Expression of Anti-Lipopolysaccharide Factor Isoform 3 in Chlamydomonas reinhardtii Showing High Antimicrobial Activity

Antimicrobial peptides are a class of proteins with antibacterial functions. In this study, the anti-lipopolysaccharide factor isoform 3 gene (ALFPm3), encoding an antimicrobial peptide from Penaeus monodon with a super activity was expressed in Chlamydomonas reinhardtii, which would develop a microalga strain that can be used for the antimicrobial peptide production. To construct the expression cluster, namely pH2A-Pm3, the codon optimized ALFPm3 gene was fused with the ble reporter by 2A peptide and inserted into pH124 vector. The glass-bead method was performed to transform pH2A-Pm3 into C. reinhardtii CC-849. In addition to 8 μg/mL zeocin resistance selection, the C. reinhardtii transformants were further confirmed by genomic PCR and RT-PCR. Western blot analysis showed that the C. reinhardtii-derived ALFPm3 (cALFPm3) was successfully expressed in C. reinhardtii transformants and accounted for 0.35% of the total soluble protein (TSP). Furthermore, the results of antibacterial assay revealed that the cALFPm3 could significantly inhibit the growth of a variety of bacteria, including both Gram-negative bacteria and Gram-positive bacteria at a concentration of 0.77 μM. Especially, the inhibition could last longer than 24 h, which performed better than ampicillin. Hence, this study successfully developed a transgenic C. reinhardtii strain, which can produce the active ALFPm3 driven from P. monodon, providing a potential strategy to use C. reinhardtii as the cell factory to produce antimicrobial peptides.     

高能湿法球磨制备鱼胶原肽螯合钙及其理化特征

为了拓展胶原肽螯合钙的制备工艺,以鱼胶原肽和CaCl2为原料,以高能湿法球磨的方法制备了鱼胶原肽螯合钙,并采用FT-IR红外图谱、X-衍射、EDS能谱等技术手段对其理化特性和结构进行分析。结果显示,随着球磨时间从15 min延长到240 min,鱼胶原肽的平均粒径逐渐降低,ZETA电位绝对值、分子量和pH值逐渐增加,鱼胶原肽对钙的螯合率逐渐从74.21%增加到85.42%。鱼胶原肽螯合钙与鱼胶原肽的主体化学结构相似,但是球磨后鱼胶原肽的-COO-1 VAS吸收峰(1 643.89 cm~(-1))向短波数方向移动到1 540～1 555 cm~(-1),而-NH2的特征吸收峰(3 313.9 cm~(-1))向长波数方向移动到3 337.12～3 380.59 cm~(-1)。球磨后鱼胶原在2θ=20°处的特征宽吸收峰消失,而在2θ=26、28、48和58°处出现多个尖峰和弥散峰,表明其从无定形结构向兼具结晶和无定形结构转变。鱼胶原肽螯合钙中钙元素比例逐渐增加,证实鱼胶原肽对钙的螯合率增加。研究表明,高能湿法球磨可应用于鱼胶原肽螯合钙的制备,该方法具有加工工艺简单、无污染和螯合率高等优点。     

Identifying and Characterizing a Novel Peritrophic Matrix Protein (MdPM-17) Associated With Antibacterial Response From the Housefly,Musca domestica (Diptera: Muscidae)

Peritrophic matrix/membrane (PM) critically prevents the midgut of insects from external invasion by microbes. The proteins in the peritrophic membrane are its major structural components. Additionally, they determine the formation and function of this membrane. However, the role of PM proteins in immune regulation is unclear. Herein, we isolated a novel PM protein (MdPM-17) from Musca domestica larvae. Further, the function of MdPM-17 in regulating host innate immunity was identified. Results showed that the cDNA of MdPM-17 full is 635 bp in length. Moreover, it consists of a 477-bp open reading frame encoding 158 amino acid residues. These amino acid residues are composed of two Chitin-binding type-2 domain (ChtBD2) and 19 amino acids as a signal peptide. Moreover, tissue distribution analysis indicates that MdPM-17 was enriched expressed in midgut, and moderate levels in the fat body, foregut, and malpighian tubule. Notably, MdPM-17 recombinant protein showed high chitin-binding capacity, thus belongs to the Class III PM protein group. MdPM-17 protein silencing via RNA interference resulted in the expression of antimicrobial peptide (defensin, cecropins, and diptericin) genes, and this occurred after oral inoculation with exogenous microbes Escherichia coli (Enterobacteriales:Enterobacteriaceae), Staphylococcus aureus (Bacillales:Staphylococcaceae), and Candida albicans (Endomycetales:Saccharomycetaceae)). Therefore, all the antimicrobial peptide (AMP) gene expression levels are high in MdPM-17-depleted larvae during microbial infection compared to controls. Consequently, these findings indicate that MdPM-17 protein is associated with the antibacterial response from the housefly.     

拖拉机动量飞轮主动防侧翻控制与模型试验研究

针对拖拉机侧翻致死致伤事故仍时有发生的问题，基于旋转刚体加速时的反向施矩原理，以反作用动量飞轮为执行元件，提出了从主动安全角度解决拖拉机侧翻问题的研究方法。通过构建拖拉机动力学系统数学模型，解析了整机侧翻行为的动态演变机理。为保证拖拉机主体结构的完整性，将动量飞轮置于拖拉机前部，取代传统静态配重的同时可主动提供防侧翻力矩。应用Matlab/Simulink软件，对反作用飞轮的回稳过程进行了基于PID控制的有效性仿真分析，同时设计并搭建了比例模型试验平台，对主动施矩飞轮的回稳控制效果进行了试验验证。结果表明，装备飞轮的拖拉机与无控制组对比，在一次试验中可多次实现整机的防侧翻控制，使整机防侧翻性能得到明显改善，且不同行驶工况下的试验数据与仿真结果的相关性较强，充分验证了本文拖拉机侧翻动力学模型的有效性。     

铁、锌配施对藿香生长、产量及有效成分的影响

为探究铁、锌不同浓度配施对藿香生长、产量及有效成分的影响,以藿香为试验材料,采用双因素随机区组设计,研究铁锌配施对藿香生长、产量、各部位铁锌分配及挥发油含量的影响。结果表明,叶面喷施铁锌肥能显著影响藿香的生长发育、产量和挥发油成分,显著影响藿香的抗氧化酶活性,显著提高淀粉、还原糖、可溶性糖和可溶性蛋白含量,通过影响氮代谢相关酶活性间接影响植株氮代谢过程。叶面施铁对藿香产量的影响大于叶面施锌,铁锌配施的增产效果优于单独施用铁、锌肥;综合考虑藿香的生长和产量指标,认为Fe₁Zn₁(0.2%铁,0.1%锌)为铁锌配施的最优处理组合。施用锌铁肥能够增加藿香各部位的铁锌含量,锌在不同部位的含量呈叶>根>茎的变化趋势,铁在不同部位的含量呈根>叶>茎的变化趋势,锌主要集中在藿香叶片,而铁则主要集中在藿香根部。高浓度的叶面施铁有助于叶片Ca含量的累积。铁锌配施能显著提高藿香挥发油含量,并影响挥发油成分,其中D-柠檬烯含量最高,在Fe₁Zn₂处理时达到最大值(22.99%)。综上,合理配施铁锌肥能提高藿香主要化学成分的含量,但过高浓度的施用增产效果并不明显。本研究结果为铁锌肥在藿香生产中的应用提供了理论依据。     

基于毛细管网的日光温室主动式集放热系统研究

为了对比日光温室传统保温蓄热后墙与基于毛细管网的主动式集放热系统（AHSCTM）的集放热性能，对AHSCTM的集放热性能进行了测试，构建了AHSCTM水温模型，利用一维差分法对相同环境条件下的外保温复合墙（370mm黏土砖和100mm聚苯乙烯板复合而成）日间储热量和夜间放热量进行了模拟。结果表明，AHSCTM的日间储热量和夜间放热量分别为相同条件下外保温复合墙的84.4%~111.3%和74.8%~100.7%，AHSCTM的COP（Coefficient of performance）为1.1~2.4。在夜间运行期间，AHSCTM放热量是相同时间段内外保温复合墙的98.2%~172.5%。因此，与外保温复合墙相比，AHSCTM有利于提高室内最低气温。改进AHSCTM的日间储热量和夜间放热量得到大幅提升，分别较外保温复合墙高67.6%~112.1%和69.0%~128.3%，COP可达2.8~7.0。改进AHSCTM的储放热性能优于外保温复合墙，说明利用改进AHSCTM配合保温墙体替代传统保温蓄热后墙是可行的。     

不同氮素水平下双季稻株型与冠层内光截获特征研究

本文旨在阐明双季稻株型与冠层内光合有效辐射截获的时空分布特征。选用4个不同株型早、晚稻品种,设置4个不同施氮水平,系统观测其植株形态和冠层内光合有效辐射截获率(IPAR)的时空分布状况。结果表明,施氮水平对早、晚稻株高、穗长、叶长和叶基角均有显著影响,均表现为随施氮水平的增加而增大;早、晚稻孕穗期的分层叶面积指数(LAI)和向上累积LAI大于抽穗后12 d,分层LAI呈冠层中部大于上部和下部的分布特征,最大分层LAI出现在0.58相对高度处;冠层上中部分层LAI和向上累积LAI随施氮水平的增加而增大;向上累积LAI随相对高度呈S型曲线分布,可用Logistic方程定量描述(R~2 0.99);早、晚稻孕穗期的冠层IPAR大于抽穗后12 d,且随施氮水平的增加而增大,其日变化表现为正午较小,早晚较大;株型紧凑的早、晚稻品种,冠层IPAR低;冠层IPAR与向下累积LAI之间的关系可用方程IPAR=a (1-e~(-b×LAI))定量描述(R~2 0.88);冠层内IPAR的三维空间分布表现为冠层上中部水平面上IPAR较低,光斑变化大,冠层下部水平面上IPAR较高,光斑变化较平缓,同一冠层高度水平面上的IPAR呈不均匀分布。研究结果可为双季稻高产栽培及理想株型的优化设计提供支撑。     

可可毛色二孢全基因组分泌蛋白的预测及分析

【目的】可可毛色二孢（Lasiodiplodia theobromae）是一种世界性分布的重要植物病原真菌,可引起严重的葡萄溃疡病（Botryosphaeria dieback）,影响果木品质并造成巨大的经济损失。本研究预测并分析可可毛色二孢基因组范围内的分泌蛋白,并明确其基本特征,为该病菌分泌蛋白致病机理的研究打下基础。【方法】依据已公布的可可毛色二孢全基因组序列,利用信号肽预测软件SignalP v5.0、跨膜结构分析软件TMHMM v2.0、细胞器定位分析软件ProtComp v9.0、GPI锚定预测软件big-PI Fungal Predictor和亚细胞器定位分析软件TargetP v2.0生物信息学软件对该菌中的典型分泌蛋白进行筛选。对分泌蛋白N端信号肽的长度、氨基酸使用频率及其切割位点进行统计分析。依据蛋白序列的同源性,应用BLASTP程序对分泌组蛋白进行功能注释分析,预测其生物学功能。采用蔗糖酶缺陷的酵母分泌系统,对所选分泌蛋白的信号肽进行活性检测。利用qRT-PCR方法检测所选分泌蛋白基因在可可毛色二孢侵染葡萄中的表达情况。【结果】在可可毛色二孢全基因组编码蛋白中共筛选获得552个潜在的具有典型信号肽的分泌蛋白,占全基因组预测蛋白总数的4.3%,其编码蛋白长度集中于101—400 aa。信号肽统计分析表明,其信号肽长度以18—20 aa的序列最为集中,信号肽长度为20 aa的蛋白数量最多。信号肽中使用频率最高的氨基酸为丙氨酸;非极性、疏水的氨基酸使用频率最高,占氨基酸总数的60.2%。其信号肽的-3至-1位置上的氨基酸相对保守,切割位点属于A-X-A类型,可被Sp I型信号肽酶识别并切割。336个分泌蛋白具有功能注释,其功能较多集中于细胞壁降解有关的酶类以及致病相关蛋白,并且这些蛋白在分子量、等电点、脂肪族氨基酸指数等方面均存在差异。通过蔗糖酶缺陷的酵母分泌系统证实,挑选的9个分泌蛋白信号肽均具有分泌活性。qRT-PCR检测结果表明,所选分泌蛋白基因在该病菌侵染初期的表达发生变化。【结论】利用生物信息学分析技术从可可毛色二孢全基因组中共预测获得552个经典分泌蛋白。其信号肽氨基酸长度分布广泛,氨基酸组成中非极性、疏水的氨基酸使用频率最高。功能注释主要集中在细胞壁组分降解相关的酶类、致病侵染相关的坏死诱导相关蛋白以及几丁质结合蛋白等。     

沙棘果实中主要活性成分质量分布

沙棘作为一种抗逆性、适应性、萌蘖性较强的植物资源，被广泛应用于黄土丘陵地区荒山绿化、土壤改良、水土保持和砒砂岩治理。沙棘不仅具有良好的生态价值，其果实中富含多种生物活性物质。本文凝练了沙棘果实中活性化合物的构成、分布及活性特征，并对沙棘果实活性物质的研究进行展望。基于相关文献，针对黄酮、花青素、酚酸、有机酸、肌醇、维生素、类胡萝卜素、不饱和脂肪酸、甾醇等广泛存在于沙棘果实中的化学成分进行总结分析，提供沙棘果实中上述活性物质分布情况的综合信息。研究表明，针对沙棘果实中活性物质的构成、分布和活性特征的研究较为丰富，但在部分领域仍缺乏研究，如栽培技术条件对活性物质的影响，活性物质与沙棘果实加工中异味的关联性等。本文为沙棘果实在食品、药品及保健品行业中的综合利用提供了理论支撑，为促进沙棘资源在经济中的全面发展提供参考。      

Modulation of plant root traits by nitrogen and phosphate: transporters,long-distance signaling proteins and peptides,and potential artificial traps

As sessile organisms, plants rely on their roots for anchorage and uptake of water and nutrients. Plant root is an organ showing extensive morphological and metabolic plasticity in response to diverse environmental stimuli including nitrogen (N) and phosphorus (P) nutrition/stresses. N and P are two essential macronutrients serving as not only cell structural components but also local and systemic signals triggering root acclimatory responses. Here, we mainly focused on the current advances on root responses to N and P nutrition/stresses regarding transporters as well as long-distance mobile proteins and peptides, which largely represent local and systemic regulators, respectively. Moreover, we exemplified some of the potential pitfalls in experimental design, which has been routinely adopted for decades. These commonly accepted methods may help researchers gain fundamental mechanistic insights into plant intrinsic responses, yet the output might lack strong relevance to the real situation in the context of natural and agricultural ecosystems. On this basis, we further discuss the established—and yet to be validated—improvements in experimental design, aiming at interpreting the data obtained under laboratory conditions in a more practical view.