猪GRP78基因克隆、生物信息学分析及组织表达谱研究

本研究旨在克隆猪葡萄糖调节蛋白78（glucose-regulated protein 78，GRP78）基因，并进行生物信息学分析，探讨其在猪不同组织中的表达情况。根据GenBank上公布的猪GRP78基因序列（登录号：XM_001927795.6）设计引物，PCR扩增及测序获得猪GRP78基因CDS序列，使用在线软件分析GRP78的理化性质、跨膜结构、信号肽、疏水性、保守结构域、二级结构和三级结构，并进行同源性比对及系统进化树构建，利用实时荧光定量PCR方法检测猪GRP78基因在各组织中的表达情况。结果显示，猪GRP78基因CDS区长1 965 bp，可编码654个氨基酸。生物信息学分析表明，GRP78理论分子质量为72.3 ku，等电点（pI）为5.06，半衰期为30 h，其水溶液在280 nm处的消光系数为30 495，肽链N端为蛋氨酸（Met），不稳定系数为32.39，属于稳定蛋白。脂肪系数为85.40，总平均疏水指数为-0.496，无跨膜结构，存在信号肽序列，说明该蛋白属于分泌型蛋白；GRP78蛋白只包含1个超家族保守结构域：HSP70结构域。蛋白二级结构分析显示，GRP78蛋白中α-螺旋、延伸链、β-转角和无规则卷曲分别为40.06%、20.49%、8.10%和31.35%。同源性比对结果显示，猪GRP78基因与人（登录号：NM_005347.4）、小鼠（登录号：NM_001163434.1）、大鼠（登录号：NM_013083.2）、山羊（登录号：XM_005687138.3）、牛（登录号：NM_001075148.1）核苷酸序列的同源性分别为93%、91%、90%、95%、95%，氨基酸序列的同源性分别为99%、98%、98%、99%、99%，各个物种之间GRP78基因保守性较高。实时荧光定量PCR结果显示，GRP78基因在心脏、肝脏、脾脏、肺脏、肾脏、小肠、胃、卵巢、输卵管、乳腺、小脑、大脑、垂体等组织中均有表达，在心脏、脾脏、肺脏中相对高表达。本研究结果为今后深入研究GRP78基因的生物学功能提供了基础材料。     

Liver histology and histomorphometry in hybrid sorubim (Pseudoplatystoma reticulatum × Pseudoplatystoma corruscans) reared on intensive fish farming

This study aimed to characterize the liver histology and histomorphometry in sorubim hybrid of different categories (nursery, growth and grow‐out) reared on fish farming. The categories were defined considering body weight (BW): nursery category (n = 5): BW = 37.06 ± 6.00 g (31.6–45.3 g); growth category (n = 5): BW = 310.40 ± 53.80 g (242.1–376.4 g) and grow‐out category (n = 5): BW = 874.28 ± 27.59 g (846.2–913.1 g). Liver fragments were processed to paraffin inclusion, and sections were stained by haematoxylin and eosin (H&E), PAS (Periodic Acid Schiff) and Perl's staining to histology, histomorphometry and density volumetric of liver structures; glycogen analysis and to detect ferric irons (Fe³⁺) respectively. The hepatosomatic index decreased between the categories (P < 0.01). The percentage of PAS‐positive hepatocytes in the nursery category was higher (P < 0.05) in relation to the growth and grow‐out categories. The hepatocytes from all fish were positive to Perl's staining. The density volumetric of liver structures did not differ among categories except to blood vessels were higher (P < 0.01) in the nursery and growth. The area (μm²) and perimeter (μm) of hepatocytes, and the area (μm²), perimeter (μm) and volume (μm³) of the nuclei from grow‐out fish were lower (P < 0.01) than those from the nursery and growth categories. Changes in morphometric characteristics of hepatocytes may result from metabolic changes associated with body growth surubins; therefore, these morphometric characteristics of liver tissue can be used as functional biomarkers for the assessment of fish health and nutrition status.     

The effects of diet supplemented with Lactobacillus rhamnosus on tissue parameters of rainbow trout,Oncorhynchus mykiss (Walbaum)

This study was carried out to establish the effects of a 6 week treatment with the diet supplemented with L. rhamnosus in concentrations of 10⁷ CFU g^?1 (G1 group) and 10⁸ CFU g^?1 (G2 group) on the condition expressed by condition factors (Fulton's, Clark's and B), intestinal microbiology, haematological, histological and selected antioxidative parameters of rainbow trout. A significantly higher condition factors were found in G1 group indicating that higher concentration of probiotic (10⁸ CFU g^?1) did not result in the better condition. Cholesterol and urea levels were significantly higher in both G1 and G2 groups, albumin in G1 and creatinine in G2 group with respect to control. A significantly higher liver TBARS level was observed in G2 group. The feeding with supplemented probiont apparently changed the resident microbiota. Three weeks after withdrawal of the supplemented feed, the microflora mostly reverted to the control composition, although L. rhamnosus in faecal matter of fish remained inherent. The epithelial structure of the proximal and distal intestine revealed the increased absorptive area in both treated groups, as well as the increase in the mucin‐secreting goblet cells. The L. rhamnosus‐treated groups demonstrated the capacity for the augmentation of the innate host defence.     

拟穴青蟹Cactus基因的cDNA克隆、序列及生物学功能

为获取拟穴青蟹Cactus基因cDNA全长、分析基本生物学信息,并初步探索其在病原物刺激下的免疫反应,实验采用RACE技术获得了拟穴青蟹Cactus(SpCactus)基因的cDNA全长序列,其cDNA全长为2035 bp,开放阅读框(ORF)1311 bp,编码436个氨基酸,分子量为46.01 ku。对蛋白理化性质进行预测发现,SpCactus为亲水性蛋白,等电点pI为4.91。经预测,SpCactus与其他物种的IκB蛋白具有相似的功能结构域。同源性比对结果显示,SpCactus与凡纳滨对虾和中国明对虾的Cactus蛋白同源性均高达62%,相似度为73%。系统进化树分析显示,SpCactus与甲壳动物聚为一支,与无脊椎动物聚为一大支。实时荧光定量PCR(RT-PCR)检测发现,SpCactus基因在拟穴青蟹不同组织中均有表达,肌肉中的表达量最高,其次为心脏、眼柄、血液和鳃,肝胰腺中的表达量最低。LPS和金黄色葡萄球菌刺激均能显著诱导SpCactus基因的表达。本实验成功扩增了SpCactus基因全长,并进行了生物信息学分析、理化性质预测,初步探讨了其生物学功能,为进一步研究其在免疫反应中的生物学功能提供参考依据。     

牦牛CRH基因克隆及其在生殖轴中的表达研究

为了探讨促肾上腺皮质激素释放激素（corticotropin-releasing hormone，CRH）基因在牦牛繁殖中的调控作用，试验分别采集5头成年母牦牛和5头成年母黄牛的下丘脑、脑垂体前叶、输卵管、卵巢和子宫等组织，通过RT-PCR技术及生物信息学软件对牦牛CRH基因编码区进行扩增、克隆及序列分析，并构建系统进化树；采用实时荧光定量PCR法检测CRH基因的组织表达。结果表明，牦牛CRH基因编码区全长573 bp，编码190个氨基酸。牦牛与黄牛、猫、鸡、人、小鼠、大鼠和猪的CRH基因核苷酸同源性分别为99.8%、43.3%、36.1%、46.2%、39.0%、38.5%和56.4%。系统进化树表明，牦牛与黄牛亲缘关系最近，并与其他物种类聚，符合物种进化规律。CRH蛋白分子式为C₉₂₀H₁₅₀₃N₂₇₉O₂₆₃S₃，分子质量为20776.95 u，理论等电点为10.95，其氨基酸组成中亮氨酸（L）、脯氨酸（P）、丙氨酸（A）和精氨酸（R）所占比例较高，分别为15.8%、12.6%、12.1%和10.5%。CRH蛋白为不稳定亲水蛋白，存在信号肽和跨膜结构。CRH蛋白二级结构中α-螺旋、β-转角、延伸链和无规则卷曲分别占41.05%、1.05%、8.42%和49.48%，三级结构分析结果与其相一致。CRH基因mRNA在牦牛下丘脑中表达量最高，显著高于其他组织（P<0.05），在子宫中表达量最低。牦牛脑垂体前叶CRH基因mRNA表达量极显著高于黄牛（P<0.01），在卵巢、输卵管中表达量显著高于黄牛（P<0.05），而两个品种在下丘脑、子宫中的表达量差异不显著（P>0.05）。提示CRH基因在牦牛繁殖调控中具有重要作用。     

水力侵蚀对砒砂岩区土壤有机碳空间变异性的影响

以侵蚀剧烈的砒砂岩区为研究对象，应用地统计学Kriging插值法准确描述水力侵蚀下土壤有机碳空间动态迁移过程来探究侵蚀与碳平衡的关系并为有效治理砒砂岩区坡面水土流失提供科学的参考。结果表明：（1）降雨前后各土层有机碳均随着土层增加而减少，表现为中等变异、空间自相关均为正相关且0—10 cm土层自相关性最大。降雨后0—20 cm土层有机碳含量减少而20—40 cm土层增加，且各土层变异程度均较雨前减弱。（2）降雨前后土壤有机碳分别与高斯模型，球状模型较为拟合，且变程值均随土层增加而增大。降雨前后0—10 cm土层表现均为强烈空间相关性且雨后结构比增大，而10—40 cm均为中等相关性雨后结构比减小。（3）降雨后空间异质性减弱斑块面积变大，总体表现为同一土层坡上侵蚀坡下沉积，而不同土层下0—20 cm土层表现为侵蚀，20—40 cm土层表现为沉积。（4）降雨前后土壤有机碳均与黏粒呈现较显著正相关，其中降雨前0—10 cm土层有机碳与黏粒为极显著正相关，降雨后各土层相关性增强均为极显著正相关。但降雨前后土壤有机碳与粉粒相关性均较小且不显著，与砂粒则基本表现为负相关。     

近35年红壤稻区土壤肥力时空演变特征—以进贤县为例

【目的】明确红壤稻区典型县域农业生产中土壤养分的变化特征以及当前土壤肥力水平,为红壤稻田土壤培肥改良提供依据。【方法】通过数据收集和野外采样分析得到江西省进贤县1982年、2008年和2017年3个时期稻田耕层土壤属性的数据,统一选取土壤pH、有机质、碱解氮、有效磷和速效钾作为土壤综合肥力评价指标,首先对3个时期各项肥力指标进行常规统计和差异性分析,采用主成分分析找出不同时期肥力差异的关键因子并确定权重,通过隶属度函数得到各项肥力指标的隶属度值,将各项肥力指标的权重和隶属度值加乘得到土壤综合肥力指数,最后结合土壤各项肥力指标和综合肥力指数的GIS空间分布图探究该区域稻田土壤肥力时空演变特征。【结果】1982—2017年进贤县稻田土壤有机质、碱解氮、有效磷和速效钾均呈不同程度的上升趋势,土壤pH呈下降趋势。1982、2008和2017年3个时期进贤县土壤pH的平均值分别为5.9、5.1、4.8,年均下降0.03个单位;35年来土壤pH整体由西部向东南和西北下降速率逐渐减低,2017年99%的稻田土壤处于酸性水平（4.5—5.5）。35年间稻田土壤有机质含量的平均值由28.1 g·kg^-1上升至36.8 g·kg^-1,1982—2008年和2008—2017年土壤有机质年均增加速率分别为0.21和0.31 g·kg^-1,2017年土壤有机质含量在30—40 g·kg^-1之间的稻田占比达94%,1982—2017年土壤有机质整体由东北向西南上升速率逐渐降低。1982—2017年土壤有效磷含量的平均值由7.0 mg·kg^-1上升至32.1 mg·kg^-1,2017年进贤县以土壤有效磷含量在20—40 mg·kg^-1的稻田为主,占比75%。1982—2017年稻田土壤速效钾累积缓慢,1982—2008年和2008—2017年土壤速效钾年均增加速率分别0.58和0.53 mg·kg^-1,2017年进贤县稻田土壤速效钾含量平均值为73.2 mg·kg^-1。稻田土壤碱解氮在1982—2008年和2008—2017年两个阶段增长均呈先快后慢的趋势,前后两个阶段的年增长速率分别为1.24和0.29 mg·kg^-1,1982—2017年进贤县土壤碱解氮含量东南地区上升速率高,西北地区上升速率低。1982、2008和2017年进贤县稻田土壤综合肥力指数的平均值分别为0.43、0.50和0.55。3个时期稻田土壤肥力指标综合得分分别为：碱解氮有效磷>pH>速效钾>有机质（1982年）;pH>有效磷>速效钾>有机质>碱解氮（2008年）;速效钾>有效磷>pH>碱解氮>有机质（2017年）。【结论】经过35年的长期耕作,进贤县稻田土壤肥力得到改善。当前进贤县稻田土壤仍存在碱解氮过量、速效钾亏缺、土壤酸化严重等问题。土壤碱解氮、pH和速效钾分别为1982年、2008年和2017年3个时期造成进贤县稻田土壤肥力空间分布差异性的关键因素。     

Soil carbon change across ten New South Wales farms under different farm management regimes in Australia

This paper examines the potential influence of soil management and land use on soil carbon on cropping farms in New South Wales (NSW), Australia. Soil organic carbon (SOC) data from ten farms spatially distributed across NSW were examined on two occasions. Soil cores to a depth 0–30 cm were measured for SOC and, as expected, SOC in the A horizon (1.16%) was significantly (p < .001) greater than in the B horizon (0.74%) of all profiles. Analysis of the 2013 and 2015 SOC data indicated that in many ways, the results runs counter to other SOC studies in Australia. Importantly, the mean SOC concentration in these agricultural soils was significantly (p < .001) less under cropping (2013-1.05%, 2015-0.97%) than in native sites (2013-1.20%, 2015-1.16%). Out of the total of 35 sites sampled from 10 farms, SOC in 49% of sites did not change significantly over 2 years, in 17% it increased significantly, whereas in 34% it decreased. Further, a clear implication of drought on SOC was seen on sites that were uncropped based on a critical value for a 95% confidence interval (p < .05) and complemented by the significant correlation (p < .05) between average annual precipitation deficit (ANPD) and SOC across the state with R² = 0.39. The mean SOC was found to be directly proportional to standard deviation and standard error. In terms of spatial variability, the C0 (nugget) value was greatest for farms with a large mean SOC and the average variogram in this study has a range of approximately 200 m which is potentially useful in determining sampling spacing for soil carbon auditing purpose. Similar empirical data over more years are required to better estimate SOC levels and to determine whether at a farm scale, factors such as land management, land use and climate can be related to soil carbon change and variability.     

Unravelling the relationship between a seasonal environment and the dynamics of forage growth in grazed swards

It is well reported in the scientific literature that pastures can have similar net forage accumulation when managed with contrasting structures. However, we hypothesized that the dynamics of forage accumulation in grazed swards is linked to seasonal-environmental conditions. Marandu palisadegrass (Brachiaria brizantha [Hochst. ex A. Rich.] was used as the forage species model. The experimental treatments were four grazing heights (10, 20, 30 and 40 cm) allocated to experimental units according to a randomized complete block design with four replicates and evaluated throughout four contrasting environmental seasons (summer, autumn, winter–early spring and late spring). Under rainy and warm periods, greater net forage accumulation was observed in pastures maintained taller; on the contrary, during the mild and dry periods, net forage accumulation rate reduced as grazing height increased. Such patterns of responses were related to compensations between tiller population density and tissues flows during summer and late spring and the reduced capacity of taller canopies to compensate lower population with greater growth rates during autumn and winter–early spring. Grazed swards changed their patterns of forage growth as they transitioned from favourable to more abiotic stressful conditions, suggesting that seasonal adjustments in grazing intensities are necessary in order to maximize forage production.