中华补血草内生与根际具ACC脱氨酶活性细菌的筛选及其生物多样性

【目的】获得江苏沿海滩涂盐生药用植物中华补血草内生及根际具有1-氨基环丙烷-1-羧酸(ACC)脱氨酶活性的细菌,研究其遗传多样性和潜在促生活性。【方法】从中华补血草和根际土壤分离筛选具有ACC脱氨酶活性的菌株,对其ACC脱氨酶活性定量检测,通过16S r RNA基因序列分析确定菌株系统发育地位。同时研究其固氮、溶磷、产植物生长素吲哚乙酸(IAA)及耐盐能力。【结果】分离筛选获得18株具有ACC脱氨酶活性的内生与根际细菌,定量检测发现其中有13株菌的ACC脱氨酶含量在20 nmolα-KA/(mg Pr·h)以上,有11株菌可以固氮,7株菌能够解磷,9株菌产生IAA。菌株的Na Cl盐耐受范围多数在0–13%之间。16S r RNA基因测序表明,活性菌株分属于7个属,多样性丰富,节杆菌属(Arthrobacter)为优势类群。其中菌株KLBMP 5180为节杆菌属的潜在新种。【结论】江苏沿海滩涂盐生药用植物中华补血草共生环境中具有丰富多样的具ACC脱氨酶活性的菌株,并存在潜在新物种资源,具有进一步研究价值。     

A simple and biosafe method for isolation of human umbilical vein endothelial cells

Human umbilical vein endothelial cells (HUVEC_S) are used as an irreplaceable tool for the study of vascular diseases. However, the technicians who isolate HUVECs are largely exposed to potential infectious threats. Here we report the development of a specialized instrument to protect researchers from known or unknown infectious agents when they operate on human umbilical cords. This instrument can be assembled by common laboratory supplies and adapted to accommodate umbilical cords of different lengths. When the cord is enclosed within the instrument, the risk of sample contamination and operator infection is greatly reduced. Using our instrument, endothelial cells were successfully isolated from human umbilical veins without contamination. The cells were verified by their cobblestone-like morphology and by immunofluorescence staining (Factor VIII and CD31 positivity and α-SMA negativity). Our instrument simplifies and optimizes the cell extraction process, and most importantly elevates the biosafety to a higher level during the isolation of human umbilical vein endothelial cells.     

Polymerase chain reaction–hybridization method using urease gene sequences for high-throughput Ureaplasma urealyticum and Ureaplasma parvum detection and differentiation

In this article, we discuss the polymerase chain reaction (PCR)–hybridization assay that we developed for high-throughput simultaneous detection and differentiation of Ureaplasma urealyticum and Ureaplasma parvum using one set of primers and two specific DNA probes based on urease gene nucleotide sequence differences. First, U. urealyticum and U. parvum DNA samples were specifically amplified using one set of biotin-labeled primers. Furthermore, amine-modified DNA probes, which can specifically react with U. urealyticum or U. parvum DNA, were covalently immobilized to a DNA–BIND plate surface. The plate was then incubated with the PCR products to facilitate sequence-specific DNA binding. Horseradish peroxidase–streptavidin conjugation and a colorimetric assay were used. Based on the results, the PCR–hybridization assay we developed can specifically differentiate U. urealyticum and U. parvum with high sensitivity (95%) compared with cultivation (72.5%). Hence, this study demonstrates a new method for high-throughput simultaneous differentiation and detection of U. urealyticum and U. parvum with high sensitivity. Based on these observations, the PCR–hybridization assay developed in this study is ideal for detecting and discriminating U. urealyticum and U. parvum in clinical applications.     

Metabolic regulation of ammonia emission in different senescence phenotypes of Nicotiana tabacum

In order to reveal the character of ammonia emission in senescent tobacco (Nicotiana tabacum), the content of NH₄⁺, total nitrogen, and soluble protein, and the activities of nitrogen metabolism-related enzymes were measured in leaves of a quick-leaf-senescence phenotype ZY90 and a slow-leaf-senescence phenotype NC89. Compared with NC89, ZY90 had a higher NH₄⁺ accumulation, a lower glutamine synthetase activity, and a significantly higher stomatal ammonia compensation point, and ammonia emission during 40 to 60 d after leaf emergence. During senescence, the quick-leafsenescence phenotype was characterized by nitrogen re-transfer by ammonia emmission, whereas the slow-leafsenescence phenotype by nitrogen re-assimilation. The ammonia emission was primarily regulated by glutamine synthetase activity, apoplastic pH, and NH₄⁺ content.     

A maize ADP‐ribosylation factor ZmArf2 increases organ and seed size by promoting cell expansion in Arabidopsis

ADP‐ribosylation factors (ARFs) are small GTP‐binding proteins that regulate a wide variety of cell functions. Previously, we isolated a new ARF, ZmArf2, from maize (Zea mays). Sequence and expression characteristics indicated that ZmArf2 might play a critical role in the early stages of endosperm development. In this study, we investigated ZmArf2 function by analysis of its GTP‐binding activity and subcellular localization. We also over‐expressed ZmArf2 in Arabidopsis and measured organ and cell size and counted cell numbers. The expression levels of five organ size‐associated genes were also determined in 35S::ZmArf2 transgenic and wild‐type plants. Results showed that the recombinant ZmArf2 protein purified from Escherichia coli exhibited GTP‐binding activity. Subcellular localization revealed that ZmArf2 was localized in the cytoplasm and plasma membrane. ZmArf2 over‐expression in Arabidopsis showed that 35S::ZmArf2 transgenic plants were taller and had larger leaves and seeds compared to wild‐type plants, which resulted from cell expansions, not an increase in cell numbers. In addition, three cell expansion‐related genes, AtEXP3, AtEXP5 and AtEXP10, were upregulated in 35S::ZmArf2 transgenic lines, while the expression levels of AtGIF1 and AtGRF5, were unchanged. Collectively, our studies suggest that ZmArf2 has an active GTP‐binding function, and plays a crucial role in growth and development in Arabidopsis through cell expansion mediated by cell expansion genes.