药用植物灯盏花种子储存条件及萌发特性研究

该文以云南春季野外采收灯盏花种子为材料,研究了不同储存湿度、储存温度、储存时间及不同光照、温度、吸胀条件等对其萌发率的影响。结果表明:(1)降低储存湿度(15%RH)和储存温度(-20、4℃)有利于种子保存,种子寿命可延长2 a多,萌发率在80%以上;高温(35、45℃)和高湿(60%RH)加快了种子衰老,不利于保存,种子为短命种子。(2)光照与否对种子萌发率无显著影响,为光中性种子,但光照有利于种子萌发后幼苗的形成。(3) 25℃为该种子萌发的最适宜温度,萌发率可达84.37%。(4)生产上播种时,尽量避免低温(4℃)和高温(30、35℃),有利于提高出苗率。(5)该种子对吸胀冷害不敏感,为抗冷型种子。因此,在种子研究和药材生产上,成熟种子采收后应及时干燥并密封存于低温下,来年春季及早播种;在育苗生产上,应提供适宜的光照条件,设置单独的恒温(25℃)育苗间,并避免低温季播种。通过研究灯盏花种子的适宜储存条件和萌发特性,为该珍稀药用物种的合理储存和高产栽培提供有效指导。     

Development of high‐resolution DNA barcodes for Dioscorea species discrimination and phylogenetic analysis

The genus Dioscorea is widely distributed in tropical and subtropical regions, and is economically important in terms of food supply and pharmaceutical applications. However, DNA barcodes are relatively unsuccessful in discriminating between Dioscorea species, with the highest discrimination rate (23.26%) derived from matK sequences. In this study, we compared genic and intergenic regions of three Dioscorea chloroplast genomes and found that the density of SNPs and indels in intergenic sites was about twice and seven times higher than that of SNPs and indels in the genic regions, respectively. A total of 52 primer pairs covering highly variable regions were designed and seven pairs of primers had 80%–100% PCR success rate. PCR amplicons of 73 Dioscorea individuals and assembled sequences of 47 Dioscorea SRAs were used for estimating intraspecific and interspecific divergence for the seven loci: The rpoB‐trnC locus had the highest interspecific divergence. Automatic barcoding gap discovery (ABGD), Poisson tree processes (PTP), and generalized mixed Yule coalescence (GMYC) analysis were applied for species delimitation based on the seven loci and successfully identified the majority of species, except for species in the Enantiophyllum section. Phylogenetic analysis of 51 Dioscorea individuals (28 species) showed that most individuals belonging to the same species tended to cluster in the same group. Our results suggest that the variable loci derived from comparative analysis of plastid genome sequences could be good DNA barcode candidates for taxonomic analysis and species delimitation.     

Efficient and green aqueous−solid system for transphosphatidylation to produce phosphatidylhydroxybutyrate: Potential drugs for central nervous system's diseases

The synthesis of nonnatural phospholipid, phosphatidylhydroxybutyrate (PB), was firstly introduced by phospholipase D (PLD)-mediated transphosphatidylation of phosphatidylcholine (PC) with sodium γ-hydroxybutyrate (NaGHB) in the aqueous–solid system. Nanoscale silicon dioxide (NSD) was employed as a carrier to provide an “artificial interphase” between PC and PLD. Special attention has been paid to the effect of the PC coverage on the surface area of hybrids of NSD-PC, the PC loading and the yield of PB. Results indicated that the highest PC loading of 98.3% and the highest PB yield of 97.3% were achieved. In addition, the free PLD in the aqueous–solid system showed the greater stability and pH tolerance than that in the traditional liquid–liquid system. The operational stability of free PLD solution was investigated. The yield of PB remained 70.7% after being used for five batches. The authors provide a new idea for drug design and the potential source of PB for medical experiments. PB is a potential drug and may have the excellent performance in the treatment of central nervous system's diseases. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2726, 2019     

ERK1/2-p90RSK-mediated phosphorylation of Na+/H+ exchanger isoform 1. A role in ischemic neuronal death

The function and regulation of Na(+)/H(+) exchanger isoform 1 (NHE1) following cerebral ischemia are not well understood. In this study, we demonstrate that extracellular signal-related kinases (ERK1/2) play a role in stimulation of neuronal NHE1 following in vitro ischemia. NHE1 activity was significantly increased during 10-60 min reoxygenation (REOX) after 2-h oxygen and glucose deprivation (OGD). OGD/REOX not only increased the V(max) for NHE1 but also shifted the K(m) toward decreased [H(+)](i). These changes in NHE1 kinetics were absent when MAPK/ERK kinase (MEK) was inhibited by the MEK inhibitor U0126. There were no changes in the levels of phosphorylated ERK1/2 (p-ERK1/2) after 2 h OGD. The p-ERK1/2 level was significantly increased during 10-60 min REOX, which was accompanied by nuclear translocation. U0126 abolished REOX-induced elevation and translocation of p-ERK1/2. We further examined the ERK/90-kDa ribosomal S6 kinase (p90(RSK)) signaling pathways. At 10 min REOX, phosphorylated NHE1 was increased with a concurrent elevation of phosphorylation of p90(RSK), a known NHE1 kinase. Inhibition of MEK activity with U0126 abolished phosphorylation of both NHE1 and p90(RSK). Moreover, neuroprotection was observed with U0126 or genetic ablation or pharmacological inhibition of NHE1 following OGD/REOX. Taken together, these results suggest that activation of ERK1/2-p90(RSK) pathways following in vitro ischemia phosphorylates NHE1 and increases its activity, which subsequently contributes to neuronal damage.     

High performance liquid chromatographic determination of 1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]-ethane (BBSKE), a novel organoselenium compound, in dog plasma using pre-column derivatization and its application in pharmacokinetic study

A novel HPLC-UV method with pre-column derivatization by using 2-mercaptoethanol was established for determination of 1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]-ethane (BBSKE) in dog plasma. The derivatives were identified by mass spectrometry. The method had a good linear range of 0.05-2 microg/ml (r(2)=0.9995). The lower limit of quantification (LOQ) was 0.05 microg/ml. The precision and accuracy were less than 7%. After dosing of BBSKE (30 mg/kg, p.o. and 0.79 mg/kg, i.v.) in dogs, AUC(0-t) were 5.72+/-2.42 and 1.35+/-0.41 microg h/ml; t(1/2) were 4.6+/-2.1 and 1.7+/-0.6h, respectively. The method was successfully applied to the pharmacokinetic study in dogs.     

Antisense RNA-mediated suppression of Bmi-1 gene expression inhibits the proliferation of lung cancer cell line A549

The oncogene Bmi-1 regulates cell proliferation and senescence. It is reported that it controlled the self-renewal of leukemic and breast cancer stem cell and was overexpressed in some solid tumors and hematologic malignancies. In this study, the effects of inactivation of Bmi-1 mediated by a plasmid-expressing antisense Bmi-1 RNA on the proliferation of lung cancer cell line A549 were investigated. As a result, when the plasmid was stably introduced into the cell line, the Bmi-1 protein level was specifically downregulated, and the cell proliferation was significantly inhibited as shown by the cell growth curve and colony forming assay. The cells were found mostly in the phase of G(0)/G(1) and cells in S phase were significantly decreased. Our results suggest that targeting Bmi-1 might be a therapeutic potential for the treatment of non-small-cell lung cancer.     

Highly efficient genome editing in Xanthomonas oryzae pv. oryzae through repurposing the endogenous type I-C CRISPR-Cas system

Efficient and modular genome editing technologies that manipulate the genome of bacterial pathogens will facilitate the study of pathogenesis mechanisms. However, such methods are yet to be established for Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of rice bacterial blight. We identified a single type I-C CRISPR-Cas system in the Xoo genome and leveraged this endogenous defence system for high-efficiency genome editing in Xoo. Specifically, we developed plasmid components carrying a mini-CRISPR array, donor DNA, and a phage-derived recombination system to enable the efficient and programmable genome editing of precise deletions, insertions, base substitutions, and gene replacements. Furthermore, the type I-C CRISPR-Cas system of Xoo cleaves target DNA unidirectionally, and this can be harnessed to generate large genomic deletions up to 212 kb efficiently. Therefore, the genome-editing strategy we have developed can serve as an excellent tool for functional genomics of Xoo, and should also be applicable to other CRISPR-harbouring bacterial plant pathogens.     

油葵苗期抗旱相关性状的QTL定位及候选基因筛选

干旱是限制向日葵生长发育的重要因素之一。为探究向日葵苗期抗旱性分子机制,该研究以向日葵K55与K58杂交构建的150个F₇重组自交系群体为材料,对其在正常浇水和干旱胁迫两种水分处理条件下的叶片相对电导率、叶绿素含量、叶面积、叶片相对含水量、根长进行表型测定,利用前期建立的SNP、SSR分子标记遗传连锁图谱,通过复合区间作图法对5个抗旱相关的性状进行QTL定位。结果表明：(1)共定位到向日葵QTL位点11个,其中正常浇水条件下5个,干旱胁迫条件下6个,表型贡献率为0.768%~7.547%,且5号连锁群上定位到的QTL位点最多(3个)。(2)QTL置信区间内共筛选到62个与干旱相关的候选基因,包括位于qLA 8 1上的rna23019、rna23004、rna22661、rna22193、rna23294、rna22783和位于qCC 13 1上的rna40140,这些基因可作为后续基因克隆及功能研究的重点候选基因。该研究结果为向日葵抗旱性研究及其遗传改良奠定了基础。     

矮化香蕉及其野生型GA3ox基因的结构特点和表达分析

香蕉的矮化突变是香蕉无性繁殖后代最常见的表型变异之一,但其变异的分子调控机理目前尚未研究清楚; 而内源赤霉素是影响植物株高的重要激素之一,GA3-氧化酶是赤霉素生物合成后期的关键酶。为探究GA3-氧化酶编码基因对香蕉矮化的分子调控机理,该研究以威廉斯B6矮化突变体及其野生型亲本为材料,通过RT-PCR技术克隆得到矮化香蕉及其野生型亲本GA3ox基因的全长cDNA序列,并对其推测的氨基酸序列进行比对分析,同时利用qRT-PCR技术对GA3ox基因在不同组织中的表达水平差异进行分析。结果表明:(1)矮化香蕉GA3ox-A和野生型香蕉GA3ox-G的ORF长度均为864 bp,均编码287个氨基酸,经序列比对分析发现两条氨基酸序列之间存在5个位点的差异,从而产生具有不同性质的蛋白质。(2)氨基酸序列同源性分析表明,矮化香蕉GA3ox的氨基酸序列与油棕、海枣、椰子的同源性最高。(3)qRT-PCR显示,GA3ox基因在矮化香蕉叶片和茎秆中的表达水平整体上低于野生型,其中GA3ox在野生型茎秆中的表达水平是矮化植株的2.2～32倍。综上推测,GA3ox基因可能对香蕉茎杆的矮化变异具有重要的调控作用。该研究结果为揭示香蕉矮化突变的分子机制与筛选优良矮化香蕉株系奠定了基础。