大豆主要过敏原Gly m Bd30K蛋白单克隆抗体的制备与应用

利用大豆主要过敏原Gly m Bd 30K蛋白抗原表位蛋白为免疫原免疫BALB/c小鼠,取免疫小鼠脾细胞与小鼠骨髓瘤NS-1细胞融合.采用半固体培养基法和有限稀释法相结合的方法快速筛选获得稳定分泌的特异性杂交瘤细胞,用杂交瘤细胞株诱生小鼠腹水,应用蛋白A亲和层析法进行抗体纯化.采用Ig类与亚类鉴定试剂盒鉴定该单克隆抗体...     

Nitrate leaching in a silage maize field under different irrigation and nitrogen fertilizer rates

Quantification of the interactive effects of nitrogen (N) and water on nitrate (NO₃) loss provides an important insight for more effective N and water management. The goal of this study was to evaluate the effect of different irrigation and nitrogen fertilizer levels on nitrate-nitrogen (NO₃-N) leaching in a silage maize field. The experiment included four irrigation levels (0.7, 0.85, 1.0, and 1.13 of soil moisture depletion, SMD) and three N fertilization levels (0, 142, and 189 kg N ha⁻¹), with three replications. Ceramic suction cups were used to extract soil solution at 30 and 60 cm soil depths for all 36 experimental plots. Soil NO₃-N content of 0-30 and 30-60-cm layers were evaluated at planting and harvest maturity. Total N uptake (NU) by the crop was also determined. Maximum NO₃-N leaching out of the 60-cm soil layer was 8.43 kg N ha⁻¹, for the 142 kg N ha⁻¹ and over irrigation (1.13 SMD) treatment. The minimum and maximum seasonal average NO₃ concentration at the 60 cm depth was 46 and 138 mg l⁻¹, respectively. Based on our findings, it is possible to control NO₃ leaching out of the root zone during the growing season with a proper combination of irrigation and fertilizer management.     

栽培大豆蛋白亚基11S/7S组成及过敏蛋白缺失分析

以175份中国大豆品种资源为材料,利用SDS-PAGE电泳和Western杂交技术,分析蛋白亚基11S和7S的含量,以及Gly m Bd 28K和 Gly m Bd 30K两种过敏蛋白的缺失情况。结果表明,参试品种的11S和7S的含量呈极显著负相关（r＝－0.95）,11S/7S比值范围为0.77～4.67,并鉴定出1份自然缺失β亚基的材料。在175个品种中没有检测到缺失30     

Ecological intensification of cropping systems enhances soil functions,mitigates soil erosion,and promotes crop resilience to dry spells in the Brazilian Cerrado

Water scarcity threatens global food security and agricultural systems are challenged to achieve high yields while optimizing water usage. Water deficit can be accentuated by soil physical degradation, which also triggers water losses through runoff and consequently soil erosion. Although soil health in cropping systems within the Brazilian Cerrado biome have been surveyed throughout the years, information about soil erosion impacts and its mitigation are still not well understood; especially concerning the role of cropping system diversification and its effects on crop yield. Thus, the aim of this study was to assess whether ecological intensification of cropping systems –inclusion of a consorted perennial grass and crop rotation– could promote soil coverage and consequently decrease water erosion and soil, water, and nutrient losses. This work studied the effects of crop rotation and consorted Brachiaria, along with different levels of investment in fertilization on soil physical quality and on soil, water, and nutrient losses, and crop yields. Results proved that soybean monoculture (SS) is a system of low sustainability even under no-till in the Brazilian Cerrado conditions. It exhibited high susceptibility to soil, water, and nutrient losses, causing low crop yields. Our results showed that water losses in SS cropping system were approximately 10% of the total annual rainfall, and total K losses would require an additional 35% of K application. Conversely, ecological intensification of cropping systems resulted in enhanced soil environmental and agronomic functions, increased grain yield, and promoted soil and water conservation: high soil cover rate, and low soil, water and nutrient losses. Ecological intensification proved to be an adequate practice to boost crop resilience to water deficit in the Brazilian Cerrado.     

抗Gly m Bd 28K单克隆抗体的制备及其免疫学特性

利用Gly m Bd 28K天然蛋白免疫BALB/c雌性小鼠,取小鼠脾脏细胞与杂交瘤细胞融合,筛选出2株能稳定分泌抗Gly m Bd 28K单克隆抗体的杂交瘤细胞株1E3和2F4。体内诱生腹水法大量制备单克隆抗体,经纯化后,得到2株均为IgG1型的单克隆抗体mAb1E3和mAb2F4。间接ELISA方法测得mAb1E3的效价达到1∶4 10×105,对Gly m Bd 28K蛋白的半数抑制率（IC50）为23.99 μg·L-1,与花生蛋白、核桃蛋白等的交叉反应率均低于0.1%。该单克隆抗体的制备为Gly m Bd 28K致敏蛋白的检测以及抗原表位的鉴定奠定了基础。     

大豆主要过敏原Gly m Bd 30K基因的克隆及其原核表达载体的构建

利用RT-PCR克隆大豆主要过敏原Gly m Bd 30K的全长基因,根据序列设计带有酶切位点的特异性引物,扩增大豆GlY m Bd 30K的完整开放阅读框,与pET一28a载体连接,构建原核表达载体.结果表明:克隆了大豆主要过敏原Gly m Bd 30K基因,且构建了其原核表达载体.该基因含有长度为1 140 bp的开放阅读框,编码379个氨基酸.该蛋白质的相对分子质量为42 758,等电点为5.08.序列同源性分析发现其与数据库中已知的Gly m Bd30K基因同源性很高,因此认为其是大豆的过敏原基因,在GenBank数据库中的登录号为EU883600.克隆的大豆主要过敏原Gly m Bd 30K基因及构建的原核表达载体,为大豆主要过敏原Gly m Bd 30K的重组表达和免疫活性鉴定等奠定基础.     

利用大豆核心种质部分样本鉴定28K和30K过敏蛋白缺失材料

大豆核心种质是筛选优异基因的重要物质基础。本研究以栽培大豆(G. max)核心种质经聚类随机选择样本为主,以栽培大豆保留种质和野生大豆（G. soja）为对照,利用小鼠单克隆抗体Gly m Bd 30K(F5) 和Gly m Bd 28K(C5)分别检测大豆的30K和28K过敏蛋白抗原,目的是发掘过敏蛋白缺失的品种资源,明确其分布规律和特点,检测大豆核心种质的代表性,为资源和育种研究提供参考。鉴定结果表明,在参试的60份野生大豆和421份栽培大豆中没有发现30K过敏蛋白缺失的种质,但28K过敏蛋白缺失率分别为13.3%和37.8%。栽培大豆核心种质28K过敏蛋白缺失比率的变化趋势与栽培大豆保留种质总体规律基本相同,在3个生态区的缺失比率差异均不显著,说明核心种质在28K过敏蛋白缺失这个性状上具有代表性。SSR标记分析发现,在缺失过敏蛋白的种质中,80%相似系数在0.44以下,表明这些种质遗传基础较为广泛,可充分利用类群内或类群间差异较大的材料作为亲本配制杂交组合,培育缺失28K过敏蛋白优质品种。     

二穗短柄草Bd21的形态学观察

二穗短柄草（Brachypodium distachyon （L.） Beauv）属于早熟禾亚科。Bd21作为二穗短柄草不同生态型中的一种,在个体大小、基因组大小、生长条件和生长周期等方面与拟南芥类似,并且具有禾本科植物许多共性,如抗病性、颖果等。Bd21的这些特点说明它是一种合适的禾本科模式植物。采用土培和水培的方法观察二穗短柄草的生长特性,利用扫描电镜观察生长锥的发育情况,结果为进一步研究二穗短柄草发育的分子机理提供重要依据。     

大豆主要过敏原Gly m Bd 30K的抗原表位区基因的克隆表达、纯化及免疫原性鉴定

根据文献并结合生物信息学方法选取大豆主要过敏原Gly m Bd 30k的抗原表位区,采用PCR方法扩增出该抗原表位区基因片段,并通过酶切连接分别构建单体的pET表达载体(pET-sGly)和二聚体的pET表达载体(pET-dGly),重组质粒转化到大肠杆菌BL21(DE3)plysS,经IPTG诱导后进行SDS-PAGE分析;同时用Ni2+离子亲和层析柱纯化表达的sGly和dGly抗原表位蛋白,用western-blotting和ELISA检测重组蛋白的免疫原性。结果表明:表达的单体sGly蛋白以可溶性表达为主,而其二聚体dGly蛋白以包涵体形式存在,纯化的sGly和dGly蛋白都具有较好的免疫原性,重组蛋白sGly的抗原性更佳。成功构建的大豆主要过敏原Gly m Bd 30k蛋白的抗原表位区单体sGly蛋白及其二聚体dGly蛋白的工程菌,为研制大豆主要过敏原的单克隆抗体以制备用于大豆主要过敏原检测的试剂奠定了基础。