玉米磷转运蛋白基因ZmPht3;1的克隆和功能分析

Pht3(phosphate transporter 3)磷转运子家族属于一类低亲和力磷转运蛋白,在调节植株体内磷素的动态平衡中发挥重要作用。为了初步探讨玉米中Zm Pht3;1基因的结构特征及其磷饥饿的响应机制,利用同源克隆的方法从耐低磷玉米自交系Mo17中分离得到Zm Pht3;1基因,并运用实时荧光定量PCR和亚细胞定位的方法对其进行深入研究。结果表明,Zm Pht3;1的编码区全长1 101 bp,编码366个氨基酸,含有典型的线粒体转运家族(mitochondrial carrier family,MCF)结构特征与6个疏水跨膜结构。荧光定量PCR分析表明,该基因在两个极端材料的根系与叶片中均有表达,而表达模式差异显著,在耐低磷玉米自交系Mo17的根系和叶片中表现为缺磷胁迫前期的一般性反应和后期的特异性反应。转化烟草的亚细胞定位结果显示,Zm Pht3;1主要分布于细胞膜上,可能是一个双亲和转运体,在玉米响应磷饥饿胁迫过程中发挥重要的适应性调节作用。     

玉米JAZ家族基因ZmJAZ4的克隆及功能分析

JAZ(Jasmonate ZIM-domain)蛋白是植物特有的一类转录因子,通过抑制茉莉素调控基因的表达,在植物的生长发育及非生物胁迫等方面发挥重要的功能。从玉米B73自交系中克隆到一个新的JAZ家族基因Zm JAZ4,该基因c DNA全长为651bp,编码蛋白含有216个氨基酸,分子量约为23.1 k D,p I为10.78,属于碱性蛋白。Real-time RT-PCR结果表明,Zm JAZ4主要在茎端分生组织、雄穗、发育早期的种子以及胚乳中表达。系统进化分析显示,Zm JAZ4与At JAZ10转录因子相似性较高。亚细胞定位试验表明,Zm JAZ4定位于细胞核内。Zm JAZ4在酵母细胞中不具有转录激活活性。激素及胁迫处理表明,Zm JAZ4在地上部的表达受PEG、Na Cl、SA、GA和ABA诱导,而在地下部的表达受到ABA和GA诱导。结果分析表明,Zm JAZ4可能是一个重要的转录调控因子,参与调控多种激素信号通路及非生物胁迫响应。     

高等植物硝酸盐转运蛋白的功能及其调控机制

硝酸盐是植物从土壤中吸收的重要无机氮素形态。植物为适应含有不同浓度NO3-的土壤环境,进化出了高亲和硝酸盐转运系统(HATS)和低亲和硝酸盐转运系统(LATS),两个基因家族NRT1和NRT2家族分别参与了LATS和HATS的NO3-的吸收和转运。近年来,随着分子生物学技术和植物基因组学的快速发展,研究人员克隆出了大量参与硝酸盐吸收和转运的基因,并对这些基因的功能进行了深入研究,逐渐形成了复杂的硝酸盐调控网络。综述了植物中硝酸盐转运蛋白基因的克隆、表达及调控,并对进一步的研究作了展望,这些结果对于理解植物硝酸盐吸收的调控机制具有重要作用。     

玉米分子伴侣基因ZmBiP1的表达模式和功能研究

以抗旱玉米自交系旱21为材料,克隆获得玉米分子伴侣基因Zm Bi P1,通过荧光定量PCR技术研究Zm Bi P1基因的表达模式,并将Zm Bi P1基因转入拟南芥中研究其功能。荧光定量PCR结果表明,Zm Bi P1基因在玉米自交系旱21的不同组织器官中均有表达,且在子房中表达量最高。Zm Bi P1基因受甘露醇胁迫诱导上调表达,受盐胁迫诱导下调表达。转基因拟南芥功能验证结果表明,过表达Zm Bi P1基因导致拟南芥在种子萌发时期对盐和甘露醇胁迫的耐受能力减弱。这些结果表明Zm Bi P1基因可能是逆境反应信号传递途径的负调节因子。     

玉米棉子糖合成酶(ZmRAFS)基因响应干旱胁迫的转录调控研究

玉米棉子糖合成酶基因ZmRAFS受干旱诱导表达,但其响应干旱的表达调控机理仍不清楚。本研究通过RNA-seq数据分析及相关实验,发现转录因子ZmHB7在玉米幼苗中响应干旱和ABA处理。通过PCR方法克隆了ZmHB7编码区片段,并构建了其原核表达载体和植物表达载体。体外诱导ZmHB7蛋白表达,纯化回收并制备相应抗体。Western blot实验证实了ZmHB7蛋白在玉米叶肉原生质体中正确表达。将ZmHB7和GFP植物表达载体分别与Zm RAFS基因全长启动子片段驱动的双荧光素酶报告载体转入玉米原生质体中,酶活检测结果显示,ZmHB7转录因子能够激活ZmRAFS启动子驱动的海参荧光素酶基因(Rluc)的表达。逐步截短Zm RAFS基因启动子,将Zm RAFS不同长度启动子片段驱动的双荧光素酶报告载体分别与GFP或ZmHB7植物表达载体转入玉米原生质体中,酶活检测结果发现,ZmHB7仍能上调ZmRAFS基因,这一结果说明ZmHB7可能间接调控ZmRAFS基因的表达。本文初步探索了ZmRAFS基因响应干旱的分子调控机制,为进一步深入研究棉子糖合成酶基因Zm RAFS的表达调控奠定了基础。     

结缕草转录因子基因ZjDREB4.1克隆和逆境表达模式

以日本结缕草(Zoysia japonica)‘Meyer’品种为材料,克隆获得了1个DREB(dehydration responsive element binding)类转录因子基因,命名为Zj DREB4.1。该基因编码区长651 bp,编码216个氨基酸,推测的蛋白质Zj DREB4.1分子量为22.9 k D,等电点p I为5.74,第50-110位氨基酸组成一个典型的AP2保守结构域。在基因的系统发生树中,Zj DREB4.1蛋白与拟南芥At TINY蛋白和玉米Zm DBF2蛋白聚为一支,属于DREB亚家族A-4组。在叶组织中Zj DREB4.1为组成型表达,受低温诱导上调表达,在干旱和高盐胁迫下表达先下调后恢复至正常水平。     

蒺藜苜蓿MtROP10基因的克隆及其功能初探

目的:克隆并研究蒺藜苜蓿ROP基因的功能,为研究该基因家族在共生途径中的作用提供依据.方法:采用RACE方法,从蒺藜苜蓿中克隆MtROP基因,利用生物信息软件比对同源性及ROP蛋白特征结构分析,利用RT-PCR方法分析该基因的组织特异性表达,构建该基因的过表达载体并转化拟南芥.结果:获得了蒺藜苜蓿ROP家族中与拟南芥ROP10高度同源的MtROP10全长序列.氨基酸编码序列具有明显的ROP家族蛋白的结构域特征.该基因在花中高表达,根中低表达.拟南芥中过表达MtROP10,可导致根毛变粗、变短、分叉.结论:MtROP10属于植物ROP家族蛋白,可能在根毛的极性生长方面具有较为重要的功能.     

玉米扩展蛋白Expansin基因家族定位及基因表达模式分析

扩展蛋白（Expansin）是一类能够使植物细胞壁松弛的活性蛋白,在植物生长发育过程中起着重要作用。利用PLAZA、NCBI、MaizeGDB、Uniprot、PLEXdb等基因组数据库,获得玉米Expansin家族的基因序列、染色体基因座位、蛋白质序列以及长度,构建玉米Expansin基因家族系统进化树,进行基因组织表达谱的分析。结果表明,玉米基因组中含有93个Expansin基因,分布于玉米的9条染色体上;多数Expansin具有250~300个氨基酸;玉米Expansin基因家族有40个Expansin A（EXPA）、47个Expansin B（EXPB）、6个Expansin-like A（EXLA）,未发现Expansin-like B（EXLB）;44个玉米Expansin基因在不同玉米组织中特异表达。该研究结果不仅为玉米扩展蛋白的深入研究奠定了基础,而且为其他研究人员对基因信息的获取提供了参考。     

人FGF-21基因的克隆、表达及其调节脂肪细胞糖代谢的活性

成纤维细胞生长因子FGF-21是FGF家族的一个新成员, 最近的研究发现其具有调节血糖的作用, 有望成为治疗2型糖尿病的基因药物。文章应用RT-PCR技术, 从成人肝脏中克隆人源的FGF-21成熟蛋白基因, 并将其克隆到T载体上, 经测序鉴定后, 将其亚克隆到原核表达载体pSUMO上, 转入大肠杆菌Rosetta(DE3)中。鉴定阳性克隆后, 用IPTG诱导FGF-21表达, 并用Ni-NTA柱进行亲和层析纯化。以3T3-L1脂肪细胞的葡萄糖吸收实验来鉴定FGF-21表达产物促进糖吸收的活性。结果表明, FGF-21成熟蛋白基因大小为546 bp, 测序结果与GenBank数据库中的序列一致。SDS-PAGE与Western blotting结果表明: 人源FGF-21成熟蛋白大小19.4 kDa, 经3T3-L1脂肪细胞的葡萄糖吸收实验验证其具有促进葡萄糖吸收的生物活性, 并且GLUT1是FGF-21发挥生物学作用的终末执行单位。     

高粱高亲和硝酸盐转运蛋白NRT2/3基因家族鉴定、表达与DNA变异分析

硝态氮是作物吸收无机氮素的主要形态,硝酸盐转运蛋白2(nitrate transporter 2,NRT2)作为高亲和性的转运蛋白,以硝酸盐作为特异性底物,在可利用的硝酸盐受限时,高亲和性转运系统被激活,在硝酸盐吸收、转运过程中发挥着重要作用。大多数NRT2不能单独转运硝酸盐,需在硝酸盐同化相关蛋白2(nitrate assimilation related protein 2,NAR2)的协助下才能完成硝酸盐的吸收或转运。作物氮利用效率受环境条件影响,品种间存在差异,因此培育高氮素利用效率品种有重大意义。高粱(Sorghum bicolor)具有耐贫瘠特性,对土壤中的氮素吸收和利用效率较高。本研究结合高粱基因组数据库对NRT2/3基因家族成员基因结构、染色体定位、理化性质、二级结构与跨膜结构域、信号肽与亚细胞定位、启动子区顺式作用元件、系统进化、单核苷酸多态性(single nucleotide polymorphism,SNP)的识别与注释及选择压力进行了全面分析。通过生物信息学分析,筛选出5个NRT2s(命名为SbNRT2-1a、2-1b、SbNRT2-2–4)基因和2个NAR2s(SbNRT3-1–2)基因,较谷子略少。分布在3条染色体上,分为4个亚家族,同一亚族中基因结构高度相似;高粱NRT2/3亲水性平均值均为正值,表明均为疏水性蛋白;α-螺旋和无规则卷曲占二级结构总量的比例大于70%;亚细胞定位均在质膜上,其中NRT2s蛋白不含信号肽,NRT3s蛋白含信号肽;进一步对其跨膜结构域进行分析,发现NRT2s家族成员跨膜结构域个数均大于10个,而NRT3s家族成员跨膜结构域个数为2个;高粱与玉米(Zea mays)NRT2/3s的共线性较好;蛋白结构域显示存在MFS_1和NAR2蛋白结构域,可执行高亲和力硝酸盐转运;系统进化树分析可知,高粱与玉米和谷子的NRT2/3基因亲缘关系更近;基因启动子顺式作用元件分析发现,SbNRT2/3基因的启动子区均具有数个植物激素和逆境应答元件,可以响应高粱生长和环境变化;基因表达热图显示低氮条件下在根诱导表达的是SbNRT2-1a、SbNRT2-1b和SbNRT3-1,推测可在高粱根部表达并调控对硝酸盐的吸收或转运过程。在SbNRT2-4和SbNRT2-1a等发现多个非同义SNP变异;选择压力分析表明,高粱NRT2/3基因家族在进化过程中受纯化选择作用。SbNRT2/3基因表达及蚜虫侵染影响与基因在不同组织中的表达分析结果一致,SbNRT2-1b和SbNRT3-1在感染蚜虫品系5-27sug根部表达显著,高粱蚜虫侵染叶片显著降低了SbNRT2-3、SbNRT2-4和SbNRT3-2的表达水平。本研究初步对高粱全基因组NRT2/3基因家族进行鉴定、表达与DNA变异分析,为高粱氮高效研究提供了基础。     

形态对不同小麦基因型氮素吸收的光合作用的影响

利用水培试验,研究了3个小麦基因型对不同形态N素吸收和积累的差异,结果表明,在不同N浓度优势,与次敏感型莱州953和钝感到江东门相比,敏感型扬麦158不仅具有较强的NO3^-和NH4^ 吸收能力,而且具有最强的增铵营养吸收能力,增铵营养促进了扬麦158和莱州953对NO3^-和NH4^ 的吸收,但在一定程度上抑制了江东门对NO3^-的吸收,与NO3^-营养及NH4^ 营养相比,增铵营养显著提高了杨表158和莱州953的全株、地上部N积累量和叶片合速率,而对江东门影响较小,因此,增铵营养促进了植株的N吸收,提高了N积累和光合速率,从而促进了小麦生长。     

Expression and localization of CLC chloride transport proteins in the avian retina

Members of the ubiquitously expressed CLC protein family of chloride channels and transporters play important roles in regulating cellular chloride and pH. The CLCs that function as Cl(-)/H(+) antiporters, ClCs 3-7, are essential in particular for the acidification of endosomal compartments and protein degradation. These proteins are broadly expressed in the nervous system, and mutations that disrupt their expression are responsible for several human genetic diseases. Furthermore, knock-out of ClC3 and ClC7 in the mouse result in the degeneration of the hippocampus and the retina. Despite this evidence of their importance in retinal function, the expression patterns of different CLC transporters in different retinal cell types are as yet undescribed. Previous work in our lab has shown that in chicken amacrine cells, internal Cl(-) can be dynamic. To determine whether CLCs have the potential to participate, we used PCR and immunohistochemical techniques to examine CLC transporter expression in the chicken retina. We observed a high level of variation in the retinal expression levels and patterns among the different CLC proteins examined. These findings, which represent the first systematic investigation of CLC transporter expression in the retina, support diverse functions for the different CLCs in this tissue.     

Short-term nitrogen-induced modulation of phosphoenolpyruvate carboxylase in tobacco and maize leaves

Untransformed maize and tobacco plants and tobacco plants constitutively expressing nitrate reductase were grown with sufficient NO(3)- to support maximal growth. Four days prior to treatment the tobacco plants were deprived of nitrogen. Excised maize leaves and tobacco leaf discs were fed with either 40 mM KNO(3) or 40 mM KCl (control) in the light. Phosphoenolpyruvate (PEP) carboxylase (Case) activity was measured at 0.3 mM and 3 mM PEP. The light- induced increase in PEPCase V(max) was greater in maize than tobacco. Furthermore light decreased malate sensitivity in maize (which was N-replete) but not in N-deficient tobacco. NO(3)- treatment increased PEPCase V:(max) values in both species and decreased the sensitivity to inhibition by malate, but effects of NO(3)- were much more pronounced in tobacco than maize. PEPCase kinase activity was, however, greater in maize leaves NO(3)- than in the Cl(-)-treated controls, suggesting that it is responsive to leaf nitrogen supply. A correlation between foliar glutamine content and PEPCase activity was observed. It is concluded that PEPCase is sensitive to N metabolites which favour increased flow through the anapleurotic pathway in both C(3) and C(4) plants.     

Influence of inorganic nitrogen and pH on the elongation of maize seminal roots

BACKGROUND AND AIMS: Root absorption and assimilation of inorganic nitrogen usually alters rhizosphere pH, but the immediate influence of such pH changes on root elongation as well as that of exogenous inorganic nitrogen itself has been uncertain. METHODS: A differential extensiometer that monitored on a real-time, continuous basis root elongation in an intact 3-d-old maize plant was developed. Treatments included root media at pH 6.5 or 5.6 that lacked nitrogen and ones at pH 6.5 that contained 100 mmol m(-3) NH(4)(+) or NO(3)(-). KEY RESULTS: Acidifying the root medium from pH 6.5 to 5.6 nearly doubled the elasticity of the seminal root, but slightly decreased its elongation. Plasticity of the root apex was not detectable in all treatments. The presence of ammonium or nitrate in the medium stimulated elongation by 29 % or 14 %, respectively. Addition of an osmoticum to the medium had no effect on root elongation in the absence of inorganic nitrogen, but diminished the stimulation of elongation in the presence of ammonium and nitrate. This indicates that these ions or their by-products serve partially as osmolytes. CONCLUSIONS: In nutrient solution, root elongation of a maize seedling--even one with ample nitrogen reserves--depended most strongly on exogenous inorganic nitrogen, and less so, if at all, on either the pH of the bulk nutrient solution or the mechanical properties of cell walls.     

Reaction of peroxynitrite with carbon dioxide: intermediates and determination of the yield of CO3 •– and NO2 •

CO2 catalyses the isomerization of the biological toxin ONOO- to NO3- via an intermediate, presumably ONOOCO2-, which has an absorption maximum near 650 nm. The reflection spectrum of solid NMe4+ ONOO- exposed to CO2 shows a similar band near 650 nm; this absorption decays over minutes. Stopped-flow experiments in which CO2 solutions were mixed with alkaline ONOO- solutions indicate the formation of at least one intermediate. The initial absorption at 302 nm is less than that of ONOO-, which indicates that reactions take place within the mixing time, and this absorption is dependent (but not linearly) on the ONOO- and CO2 concentrations. We found that reaction of peroxynitrite with carbon dioxide forms some trioxocarbonate(*1-) (CO3*-) and nitrogen dioxide (NO2*) radicals via homolysis of the O-O bond in ONOOCO2-. We determined the extent of radical formation by mixing peroxynitrite, carbon dioxide and nitrogen monoxide. The later reacts with CO3*- and NO2* radicals to form, effectively, three NO2- per homolysis; ONOOCO2- that does not undergo homolysis yields NO3- and CO2. Based on the NO3- and NO2- analyses, the extent of conversion to NO3- is 96 +/- 1% and that of homolysis is 3 +/- 1%, respectively, significantly less than that reported in the literature.     

不同NH4^＋／NO3^-比例的氮素营养对棉花氮素代谢的影响

The influence of different NH4^+／NO3^- ratios on nitrogen metabolism of cotton was studied under controlled hydroponics, The results showed that compared with single nitrate nutrition, solutions with 25/75, 50/50, 75/25 and 100/0 of NH4^+／NO3^- significantly increased the soluble protein accumulation in leaves and roots of cotton, and the maximum content of soluble protein in leaves and roots appeared respectively in the solution with 50/50 and 75/25 of NH4^+／NO3^- The soluble protein content in roots was increased with the increase of NH4+ percentage, but was slightly less in the solution of 100/0 than 75/25, which was probably related to the excess NH4^+ limiting boot metabolism. With the increase of NH4+ percentage, the nitrate content in petiole and the nitrate reductase activity in functional blade declined, but ammoniac nitrogen content increased in every organ of cotton. These results showed that foreign nitrogen affected the nitrogen metabolism of cotton in a different way, and the nitrogen absorption by cotton was probably related to different forms of foreign nitrogen.     

Selective recognition of mannose by the human eosinophil Charcot-Leyden crystal protein (galectin-10): a crystallographic study at 1.8 A resolution.

The role(s) of the eosinophil Charcot-Leyden crystal (CLC) protein in eosinophil or basophil function or associated inflammatory processes is yet to be established. Although the CLC protein has been reported to exhibit weak lysophospholipase activity, it shows virtually no sequence homology to any known member of this family of enzymes. The X-ray crystal structure of the CLC protein is very similar to the structure of the galectins, members of a beta-galactoside-specific animal lectin family, including a partially conserved galectin carbohydrate recognition domain (CRD). In the absence of any known natural carbohydrate ligand for this protein, the functional role of the CLC protein (galectin-10) has remained speculative. Here we describe structural studies on the carbohydrate binding properties of the CLC protein and report the first structure of a carbohydrate in complex with the protein. Interestingly, the CLC protein demonstrates no affinity for beta-galactosides and binds mannose in a manner very different from those of other related galectins that have been shown to bind lactosamine. The partial conservation of residues involved in carbohydrate binding led to significant changes in the topology and chemical nature of the CRD, and has implications for carbohydrate recognition by the CLC protein in vivo and its functional role in the biology of inflammation.     

小流域内植被类型对土壤NO3-/NH4+空间变化的影响

流域内植被类型、地形地貌特征对土壤氮循环过程有重要的作用,是影响下游水体无机氮素来源以及富营养化的关键因子。通过比较小流域内4种植被类型(落叶松人工林、油松人工林、天然阔叶次生林和农田(玉米))对土壤NO3--N和NH4+-N含量空间变化的影响,揭示流域内不同立地条件下水源涵养林与土壤无机氮变化特征之间的关系。结果表明:4种植被类型土壤NO3--N和NH4+-N含量差异显著(P<0.05);由坡上到坡下土壤NO3--N和NH4+-N含量显著降低;在土壤表层NO3--N和NH4+-N含量最高,随着土层深度增加无机氮含量减少;与水源涵养林天然植被和人工林植被相比,农田土壤NO3--N含量最高(11.86mg·kg-1),有较高的氮流失风险。     

Analyses of phylogeny, evolution, conserved sequences and genome-wide expression of the ICK/KRP family of plant CDK inhibitors

Background and Aims

The cell cycle is controlled by cyclin-dependent kinases (CDKs), and CDK inhibitors are major regulators of their activities. The ICK/KRP family of CDK inhibitors has been reported in several plants, with seven members in arabidopsis; however, the phylogenetic relationship among members in different species is unknown. Also, there is a need to understand how these genes and proteins are regulated. Furthermore, little information is available on the functional differences among ICK/KRP family members.

Methods

We searched publicly available databases and identified over 120 unique ICK/KRP protein sequences from more than 60 plant species. Phylogenetic analysis was performed using 101 full-length sequences from 40 species and intron–exon organization of ICK/KRP genes in model species. Conserved sequences and motifs were analysed using ICK/KRP protein sequences from arabidopsis (Arabidopsis thaliana), rice (Orysa sativa) and poplar (Populus trichocarpa). In addition, gene expression was examined using microarray data from arabidopsis, rice and poplar, and further analysed by RT-PCR for arabidopsis.

Key Results and Conclusions

Phylogenetic analysis showed that plant ICK/KRP proteins can be grouped into three major classes. Whereas the C-class contains sequences from dicotyledons, monocotyledons and gymnosperms, the A- and B-classes contain only sequences from dicotyledons or monocotyledons, respectively, suggesting that the A- and B-classes might have evolved from the C-class. This classification is also supported by exon–intron organization. Genes in the A- and B- classes have four exons, whereas genes in the C-class have only three exons. Analysis of sequences from arabidopsis, rice and poplar identified conserved sequence motifs, some of which had not been described previously, and putative functional sites. The presence of conserved motifs in different family members is consistent with the classification. In addition, gene expression analysis showed preferential expression of ICK/KRP genes in certain tissues. A model has been proposed for the evolution of this gene family in plants.