌株,以辣椒花药和枯草芽孢杆菌为试验材料,以穿梭表达载体pHT43和基因敲除载体pK18mobsacB为媒介,将辣椒花药中的高赖氨酸基因CFLR转化到枯草芽孢杆菌中。经过2次细胞内同源交换和培养基筛选,最终获得不带质粒抗性标记的高产L-赖氨酸枯草芽孢杆菌株CMCC(B)63501/pK18mobsacB-ΔALA::CFLR,该菌株连续传代20次后仍具有明显高于野生型的CFLR基因拷贝数,经HPLC验证,重组菌株L-赖氨酸含量比野生菌株提高了64.89%。结果表明,本研究所构建的非抗性高产L-赖氨酸枯草芽孢杆菌株产量高、遗传稳定性好、低纯化成本,具有良好的应用潜力。     

枯草芽胞杆菌Y13挥发性物质的分析及抑菌活性

枯草芽胞杆菌Bacillus subtilis Y13是一株高效广谱的生防菌，对油茶主要病害具有较好的生防效果，试验表明其发酵产生的挥发性物质对油茶炭疽病菌有较好的抑制作用。对菌株Y13产生的挥发性物质进行定性定量分析，并测定其抑菌活性，为油茶病害无公害防治及药剂开发提供理论依据。采用顶空固相微萃取技术（HS-SPME）和气相色谱-质谱联用仪（GC-MS）收集并分析Y13挥发性物质成分，购买相应挥发性物质纯品，采用双皿倒扣法测定其抑菌活性。Y13挥发性物质对10种植物病原真菌抑菌率在19.2%～83.9%，其中挥发性物质对果生炭疽菌Colletotrichum fructicola的抑菌活性最强，抑菌率达到83.9%；检测其中10种挥发性化合物纯品对果生炭疽菌的抑菌活性发现，除了正十八烷外，其他9种挥发性化合物纯品均具有不同程度的抑菌活性；筛选出的4种高效抑菌挥发性化合物纯品对7种常见的油茶病原真菌的抑菌活性检测结果表明，4种挥发性化合物纯品（10 μL/平板）对7种常见的油茶病原真菌均具有较好的抑菌活性，抑菌活性壬醛 > 苯甲醛 > 3-甲基-4-苯基吡唑 > 苯丙噻唑。Y13挥发性物质对油茶主要病害病原菌有很好的抑制作用，具有一定的研究意义和良好的开发价值。     

苏云金芽胞杆菌杀虫晶体蛋白Cry1D新基因的克隆与特性

为扩充鳞翅目害虫杀虫基因资源，本研究从苏云金芽胞杆菌BN23-5中克隆得到一个新的cry基因，并对其进行鉴定和分析。该基因为一个完整的cry1D基因，全长3501 bp，编码1166个氨基酸残基。该氨基酸序列是一个新的Cry氨基酸序列，与Cry1Db1的同源性最高，为86%，命名为Cry1Dd1（登录号为KJ728844）。将该基因插入穿梭表达载体pSTK中，转入BT无晶体突变株HD73^-中进行表达。结果表明，cry1Dd1基因能在BT无晶体突变株中表达，并形成菱形伴孢晶体。SDS-PAGE验证其分子量为132.2 kD，与预测的大小相符。生物活性测定表明，Cry1Dd1晶体蛋白对小菜蛾的幼虫具有杀虫活性，LC₅₀为13.1 μg/mL；能明显抑制甜菜夜蛾幼虫的生长；但对棉铃虫幼虫没有杀虫活性。对cry1Dd1基因序列进行分析，cry1Dd1包含8个block保守区域，这和目前其他的cry基因相似；Cry1Dd1蛋白的活性区域为N端的37~593位氨基酸残基。     

短小芽孢杆菌HB-3的分离鉴定及其淀粉酶的纯化特性研究

从湖北省襄阳市三九酿酒厂下水道污泥分离筛选得到产淀粉酶菌株HB-3,根据菌株生长形态和生理生化特征分析,初步鉴定为短小芽孢杆菌。短小芽孢杆菌HB-3在淀粉培养基上培养72 h后,淀粉酶活力达到120.0 U/mL。然后使用DEAE-52和CM-52阴阳离子交换层析柱对短小芽孢杆菌HB-3产生的淀粉酶进行了部分纯化。结果表明,短小芽孢杆菌HB-3中淀粉酶经过部分纯化后,纯化倍数为5.9,酶活力回收率为90.9%。纯化后的淀粉酶使用SDS-PAGE凝胶电泳研究,以标准蛋白为对照,计算出短小芽孢杆菌HB-3中淀粉酶分子量为56.5 kDa。同时对短小芽孢杆菌HB-3的淀粉酶进行了纯化特性研究,结果表明,该酶的酶促反应最适温度为50℃,40～60℃热稳定性良好;该酶的酶促反应最适pH为5.0,pH 4.0～6.0酸碱稳定性良好,该酶对底物淀粉的反应动力学米氏常数K_m为0.062 mmol/L,最大反应速率V_(max)为3.019 mmol/(L·min)。     

小麦赤霉病生防菌DZSG23的抗病机制

课题组前期从健康杜仲叶片中分离得到1株内生细菌枯草芽孢杆菌DZSG23,此菌可以较好地防控小麦赤霉病。为明确芽孢杆菌DZSG23在小麦中的定殖与强化抗病机制研究,利用抗生素标记法分析了DZSG23菌体在小麦组织中的定殖情况,采用荧光定量PCR技术检测不同生育期小麦穗部PR-1、AOS、ACOI等基因的表达水平变化。结果表明,DZSG23可在苗期小麦植株和小麦穗表面稳定定殖;DZSG23浇灌接种苗期小麦后的第34天,DZSG23在苗期小麦植株的根、茎和叶中的定殖量分别达到1.437×10⁶ CFU·g^-1、3.285×10³ CFU·g^-1和2.377×10³ CFU·g^-1;DZSG23喷施小麦穗表面15 d后,穗表面菌群密度仍可达7.146×10³ CFU·g^-1。此外,诱导系统抗性(ISR)信号通路研究表明,拮抗菌DZSG23可以诱导PR-1和AOS基因的上调表达,表明拮抗菌DZSG23引入小麦后可诱导小麦的水杨酸(SA)和茉莉酸(JA)信号通路,增强其抗病性。     

Potential of indigenous Bacillus spp. as probiotic feed supplements in an extruded low‐fish‐meal diet for juvenile olive flounder,Paralichthys olivaceus

A 12‐week feeding trial was designed to assess the probiotic potential of indigenous Bacillus amyloliquefaciens and/or Bacillus subtilis singly or in combination with Bacillus licheniformis in an extruded feed for olive flounder (Paralichthys olivaceus) juveniles. A high fish meal (FM) diet (control) and a low‐FM diet containing an alternative protein blend (30% FM replacement, FM30) were formulated. Three other experimental diets were prepared by inclusion of B. amyloliquefaciens (BA), B. subtilis (BS), or a mixture of B. amyloliquefaciens, B. subtilis, and B. licheniformis (BASL) into FM30 diet, with a final concentration of 10⁶ CFU/g diet. Results indicated that the FM30 diet was well tolerated by flounder, and the overall performance was not affected by dietary treatments. Lysozyme activity and total immunoglobulin level were significantly reduced in flounders when fed with the FM30 diet compared with the BASL and BA diets, respectively. The Bacillus additives neither enriched the relative abundance of the corresponding Bacillus spp. in the relevant gut microbiota of olive flounder nor modulated the presumptive gene functions of the gut microbiome. Despite the absence of growth‐promoting effect, the tested probiotics could still be economically viable for use as immunostimulants in commercial flounder diets with partial FM replacement.     

凝结芽孢杆菌对热应激肉鸡生长性能及肠道消化与吸收功能的影响

本试验旨在研究凝结芽孢杆菌对热应激肉鸡生长性能及肠道消化与吸收功能的影响。选择320只28日龄健康爱拔益加（AA）肉公鸡,随机分为5个组,每组8个重复,每个重复8只。常温对照组（TC组）肉鸡饲养于（22±1）℃的常温环境,饲喂基础饲粮;热应激组（HS组）肉鸡饲养于（34±1）℃的高温环境,饲喂基础饲粮;热应激试验组肉鸡饲养于（34±1）℃的高温环境,分别饲喂在基础饲粮中添加0.2×107(HS-BC0.2组)、1.0×107(HS-BC1.0组)、5.0×107CFU/g(HS-BC5.0组)凝结芽孢杆菌的试验饲粮。试验期14 d。结果表明：与TC组相比,HS组的末重、平均日增重（ADG）、平均日采食量（ADFI）,干物质和粗蛋白质表观代谢率,空肠食糜总淀粉酶和总蛋白酶酶活,空肠绒毛高度、绒毛高度/隐窝深度以及空肠钠/葡萄糖协同转运蛋白1(SGLT1)、基础氨基酸转运载体（r-BAT）、y+L氨基酸转运载体-1(y+LAT1) mRNA表达量均显著降低（P<0.05）,料重比（F/G）显著增加（P<0.05）。与HS组相比,HS-BC1.0和HS-BC5.0组的ADG、粗蛋...