A Nucleus-encoded Topological Specificity Factor PpMinE in Physcomitrella patens has Conserved Function Similar to Its Chloroplast-encoded Ancestor

A nucleus-encoded MinE gene, designated PpMinE, from Physcomitrella patens was identified using RT-PCR. The presence of both N- and C-terminal extensions in PpMinE protein suggested its cyanobacterial origin. The transient expression of PpMinE using green fluorescent protein fusion in tobacco （Nicotiana tabacum L.） indicated that the PpMinE was a chloroplast-targeted protein. Overexpression of PpMinE in Escherichia coli caused division site misplacement and minicell formation, suggesting evolutionary functional conservation of MinE during plant phylogenesis. According to the phylogenetic tree, PpMinE protein has a close relationship with the highland plants, which suggests that the transfer events of MinE gene from plastid to nucleus might have occurred before the origin of the land plants.

Heterologous expression of Hp BHY and Cr BKT increases heat tolerance in Physcomitrella patens

Heat stress can restrict plant growth,development,and crop yield.As essential plant antioxidants,carotenoids play significant roles in plant stress resistance.b-carotene hydroxylase(BHY)and b-carotene ketolase(BKT),which catalyze the conversions of b-carotene to zeaxanthin and b-carotene to canthaxanthin,respectively,are key enzymes in the carotenoid biosynthetic pathway,but little is known about their potential functions in stress resistance.Here,we investigated the roles of b-carotene hydroxylase and b-carotene ketolase during heat stress in Physcomitrella patens through expressing a b-carotene ketolase gene from Chlamydomonas reinhardtii(Cr BKT)and a b-carotene hydroxylase gene from Haematococcus pluvialis(Hp BHY)in the moss P.patens.In transgenic moss expressing these genes,carotenoids content increased(especially lutein content),and heat stress tolerance increased,with reduced leafy tissue necrosis.To investigate the mechanism of this heat stress resistance,we measured various physiological indicators and found a lower malondialdehyde level,higher peroxidase and superoxide dismutase activities,and higher endogenous abscisic acid and salicylate content in the transgenic plants in response to high-temperature stress.These results demonstrate that Cr BKT and Hp BHY increase plant heat stress resistance through the antioxidant and damage repair metabolism,which is related to abscisic acid and salicylate signaling.

狐米草(Spartina patens)低能重离子突变系营养成分分析

为从经低能重离子诱变的狐米草突变系中筛选出适合在滩涂生长、营养丰富的牧草株系,在前文比较了模拟滩涂环境种植的各突变系生物量和光合能力的基础上,本文进一步检测了几种主要有机和无机营养成分含量以及几种主要重金属污染元素在各突变系地上部分的积累量。结果显示,除N5、N10、N3突变系的粗蛋白含量较对照系有明显提高(分别提高了26.95%、18.87%和10.91%)、N4突变系的无氮浸出物含量略高于对照系(2.84%)外,所有突变系的有机营养含量与对照系或没有明显差别或略低;其无机营养含量均较对照系有明显提高,其中N1、N3、N5等突变系的含量相对较高。表明低能重离子束诱变改变了狐米草的经济性状,其中,N5和N3突变系因生物量大、光合能力强、营养丰富,较适合于沿海滩涂种植。

The effects of methanol extract of cleistopholis patens on the reproductive system of female Wistar rats

Objective:In Port Harcourt and its environs,extracts of Cleistopholis patens are used by traditional medicine healers for the treatment of menstrual irregularities and other gynaecological disorders.The objective of this study was therefore to determine the effects of orally administered methanol extract of the stem bark of Cleistopholis patens on the reproductive organs of non-pregnant albino(Wistar) rats.Methods:3g/kg(low dose) and 6g/kg(high dose) of the extract were administered orally,daily to two different groups of animals,respectively, over a period of 28 days.A third(control) group of animals received distilled water only,orally over the same period.Five animals from each of the groups were sacrificed on day 8,15 and 29.Venous blood samples and reproductive organs respectively were taken from each group of sacrificed animals for hormonal and histopathological analysis.Results:Results of the hormonal assay revealed a general increase in the levels of Follicle Stimulating Hormone(FSH),Luteinizing Hormone(LH),progesterone,and estrogen.The highest levels were noticed in the animals sacrificed on the 29th day(LH =5.48±0.04 IU/L;FSH =3.80±0.00 IU/ L;Progesterone =7.14±0.15 nmol/L;Estrogen = 0.168±0.002 nmol/L).These increases were statistically significant compared to those of the control animals(LH =2.90±0.00 IU /L;FSH = 1.28±0.02 IU/L; Progesterone = 3.80±0.00 nmol/L;Estrogen = 0.130±0.002 nmol/L;P【0.05),and were also dose dependent. Results of the histopathological studies showed presence of chronic inflammatory cells in the tissues of the fallopian tubes and uterus on the 29th day.However,no changes were observed in the ovaries.Conclusion: The administration of the extract produced a dose and time-dependent increase in FSH,LH,progesterone and estrogen levels.We postulate that these observed effects may have been induced by the phytoestrogens (known to have 1/1 000 th of the efficacy of natural oestrogens) in the extract.The hormonal and histopathological changes may explain the effects described by patients following ingestion of extracts of this plant in traditional medical practice.However,it remains to be determined if these effects are harmful or beneficial in disease conditions.

Maintenance of Normal Stress Tolerance in the Moss Physcomitrella patens Lacking Chloroplastic CuZn-Superoxide Dismutase

Superoxide dismutases (SODs) catalyze the dismutation of superoxide and play an important role in reducing oxidative stress in plants. Based on in-gel SOD activity staining, chloroplasts of the moss Physcomitrella patens have two CuZn-SODs as the major SOD isozymes and minor SODs, including a Fe-SOD and two Mn-SODs. To investigate the contribution of chloroplastic SODs to stress tolerance in P. patens, we generated a double mutant lacking chloroplastic CuZn-SOD genes. The mutant did not show any differences in comparison to the wild type based on the growth of protonemata on normal and high-salt media, extractable activities of the other SODs after culture on normal and high-salt media, and inhibition of Fv/Fm under stress conditions (high-salt, high-light, and high-temperature). These results indicate that chloroplastic CuZn-SODs do not play a principal role in oxidative stress tolerance in chloroplasts under the investigated conditions. These findings explain the previously reported unusual response of P. patens to copper deficiency, in which chloroplastic CuZn-SODs are preferentially inactivated but cytosolic CuZn-SODs are unaffected.

Gas Chromatography-Mass Spectrometry Study from the Leaves Fractions Obtained of <i>Vernonanthura patens</i>(Kunth) H. Rob

Preliminary phytochemical study of methanol extracts from Vernonanthura patens’s leaves located in Marcabelí (El Oro province), Ecuador. The methodology consisted of chromatographic column separations with increasing polarity solvents and the analysis of fractions by Gas Chromatography-Mass Spectrometry coupled system (GC-MS). The structure of 53 compounds was proposed. Analyzed V. patens species showed the presence of terpene and aliphatic hydrocarbons, free fatty acids and their methyl and ethyl esters, oxygen sesqui, triterpenoids and sugars. For ecuadorian species, there are no records of chemical studies.

Studies of the Volatile Compounds Present in Leaves, Stems and Flowers of <i>Vernonanthura patens</i>(Kunth) H. Rob

The study of the volatile components of the leaves, stems and flowers of Vernonanthura patens is discussed. A micro solid-phase extraction at constant temperature with a dimethylsiloxane fiber of 100 μm was performed. The compounds extracted were analyzed by gas chromatography coupled to mass spectrometry (GC-MS). 7 monoterpenes structures were assigned to leaves, and three to stems, these compounds were not detected in the flowers with the configuration of the system used. 17 sesquiterpenes were identified in the leaves;6 in stems and 2 in flowers, finding coincidence in some of them. The major components were α-humulene in leaves, bergamotene in stems and caryophyllene in flowers.

低能重离子诱导的狐米草(Spartina patens)突变系生物量与光合能力比较

为从狐米草低能重离子突变系中筛选出生物量大、光合能力强、并可在滩涂生长良好的牧草用突变系,在室外模拟沿海滩涂环境中种植狐米草,检测各突变系的叶长、叶宽、茎高、茎直径、地上部分生物量以及各突变系单位面积的光合能力等指标。结果显示:各突变系的叶片均比对照大,茎也比对照高且粗,但地上生物量只有N1、N3和N5突变系高于对照;多数突变系在光能的吸收、传递与光化学转换方面的能力要高于对照,但只有N1、N2、N3、N5和N9等5个突变系的单位面积光合速率高于对照。综合生物量与光合能力的检测结果,N1、N3和N5突变系在生物量与光合能力方面相对有较高的优势。这为适合我国沿海滩涂生长的优良狐米草牧草突变系筛选提供了依据。

希茉莉(Hamelia patens Jacq.)花粉发育时期快速检测

茜草科希茉莉（Hamelia patens Jacq.）的花粉用DAPI（4’,6-diamidino-2-phenylindole）直接染色不能观察到花粉核,本研究探索出适宜在DAPI染色前处理希茉莉花粉壁的水浴加热-氧化方法,使得希茉莉花粉核能在荧光显微镜下清晰地显示出来,从而快速检测花粉所处的发育阶段。结果表明：（1）单核花粉和二核花粉最适宜的水浴加热温度和时间分别为65℃、20~50 min和55℃、20~40 min;（2）花粉发育阶段与花朵、花药长度的对应关系为：花朵0.90~1.00 cm、花药0.50~0.60 cm时对应花粉的四分体时期,花朵1.10~1.60 cm、花药0.60~0.85 cm时对应单核花粉时期,花朵1.80~2.70 cm（花冠裂片张开前）、花药0.91~1.01 cm时对应二核花粉时期。

小立碗藓(Physcomitrella patens)植物microRNA结合靶位预测

利用857条植物miRNA序列对27546条小立碗藓mRNA序列进行搜索,预测出162个植物miRNA家族在小立碗藓中存在结合靶位。miRNA结合靶位数目和miRNA协同作用网络分析结果同时显示,miR482和miR1168在小立碗藓中结合靶位多、协同作用广,提示它们对于小立碗藓可能具有重要生物学功能。52个莱茵衣藻特有的miRNA被预测在小立碗藓中存在结合靶位,显示小立碗藓在从藻类向种子植物进化过程中处在独特演化位置。

小立碗藓泛素连接酶U-box家族的鉴定及表达分析

U-box家族编码E3泛素连接酶能够特异性识别底物,从而调控蛋白的修饰和降解过程。为鉴定小立碗藓U-box家族成员,分析其生物学特征及基因表达规律,该研究基于小立碗藓的全基因组测序数据,通过生物信息学技术对泛素连接酶U-box家族成员的理化性质、进化关系、顺式作用元件、组织表达模式、胁迫响应等进行了分析。结果表明:(1)在小立碗藓全基因组中共鉴定得到31个U-box家族成员,并且不均匀地分布于14条染色体上;其分子量大小在37.75~117.49 kDa之间,等电点介于5.32~8.55之间。(2)进化树显示,小立碗藓的U-box家族成员分布在I-IX亚家族中,其中亚家族VII含有小立碗藓成员最多,共11个(约占36.67%),说明小立碗藓在历史进化过程中具有高度保守性且功能多样化。(3)经启动子分析发现,小立碗藓U-box家族成员具有结合GA和ABA等多个激素的元件。(4)组织表达分析发现小立碗藓U-box家族成员主要在绿丝体、轴丝体和孢子体S3时期大量表达。(5)通过ABA、甘露醇和饱和LiCl处理后发现,配子体的拟茎均发生黄化且PpPUB21的表达量均被显著诱导。推测小立碗藓U-box家族成员在生长发育和响应逆境胁迫中发挥着重要作用,这为后续深入研究小立碗藓U-box家族成员的基因功能研究奠定了理论基础,也为其相关研究提供了参考。

A Transcriptome Atlas of Physcomitrella patens Provides Insights into the Evolution and Development of Land Plants

Identifying the genetic mechanisms that underpin the evolution of new organ and tissue systems is an aim of evolutionary developmental biology. Comparative functional genetic studies between angiosperms and bryophytes can define those genetic changes that were responsible for developmental innovations. Here, we report the generation of a transcriptome atlas covering most phases in the life cycle of the model bryo- phyte Physcomitrella patens, including detailed sporophyte developmental progression. We identified a comprehensive set of sporophyte-specific transcription factors, and found that many of these genes have homologs in angiosperms that function in developmental processes such as flowering and shoot branching. Deletion of the PpTCP5 transcription factor results in development of supernumerary sporangia attached to a single seta, suggesting that it negatively regulates branching in the moss sporophyte. Given that TCP genes repress branching in angiosperms, we suggest that this activity is ancient. Finally, compar- ison of P. patens and Arabidopsis thaliana transcriptomes led us to the identification of a conserved core of transcription factors expressed in tip-growing cells. We identified modifications in the expression patterns of these genes that could account for developmental differences between P. patens tip-growing cells and A. thaliana pollen tubes and root hairs.

Targeted Gene Knockouts Reveal Overlapping Functions of the Five Physcomitrella patens FtsZ Isoforms in Chloroplast Division, Chloroplast Shaping, Cell Patterning, Plant Development, and Gravity Sensing

Chloroplasts and bacterial cells divide by binary fission. The key protein in this constriction division is FtsZ, a self-assembling GTPase similar to eukaryotic tubulin. In prokaryotes, FtsZ is almost always encoded by a single gene, whereas plants harbor several nuclear-encoded FtsZ homologs. In seed plants, these proteins group in two families and all are exclusively imported into plastids. In contrast, the basal land plant Physcomitrella patens, a moss, encodes a third FtsZ family with one member. This protein is dually targeted to the plastids and to the cytosol. Here, we report on the targeted gene disruption of all ftsZ genes in R patens. Subsequent analysis of single and double knockout mutants revealed a complex interaction of the different FtsZ isoforms not only in plastid division, but also in chloroplast shaping, cell patterning, plant development, and gravity sensing. These results support the concept of a plastoskeleton and its functional integration into the cytoskeleton, at least in the moss R patens.

Computational Analysis of Drought Stress-Associated miRNAs and miRNA Co-Regulation Network in Physcomitrella patens

miRNAs are non-coding small RNAs that involve diverse biological processes. Until now, little is known about their roles in plant drought resistance. Physcomitrella patens is highly tolerant to drought; however, it is not clear about the basic biology of the traits that contribute P. patens this important character. In this work, we discovered 16 drought stress-associated miRNA （DsAmR） families in P. patens through computational analysis. Due to the possible discrepancy of expression periods and tissue distributions between potential DsAmRs and their targeting genes, and the existence of false positive results in computational identification, the prediction results should be examined with further experimental validation. We also constructed an miRNA co-regulation network, and identi- fied two network hubs, miR902a-Sp and miR414, which may play important roles in regulating drought-resistance traits. We distributed our results through an online database named ppt-miRBase, which can be accessed at http：/Poioinfor.cnu.edu.cn/ppt_miRBase/index.php. Our methods in finding DsAmR and miRNA co-regulation network showed a new direction for identifying miRNA functions.

Target deletion of the AAA ATPase PpCDC48II in Physcomitrella patens results in freezing sensitivity after cold acclimation

CDC48 is a highly conserved protein in eukaryotes and belongs to the AAA （ATPase associated with a variety of cellular activities） superfamily. It can interact with many different cofactors and form protein complexes that play important roles in various cellular processes. According to the Physcomitrella patens database, one member of the ATPases, the cell cycle gene PpCDC4811, was cloned. PpCDC48II contains two typical ATPase modules and is highly homologous to AtCDC48A. PpCDC4811 was up-regulated in mRNA levels after incubation at 0~C for 36 and 72 h. To further elucidate protein function, we disrupted the PpCDC4811 gene by transforming P. patens with the corresponding linear genomic sequences. When treated to the same freezing stress, it was found that PpCDC4811 knockout plants were less resistant to freezing treatment than wild type after acclimation. This suggested that PpCDC481I was an essential gene for low-temperature-induced freezing tolerance in P. patens cells.

基于质量源于设计理念的小儿腹痛草标准汤剂的制备工艺及质量标准

目的运用质量源于设计理念(QbD),优化小儿腹痛草标准汤剂的制备工艺,并建立其质量标准。方法根据中药质量标志物理论预测分析关键质量属性;采用失效模型与影响分析筛选关键工艺参数(CPPs);建立关键质量属性的测定方法;根据单因素实验确定初步范围后使用Box-Behnken实验优化提取工艺;以熵值法进行综合评分;建立设计空间,选取较优操作空间进行工艺验证;以最佳工艺制备15批不同产地小儿腹痛草饮片的标准汤剂,最终建立其出膏率、浸出物、薄层色谱、指纹图谱、含量及含量转移率的质量标准。结果以獐牙菜苦苷含量、龙胆苦苷含量及出膏率为关键质量属性;以浸泡时间、加水量、煎煮时间为关键工艺参数;所建立的模型差异有统计学意义;筛选的最佳工艺为:浸泡时间90 min、加水量15倍、煎煮时间30 min(第二煎20 min);出膏率21.13%~30.73%;浸出物含量在82.00%~88.00%;薄层色谱中獐牙菜苦苷斑点清晰;15批样品与参照图谱的相似度均>0.85;獐牙菜苦苷含量250.64~385.21 mg·g^(-1),转移率43.76%~77.73%;龙胆苦苷含量0.69~2.70 mg·g^(-1),转移率56.02%~105.29%。结论基于以上方法及技术筛选出小儿腹痛草标准汤剂制备工艺,可为其配方颗粒的制剂开发及质量控制提供参考。

小立碗藓PpCAD4基因敲除载体的构建及功能研究

木质素是植物抵御外界环境的重要成分,肉桂醇脱氢酶(cinnamic alcohol dehydrogenase,CAD)是木质素合成途径中的关键酶和限速酶之一。前期数据表明,在灰霉菌侵染下,PpCAD4基因上调表达,但PpCAD4功能尚未明确。本次研究利用同源重组原理构建PpCAD4.1-PTN182-PpCAD4.2敲除载体,从而破坏PpCAD4的alcohol dehydrogenase GroES-like domain(ADH_N)和zinc-binding dehydrogenase(ADH_zinc_N)结构域。利用PGE 6000介导小立碗藓原生质体转化,筛选并鉴定得到敲除PpCAD4突变体植株。qRT-PCR表明,敲除型植株中PpCAD4的表达量相对野生型减少了84%。通过对配子体的形态观察发现,PpCAD4突变株通过增加配子体中拟叶的数量,使得植株生物量增加,这表明CAD4在早期陆生植物的生长发育中起着重要作用。用灰霉菌侵染小立碗藓发现,0.5 d后野生型配子体菌丝入侵率为15%,cad4-ko菌丝入侵率为40%。侵染1 d后,Ppcad4-ko配子体菌丝侵入率是野生型的2倍。表明PpCAD4能够增加小立碗藓对真菌病原菌的抗性。

山兰研究进展

本文介绍了山兰生态适应性、植物学特性、野生驯化及营养成分方面的研究现状,旨在为山兰的栽培技术及开发利用提供理论依据。

小立碗藓细胞色素P450基因CYP73A49的克隆与功能分析

本研究克隆了一个小立碗藓(Physcomitrella patens)P450基因CYP73A49,其cDNA全长1 629 bp,预测编码542个氨基酸。为了进一步研究其生物学功能,利用同源重组技术对CYP73A49基因实施定点突变,成功构建了能诱导其功能缺失的载体,并获得了苔藓的功能缺失突变体。

应用傅立叶变换回旋共振质谱对小立碗藓新蛋白的序列分析

Two new proteins from physcomitrella patens were sequenced by nanoLC-FT-ICR tandem mass spectrometry.Eight and seven peptides were sequenced for spot 303 and spot 413 respectively.Database search was performed through the search engine Mascot (www.matrixscience.com) by MS/MS ion search program and Sequence Tag program.Blast was performed too.No statistical significant results were obtained from database search.The reliable amino acid sequences can be used for gene clone due to high mass accuracy.This research indicates that nanoLC-FT-ICR tandem mass spectrometric technique is powerful tools for new protein sequence analysis and will play an important role in plant proteomics research.