A Brachypodium distachyon calcineurin B-like protein-interacting protein kinase,BdCIPK26,enhances plant adaption to drought and high salinity stress

As sessile organisms,plants possess a complex system to cope with environmental changes.Ca^(2+)functions as a vital second messenger in the stress signaling of plants,and the CBL-interacting protein kinases(CIPKs)serve as essential elements in the plant Ca^(2+)signaling pathway.In this study,calcineurin B-like protein-interacting protein kinase 26(BdCIPK26)from Brachypodium distachyon was characterized.Overexpression of BdCIPK26 enhanced tolerance to drought and salt stress of transgenic plants.Further investigations revealed that BdCIPK26 participated in abscisic acid(ABA)signaling,conferred hypersensitivity to exogenous ABA in transgenic plants,and promoted endogenous ABA biosynthesis.Moreover,BdCIPK26 was found to maintain ROS homeostasis in plants under stress conditions.Therefore,this study indicates that BdCIPK26 functions as a positive regulator in drought and salt stress response.

Pathogenicity of Rice Blast Fungus Magnaporthe oryzae on Brachypodium distachyon

Inoculation methods for rice blast fungus Magnaporthe oryzae to Brachypodium distachyon were developed to investigate the infection process and symptom development in comparison with those on rice （Oryza sativa） and barley （Hordeum vulgare）.M.oryzae could infect leaves,sheathes,stems and panicles of B.distachyon and cause blast disease.Spraying conidial suspension on either intact seedlings or leaf segments induced typical symptoms on B.distachyon.During the intact seedling inoculation,the symptom developed on B.distachyon leaves closely resembled that on rice;but the lesions on B.distachyon had better uniformity in shapes and sizes than those on rice or barley.In the leaf segments inoculation,only initial and low-developed lesions could be found on rice,while normal symptoms on B.distachyon and barley.Inoculated with low-virulent mutants of M.oryzae,B.distachyon produced low-level symptoms.The symptom level of each mutant on B.distachyon corresponded well to that on rice.In addition,typical infection processes presented on B.distachyon leaves：forming melanized appressoria,penetrating into host epidermis and then forming hyphae in epidermal cells.According to these results,B.distachyon can be used as a candidate for studying fungus-plant interactions and as a probable source of disease resistance.

RNA-seq analysis of Brachypodium distachyon responses to Barley stripe mosaic virus infection

Barley stripe mosaic virus(BSMV) is the type member of the genus Hordeivirus. Brachypodium distachyon line Bd3-1 shows resistance to the BSMV ND18 strain, but is susceptible to an ND18 double mutant(βNDTGB1R390K, T392K) in which lysine is substituted for an arginine at position 390 and for threonine at position 392 of the triple gene block 1(TGB1) protein. In order to understand differences in gene expression following infection with ND18 and double mutant ND18, Bd3-1 seedlings were subjected to RNA-seq analyses at 1, 6, and14 days post inoculation(dpi). The results revealed that basal immunity genes involved in cellulose synthesis and pathogenesis-related protein biosynthesis were enhanced in incompatible interactions between Bd3-1 and ND18. Most of the differentially expressed transcripts are related to trehalose biosynthesis, ethylene, jasmonic acid metabolism,protein phosphorylation, protein ubiquitination, transcriptional regulation, and transport process, as well as pathogenesis-related protein biosynthesis. In compatible interactions between Bd3-1 and ND18 mutant, Bd3-1 developed weak basal resistance responses to the virus. Many genes involved in cellulose biosynthesis, protein amino acid phosphorylation,protein biosynthesis, protein glycosylation, glycolysis and cellular macromolecular complex assembly that may be related to virus replication, assembly and movement were up-regulated. Some genes involved in oxidative stress responses were also up-regulated at14 dpi. BSMV ND18 mutant infection suppressed expression of genes functioning in regulation of transcription, protein kinase, cellular nitrogen compound biosynthetic process and photosynthesis. Differential expression patterns between compatible and incompatible interactions in Bd3-1 to the two BSMV strains provide important clues for understanding mechanism of resistance to BMSV in the model plant Brachypodium.

Overexpression of a Glycosyltransferase Gene from a Metabolically Poly-Resistant Beckmannia syzigachne Population Alters Growth and Confers Herbicide Resistance to Brachypodium distachyon

Beckmannia syzigachne is a noxious weed for rice-wheat rotations in China.The B.syzigachne(AH-02)population evolved metabolic resistance to fenoxaprop-P-ethyl and mesosulfuron-methyl.To investigate the function of GT73C1 in this population,the GT73C1 gene was amplified by reverse transcription-polymerase chain reaction,and the sequence was 100%consistent with the transcriptome data.Its phylogenetic tree was displayed and annotated using FigTree v1.4.4.The plant overexpression vector of GT73C1 gene was constructed and used to transform Brachypodium distachyon plants.Furthermore,the expression of GT73C1 was significantly induced by fenoxaprop-P-ethyl and mesosulfuron-methyl,which was consistent with the findings from the whole plant bioassay.These results indicate that GT73C1 is closely related to the metabolic resistance of B.distachyon.

Host status of Brachypodium distachyon to the cereal cyst nematode

Cereal cyst nematode（Heterodera avenae, CCN） distributes worldwide and has caused severe damage to cereal crops, and a model host will greatly aid in the study of this nematode. In this research, we assessed the sensitivity of 25 inbred lines of Brachypodium distachyon to H. avenae from Beijing, China. All lines of B. distachyon were infested by secondstage juveniles（J2s） of H. avenae from Daxing District of Beijing population, but only 13 inbred lines reproduced 0.2–3 cysts/plant, showing resistance. The entire root system of the infested B. distachyon appeared smaller and the fibrous roots were shorter and less numerous. We found that a dose of 1 000 J2s of H. avenae was sufficient for nematode infestation. We showed that Koz-1 of B. distachyon could reproduce more cysts than TR2A line. Line Koz-1 also supported the complete life cycles of 5 CCN geographical populations belonging to the Ha1 or Ha3 pathotype group. Our results suggest that B. distachyon is a host for CCN.

二穗短柄草(Brachypodium distachyon)BdAD1基因的克隆、表达及功能分析

本研究利用生物信息学结合RT-PCR技术从二穗短柄草(Brachypodium distachyon)中克隆出BdAD1的c DNA基因,该基因编码一个包含500个氨基酸残基的乙醛脱氢酶家族蛋白。系统进化关系分析表明,该BdAD1蛋白序列与小麦(Triticum aestivum)、羊草(Leymus chinensis)和大麦(Hordeum vulgare)的同源蛋白具有较近的亲缘关系。BdAD1基因在植物细胞的细胞核和细胞质中均有表达,而且BdAD1蛋白兼具松柏醛脱氢酶和芥子醛脱氢酶的活性(CALDH/SALDH),可将松柏醛与芥子醛分别酶解生成阿魏酸和芥子酸,但它对松柏醛的催化效率显著高于芥子醛,因此推测BdAD1可能在苯丙烷代谢途径中对阿魏酸的合成具有重要的调控作用。

Sugarcane mosaic virus infection of model plants Brachypodium distachyon and Nicotiana benthamiana

Sugarcane mosaic virus (SCMV;genus Potyvirus, family Potyviridae) is a causal pathogen of sugarcane mosaic disease, and it is widespread in regions where sugarcane (Saccharum spp. hybrids) is grown. It is difficult to investigate the molecular mechanism of pathogen infection in sugarcane because of limited genomic information. Here, we demonstrated that SCMV strain FZ1 can systemically infect Brachypodium distachyon inbred line Bd21 and Nicotiana benthamiana through inoculation, double antibody sandwich enzyme-linked immunosorbent, transmission electron microscopy, and reverse transcription PCR assays. The leaves of Bd21 developed mosaic symptoms, while the leaves of N. benthamiana showed no obvious symptoms under the challenge of SCMV-FZ1. We concluded that B. distachyon inbred line Bd21 is a promising experimental model plant compared with N. benthamiana for study on the infectivity of SCMV. This is the first report on the SCMV infection of model plants B. distachyon inbred line Bd21 and N. benthamiana, which will shed light on the mechanism of SCMV infection of sugarcane and benefit sugarcane breeding against sugarcane mosaic disease.

Evaluation of Drought Stress-Inducible W<i>si</i>18 Promoter in <i>Brachypodium distachyon</i>

The rice Wsi18 promoter confers drought-inducible gene expression. This property makes it a useful candidate to drive relevant genes for developing drought resistant traits for different monocot crops. In this study, we showed that the Bradi2G47700 gene, the closest homologue to rice Wsi18, was upregulated in Brachypodium distachyon plants exposed to ABA and mannitol. Wsi18: uidA transgenic B. distachyon plants were produced and then subjected to ABA or mannitol treatment. The expression of uidA in three transgenic lines (line 10, 18 and 37) was significantly upregulated in plants exposed to ABA (fold increases of 5.61 ± 0.98, 2.88 ± 0.75 and 9.13 ± 1.96, respectively) compared to the same transgenic plant lines without treatment. The expression of uidA in two transgenic lines (lines 18 and 37) also showed upregulation when treated with mannitol (fold increases of 4.43 ± 1.07 and 8.47 ± 2.90, respectively) compared to the same transgenic plant lines without mannitol treatment. Moreover, GUS histochemical assay showed increased Wsi18 promoter activity in the leaves and stems of transgenic lines upon treatment with ABA or mannitol. This is the first report of the drought inducible rice Wsi18 promoter being active in B. distachyon which is a model plant for molecular biology research of various monocot plants. Taken together, the results indicate that the Wsi18 promoter and its homologue may be explored as a useful tool for drought stress-inducible gene expression in different monocot crops.

二穗短柄草(Brachypodium distachyon)BdCO基因调控开花的转录组学分析

CONSTANS(CO)是植物光周期诱导开花途径中的关键基因之一。为探究BdCO在光周期途径中的分子调控机制,本研究对野生型二穗短柄草(Brachypodium distachyon)Bd21植株、过表达BdCO基因型二穗短柄草(CO_OX)植株和BdCO基因敲除型二穗短柄草(CO_A3)植株进行转录组测序分析,对差异表达基因进行GO和KEGG功能富集分析,最后观察三种植株的开花表型。结果表明,对比Bd21 vs CO_OX和Bd21 vs CO_A3的基因表达量,分别检测到1382个和773个差异表达基因;GO功能富集分析发现,Bd21 vs CO_OX的差异表达基因主要富集在小核仁核糖核蛋白复合物、snoRNA结合和rRNA处理中,Bd21 vs CO_A3的差异表达基因主要富集在类囊体、色素结合和光合作用中;KEGG通路富集分析发现,Bd21 vs CO_OX的差异表达基因主要富集在植物激素信号转导、真核生物中的核糖体生物发生、光合作用-天线蛋白和昼夜节律-植物等通路,Bd21 vs CO_A3的差异表达基因主要富集在MAPK信号通路-植物、真核生物中的核糖体生物发生和光合作用-天线蛋白等通路;在长日照条件下,CO_OX植株的开花时间比野生型Bd21的提前约1.90 d,CO_A3植株的开花时间比野生型Bd21的延迟约7.92 d。综上,BdCO影响光周期途径相关基因的表达,同时影响二穗短柄草的开花时间,说明BdCO在二穗短柄草的光周期途径调控开花的过程中发挥重要作用。

Digital imaging approaches for phenotyping whole plant nitrogen and phosphorus response in Brachypodium distachyon

This work evaluates the phenotypic response of the model grass （Brachypodium distacbyon （L.） P. Beauv.） to nitrogen and phosphorus nutrition using a combination of imaging techniques and destructive harvest of shoots and roots. Reference line Bd21-3 was grown in pots using 11 phosphorus and 11 nitrogen concentrations to establish a dose-response curve. Shoot biovolume and biomass, root length and biomass, and tissue phosphorus and nitrogen concentrations increased with nutrient concentration. Shoot biovolume, estimated by imaging, was highly correlated with dry weight （R2 〉 0.92） and both biovolume and growth rate responded strongly to nutrient availability. Higher nutrient supply increased nodal root length more than other root types. Photochemical efficiency was strongly reduced by low phosphorus concentrations as early as 1 week after germination, suggesting that this measurement may be suitable for high throughput screening of phosphorus response. In contrast, nitrogen concentration had little effect on photochemical efficiency. Changes in biovolume over time were used to compare growth rates of four accessions in response tonitrogen and phosphorus supply. We demonstrate that a time series image-based approach coupled with mathematical modeling provides higher resolution of genotypic response to nutrient supply than traditional destructive techniques and shows promise for high throughput screening and determina- tion of genomic regions associated with superior nutrient use efficiency.

二穗短柄草根部木栓质调控基因BdMYB92的克隆及功能研究

为了探究二穗短柄草(Brachypodium distachyon)根部木栓质合成的调控机制,该研究以二穗短柄草Bd21为试验材料,利用生物信息学分析方法克隆了二穗短柄草根部木栓质合成的调控转录因子基因BdMYB92(GenBank登录号为OP497966);采用荧光定量PCR方法,分析BdMYB92基因在二穗短柄草不同组织中的表达模式以及对6种非生物胁迫处理(空气中干旱、20%PEG-6000模拟干旱、4℃中冷处理、200 mmol/L NaCl、100μmol/L ABA和机械损伤)的响应表达特征;利用双荧光素酶和酵母单杂交验证BdMYB92蛋白和BdFAR4基因启动子的互作关系。结果表明:(1)二穗短柄草BdMYB92基因cDNA全长为1343 bp,开放阅读框为990 bp,编码329个氨基酸,蛋白分子量为36.4 kD,理论等电点为5.54。(2)亚细胞定位结果显示,BdMYB92定位在细胞核。(3)荧光定量PCR分析表明,BdMYB92在二穗短柄草根中的表达量显著高于叶鞘、节、穗、节间等其他组织;6种非生物胁迫处理均能诱导BdMYB92的上调表达,且对干旱胁迫的响应最为迅速,表明BdMYB92参与二穗短柄草响应逆境的过程。(4)双荧光素酶和酵母单杂交试验结果表明,BdMYB92与木栓质合成基因BdFAR4启动子区存在相互作用,且能够直接调控木栓质合成基因BdFAR4的转录表达。研究推测,BdMYB92可能与二穗短柄草中其他的木栓质合成基因存在相互作用,进而调控木栓质在根部的沉积。

二穗短柄草SPL家族基因的鉴定、系统发育和表达分析

植物特异性SPL家族基因编码SBP(squamosa promoter-binding protein-like)保守结构,参与调节植物的生长和发育。为了解二穗短柄草中SPL家族基因的分布、结构及功能,对二穗短柄草全基因组中SPL家族成员进行了系统分析。结果表明,在二穗短柄草全基因组中鉴定出17个BdSPL基因,在5条染色体上呈不均匀分布,bd03号染色体上分布最多;根据系统发育树,17个BdSPL被分为6个组,同组成员有保守的结构和基序;其中6对BdSPL基因发生基因复制事件;在BdSPL基因启动子区域发现了各种顺式元件,如ABRE、CGTAC-motif和LTR。转录组数据和qRT-PCR分析显示,BdSPL基因在二穗短柄草花中表达量较高,而在根系中表达量较低,在不同植物激素处理下表达量有差异。

小麦抗白粉病基因pm42的EST连锁图谱构建和比较基因组学分析

目的基因精细遗传连锁图谱的构建是图位克隆的基础,小麦功能基因精细遗传连锁图谱的构建依赖于比较基因组学分析。水稻和短柄草(Brachypodium distachyon)基因组序列是小麦比较基因组学分析和功能基因精细遗传定位的重要工具。本研究利用小麦、短柄草和水稻的基因组共线性关系对小麦抗白粉病基因pm42进行比较基因组学分析,明确了pm42基因所在2BS基因组区域与短柄草第1染色体和水稻第3染色体直系同源基因组区域的对应关系,开发出与抗白粉病基因pm42连锁的EST-SSCP(expressed sequence tag-single strand conformation polymorphism)标记CD452782和BF201235,以及EST-STS(expressed sequence tag-sequence tagged site)标记CJ674042、EB513371和CV771633,构建了pm42基因EST标记遗传连锁图谱,CJ674042、BF201235、CD452782和CV771633位于pm42近端粒侧,距离pm42的遗传距离分别为1.9、12.0、19.7和25.7cM;EB513371位于pm42近着丝粒侧,与pm42的遗传距离为14.6cM。整合原有的作图数据,构建了pm42基因的高密度比较基因组学遗传连锁图谱,pm42被定位于3.3cM的区间,该区间对应于短柄草66kb的基因组区域及水稻69kb的基因组区域。该结果为抗白粉病基因pm42高密度精细遗传连锁图谱构建、分子辅助选择和基因聚合奠定了基础。

二穗短柄草叶绿体RNA编辑位点的预测及其鉴定

RNA编辑是陆生植物叶绿体转录后基因表达调控的一种重要方式。为进一步探讨单子叶植物RNA编辑功能及其发生机制,本研究通过生物信息学方法,对二穗短柄草叶绿体的81个蛋白编码基因的RNA编辑位点进行了预测和鉴定分析,结果发现78个基因存在编辑位点,共检测到176个编辑位点,都是C到U的转换。其中ndhB最多,为14个编辑位点。为验证预测结果,利用blast工具比对了NCBI的二穗短柄草EST数据库,最终确定存在于17个基因中的34个编辑位点是真实存在的,其中19个为沉默编辑,15个为有效编辑。与5个不同单子叶禾本科物种18个蛋白编码基因的RNA编辑位点的比较发现,在rpoB-206位点,只有二穗短柄草发生了编辑,且只有ndhD-295(293)为部分编辑位点。

二穗短柄草幼胚再生体系及农杆菌介导转化的初步研究

以二穗短柄草3个品系BDR018,BD21,BD21-3的幼胚为外植体,研究了幼胚大小对胚性愈伤组织诱导和再生的影响。采用携带gus和bar基因双元表达载体(pDM805)的根癌农杆菌菌株AGL1对BDR018品系的幼胚愈伤组织进行了遗传转化。本实验探讨了影响幼胚愈伤组织诱导再生及遗传转化的几个因素。结果发现,幼胚大小介于0.5～1.0mm之间的愈伤组织诱导率最高。随愈伤组织年龄的增加,BDR018,BD21,BD21-3愈伤组织的再生率下降,白化率上升。愈伤组织年龄在5～8周范围内转化效率较高,平均转化效率为38.5%。真空处理5min和0.01%的Silwet L-77处理均可提高转化效率。对转化植株进行GUS基因化学组织检测和PCR鉴定,初步证明外源基因已整合再生植株基因组中。

盐胁迫对二穗短柄草幼苗生长及不同器官中盐离子稳态的影响

采用4种浓度的NaCl溶液(50、100、150、200mmol/L)处理二穗短柄草和拟南芥(对照)幼苗,测定不同浓度盐胁迫下2种植物幼苗的生长指标和离子分布,以探讨二穗短柄草在盐胁迫下主要阳离子平衡机制。结果表明:(1)盐胁迫显著抑制二穗短柄草根叶的生物量积累。(2)根冠比数据显示,在盐胁迫条件下二穗短柄草能够更好地维系地下部分的生物量积累。(3)在4种浓度盐胁迫下,二穗短柄草叶中Na+含量低于根系,而且K+、Cl-含量和K+/Na+比值始终高于根系,说明在二穗短柄草中Na+从地下到地上的转运受到抑制,但对Cl-的转运缺乏有效的调控。(4)回归分析发现,盐胁迫下二穗短柄草和拟南芥根部Na+与K+含量变化呈正相关关系,而在叶部则不相关,说明二穗短柄草和拟南芥Na+与K+在根部具有相同的离子通道,而在叶部却具有各自独立的转运途径。

普通小麦及其近缘物种花序、小穗和小花的形态结构分析

花的发育影响着种子的发育。普通小麦是三大粮食作物之一,但对小麦花序发育的研究滞后于水稻和玉米。为揭示小麦花发育的机理,本研究采用体视镜观察、扫描电子显微镜观察和组织切片后光学显微镜观察等方法,对普通小麦及其近缘物种一粒小麦、拟斯卑尔脱山羊草、粗山羊草、二粒小麦以及二穗短柄草的花序、小穗和小花形态结构进行了系统观察和分析比较。结果表明,普通小麦与二倍体小麦、二粒小麦表现出一定差别：普通小麦每个小穗形成8~10朵小花,但在发育后期,大多数小花退化不育,每个小穗只有3~4朵小花能够形成种子;二倍体小麦和二粒小麦的小穗结构与普通小麦类似,但每个小穗发育形成3~5朵小花,小花数目明显减少。与普通小麦相似,二穗短柄草每个小穗同样分化形成多朵小花,不同的是,二穗短柄草的大多数小花发育正常,是可育的。以上这些研究结果暗示小麦具有巨大的增产潜力。

新的禾本科模式植物——二穗短柄草

水稻(Oryza sativa)是禾本科中目前唯一已完成基因组测序的模式植物,但由于它很早从冷季型禾本科中进化分离出来,故其作为禾本科其他亚科的模式植物有一定局限性,因此,需要一种新的、更适合的模式植物。二穗短柄草(Brachypodium distachyon)是一种温带禾草,归属禾本科早熟禾亚科短柄草族短柄草属,具有基因组小、株型小、自花授粉、一年生、生命周期短、有二倍体和一系列多倍体、易培植、易转化等诸多优点,尤其是与很多重要的谷类作物如小麦、大麦、燕麦、玉米、水稻、高粱等以及许多牧草与草坪草有较近的亲缘关系,因而是温带禾本科植物功能基因组学以及生物能源作物(如柳枝稷)研究的理想模式植物,作者例举二穗短柄草之所以成为新模式植物系统的依据,并介绍其在遗传进化、基因组特点、功能基因组及形态特征等方面的研究进展。

禾本科新型模式植物二穗短柄草的研究进展

二穗短柄草(Brachypodium distachyum)植株矮小,生育期短,基因组大小仅为水稻的0.71倍;和水稻相比,在进化上它与小麦等温季型禾谷类作物的亲缘关系更近,是目前较为理想的禾谷类作物模式植物。在短短几年中人们对这一模式植物在细胞遗传学、基因组学以及遗传转化等方面进行了大量研究。对已取得的研究进展进行了全面总结和综述,以期为禾谷类作物尤其是温季型禾谷类作物的相关研究提供参考。

短柄草磷转运蛋白家族基因的克隆及表达分析

植物高亲和力磷转运蛋白Pht1家族是一类H2PO4-/H＋共转运子,主要在根系中负责磷的吸收和转运,其表达受低磷调控,对该家族成员的研究有助于揭示磷的吸收和转运机制。根据水稻、拟南芥、大麦中磷转运蛋白基因的序列,预测出短柄草Pht1家族共有12个成员,分别命名为BRAdi;Pht1;1~BRAdi;Pht1;12,设计基因特异引物,对基因组和cDNA全长基因进行克隆、测序,通过对基因编码的氨基酸序列同源比对分析,构建了系统发生树,并利用实时荧光定量PCR技术对各基因成员的表达模式进行了分析。结果表明,预测到的成员具有Pht1家族的典型结构,成员间的同源性高,进化树分析将这12个同源基因分为不同的亚组,这些同源基因与大麦的同源性较高,其次是水稻,而与拟南芥的同源性最低。这些基因在不同的组织中表达量不同,在种子中的表达量最高,有5个基因在苗期根中的表达显著高于叶片。