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.

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.

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。

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.

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 co-factors 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 PpCDC48II, was cloned. PpCDC48II contains two typical ATPase modules and is highly homologous to AtCDC48A. PpCDC48II was up-regulated in mRNA levels after incubation at 0°C for 36 and 72 h. To further elucidate protein function, we disrupted the PpCDC48II gene by transforming P. patens with the corresponding linear genomic sequences. When treated to the same freezing stress, it was found that PpCDC48II knockout plants were less resistant to freezing treatment than wild type after acclimation. This suggested that PpCDC48II was an essential gene for low-temperature-induced freezing tolerance in P. patens cells.

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.

小立碗藓愈伤组织诱导和培养

小立碗藓已经成为植物功能基因组学的模式系统,其材料的大量培养是所有工作的基础。文章探讨了小立碗藓愈伤组织诱导和组织培养的基本条件,并观察了小立碗藓愈伤组织的亚显微结构。

苔藓植物小立碗藓,功能基因组学研究新的模式系统

苔藓植物具有相对简单的发育模式,单倍体的配子体在其生活史中占主导地位,作为研究植物生物学过程的模式系统具有诸多的优越性。苔藓植物小立碗藓能够高效地通过同源重组的方式将外源核酸整合到其核DNA,这就使得基因打靶在此物种中就像在小鼠胚胎干细胞和酵母中一样成为一个非常便利的技术。另外由于小立碗藓与高等植物在生物特征上有很大相似之处加之具有其他诸多优越性,它有望成为一个诱人的植物生物学和功能基因组学研究的模式系统。

有前景的模式植物小立碗藓的研究新进展

小立碗藓是在分子生物学研究方面有广阔应用前景的模式植物。该文主要综述了有关小立碗藓在功能基因组学、进化和适应性及植物生理等方面最新的研究进展。

植物分子生物学研究极具前景的模式系统——小立碗藓

小立碗藓作为植物分子生物学研究极具前景的模式系统已日益受到人们的重视,它的生活史周期短,易于培养,转基因植株易于分析,核基因组容易和外源DNA发生同源重组,这些特点使它成为研究基因功能的良好材料。一些成功的基因敲除和基因破坏已经在小立碗藓中实现,这些基因的功能也通过小立碗藓转化植株的特点得以证实。小立碗藓标签突变文库已经建立,其应用为小立碗藓基因的进一步研究打下了基础。关于小立碗藓的ESTs数据库已经建立,已有67 000条ESTs信息。

小立碗藓-灰霉菌互作过程中活性氧代谢及病程相关蛋白1转录表达的变化

为了解苔藓植物小立碗藓与病原真菌灰霉菌的互作关系,观察了灰霉菌在小立碗藓配子体中的感染行为和小立碗藓受感染后的组织病理学变化.另外,还测定了接种灰霉菌后小立碗藓配子体的过氧化氢量、过氧化物酶(POD)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)的活性以及病程相关蛋白1(PR1)基因转录表达变化.结果表明:(i)在灰霉菌接种早期小立碗藓出现类似维管束植物的过敏性反应;(ii)接种早期PR1基因表达上调和POD活性增高,说明PR1和POD可能与小立碗藓对灰霉菌的早期防御反应有关;(iii)病原菌侵染引起小立碗藓配子体内CAT、SOD活性下降和过氧化氢量升高,说明小立碗藓与灰霉菌互作中出现了氧化还原态的变化.

小立碗藓冷驯化相关基因Pp-LIM only A的克隆与表达

植物经历冷驯化后抗冻能力会有所提高。利用cDNA-AFLP方法从经过0oC冷驯化处理的小立碗藓中筛选到差异表达的Pp-LIM only A基因片段。cDNA和基因序列比较分析表明此基因含有7个内含子和8个外显子,编码由345个氨基酸残基组成的蛋白质,其中只含有一个LIM结构域,与动物蛋白质PDZ/LIM家族有很高的同源性,推测是一种新的植物LIM蛋白。实时定量PCR分析显示其在冷驯化6 h后表达量即开始明显增加,并随着冷驯化时间的延长表达量大幅度提高。Pp-LIM only A蛋白可能通过LIM结构域对细胞骨架的作用而影响了细胞膜的稳定性,本研究对其在抗冻中的作用作了进一步讨论。

小立碗藓Ran基因的克隆与表达分析

Ran是一种大量分布于细胞核内的小分子的GTP酶,在核质运输过程中具有十分重要的作用,参与调控细胞分裂及其信号传导。本研究利用基于同源序列的PCR技术,从小立碗藓(Physcomitrella patens)中成功克隆了PpRan基因的开放阅读框(ORF)序列,并对其编码的氨基酸序列进行了相关分析。利用半定量PCR技术对PpRan基因在不同组织中以及受到缺磷胁迫时的表达研究发现,PpRan基因的表达具有组织特异性,茎叶体和原丝体中表达量较高,假根中表达量比较低;PpRan基因受缺磷胁迫诱导表达,随着胁迫时间的延长表达量越来越高。这些结果表明,PpRan基因可能在小立碗藓响应缺磷胁迫时发挥重要作用。

苔藓分子生物学的一些进展

本文简述了近年来苔藓分子生物学的突破性进展。 1995年以前 ,苔藓系统学中尚无DNA序列数据 ,但到 1999年产生了 2 5 0个分类单位的 10 0 0个DNA序列 ,并分析了它们的系统发生信息。在分子遗传学研究中 ,小立碗藓Physcomitrellapatens、葫芦藓Funariahygrometrica、土生墙藓Tortularuralis和角齿藓Cer atodonpurpureus正在发展成有力的工具。表达序列靶 (ESTs)和外源基因的表达已成功地用于这些苔藓植物。同源重组在酵母和小白鼠细胞中已经确定 ,然而至今尚无任何植物模型体系。 1997年Schaefer和Zryd证明 ,小立碗藓具有有效的同源重组能力 ,至今在所有植物中是独特的 。

小立碗藓CAS基因的克隆及与拟南芥CAS基因的比较

钙信号在动植物的生长、发育过程中具有不可替代的作用.胞外钙受体(Extracellular Calcium-sensing receptor,CAS)是动植物质膜上一种跨膜离子通道蛋白,可以感受胞外Ca2+水平,充当第一信使将胞外的信号传递到细胞内.但迄今为止,仅2003年从植物拟南芥中克隆了胞外钙受体CAS基因,其起源进化我们知之甚少.本实验成功克隆了小立碗藓CAS基因,其cDNA全序列共1 574 bp,5′-非翻译区80个核苷酸,3′-非翻译区234个核苷酸,开放阅读框(ORF)1 257 bp,编码419个氨基酸,共有6个内含子.虽然藓类植物与被子植物进化距离较远,但小立碗藓CAS基因前五个内含子的插入位置以及相位与拟南芥完全相同,这说明植物中CAS基因在漫长的进化过程中非常保守,暗示被子植物胞外钙离子信号感受、传递机制起源于藓类植物.

小立碗藓细胞色素P450基因PpCYP51G1的分离及转化载体构建

从小立碗藓(Physcomitrella patens)中克隆了细胞色素P450 CYP51基因PpCYP51G1的全长编码序列,编码区长1 509 bp,预测编码488个氨基酸,蛋白二级结构含有自由卷曲、伸展片段和α-螺旋,N端含有一个跨膜区域。构建了PpCYP51G1的功能缺失载体,并利用PEG介导的原生质体转化技术获得该基因的功能缺失转化体;同时,构建了PpCYP51G1-GFP融合蛋白载体,亚细胞定位结果表明,PpCYP51G1定位在内质网中。

苔藓植物双向凝胶电泳蛋白样品制备的优化

比较了苔藓植物小立碗藓3种蛋白样品制备方法,结果分析表明,直接研磨法和三氯乙酸(TCA)/丙酮(acetone)沉淀法检测的蛋白点的数量相差不明显;直接研磨法提取蛋白所得到的2-DE图谱蛋白点模糊,分辨率低,横竖条纹干扰严重;三氯乙酸(TCA)/丙酮(acetone)沉淀法分离效果较好,分辨率有所提高。改良的酚抽提/乙酸铵沉淀法检测的蛋白点远远多于前两种方法,所得到的图谱质量最高,分辨率大大提升,蛋白点分布均匀清晰,横竖条纹干扰也很少,凝胶背景干净,是最佳的小立碗藓蛋白样品制备的方法。

基因定点整合技术及其在苔藓研究中的进展

基因定点整合技术是 2 0世纪 80年代后兴起的一种分子生物学技术 ,在精细研究基因功能 ,消除转基因沉默 ,基因治疗等有重要意义。基因定点整合技术是功能基因组学研究的重要手段。目前关于基因定点整合技术在酵母和鼠胚胎干细胞中的应用已经很成熟 ,高等植物由于同源重组频率较低而限制了它的应用 ,但小立碗藓 (Physcomitrellapatens)基因同源重组频率较高 ,基因定点整合技术得到了成功应用 ,可望成为一种新的分子生物学的模式植物。本文针对基因定点整合的原理、技术路线及进展作一综述。