羟丙基壳聚糖对小麦幼苗生理功能的影响

利用0.3%羟丙基壳聚糖溶液对小麦幼苗进行喷洒处理,在幼苗发育的5个时段对4种酶活性和叶绿素含量进行测定。结果表明,用羟丙基壳聚糖处理后,硝酸还原酶、核酮糖二磷酸(RuBP)羧化酶、超氧化物歧化酶活性比对照高出20%左右,多酚氧化酶活性变化不显著,随着幼苗发育呈现先高后低的变化,叶绿素含量处理明显高于对照30%左右,说明羟丙基壳聚糖可以促进小麦碳氮代谢,对小麦的抗性和光合作用有明显的促进作用。     

花生RuBPCase小亚基基因的克隆与序列分析

根据已知植物的核酮糖-1,5-二磷酸羧化/加氧酶(RuBPCase)小亚基基因序列设计简并引物,采用RT-PCR技术从花生叶片中克隆得到RuBPCase小亚基基因,命名为AhrbcS,Genbank登录号为KF607110,该基因编码区长度为549 bp,编码182个氨基酸.序列比对结果表明,AhrbcS编码蛋白与绿豆、菜豆、大豆、短绒野大豆和烟豆等具有较高的相似性.     

Potential for chemolithoautotrophy among ubiquitous bacteria lineages in the dark ocean

Recent studies suggest that unidentified prokaryotes fix inorganic carbon at globally significant rates in the immense dark ocean. Using single-cell sorting and whole-genome amplification of prokaryotes from two subtropical gyres, we obtained genomic DNA from 738 cells representing most cosmopolitan lineages. Multiple cells of Deltaproteobacteria cluster SAR324, Gammaproteobacteria clusters ARCTIC96BD-19 and Agg47, and some Oceanospirillales from the lower mesopelagic contained ribulose-1,5-bisphosphate carboxylase-oxygenase and sulfur oxidation genes. These results corroborated community DNA and RNA profiling from diverse geographic regions. The SAR324 genomes also suggested C(1) metabolism and a particle-associated life-style. Microautoradiography and fluorescence in situ hybridization confirmed bicarbonate uptake and particle association of SAR324 cells. Our study suggests potential chemolithoautotrophy in several uncultured Proteobacteria lineages that are ubiquitous in the dark oxygenated ocean and provides new perspective on carbon cycling in the ocean's largest habitat.     

A 3-hydroxypropionate/4-hydroxybutyrate autotrophic carbon dioxide assimilation pathway in Archaea

The assimilation of carbon dioxide (CO2) into organic material is quantitatively the most important biosynthetic process. We discovered that an autotrophic member of the archaeal order Sulfolobales, Metallosphaera sedula, fixed CO2 with acetyl-coenzyme A (acetyl-CoA)/propionyl-CoA carboxylase as the key carboxylating enzyme. In this system, one acetyl-CoA and two bicarbonate molecules were reductively converted via 3-hydroxypropionate to succinyl-CoA. This intermediate was reduced to 4-hydroxybutyrate and converted into two acetyl-CoA molecules via 4-hydroxybutyryl-CoA dehydratase. The key genes of this pathway were found not only in Metallosphaera but also in Sulfolobus, Archaeoglobus, and Cenarchaeum species. Moreover, the Global Ocean Sampling database contains half as many 4-hydroxybutyryl-CoA dehydratase sequences as compared with those found for another key photosynthetic CO2-fixing enzyme, ribulose-1,5-bisphosphate carboxylase-oxygenase. This indicates the importance of this enzyme in global carbon cycling.     

吉林省粳稻盐胁迫响应差异表型和蛋白质分析

以吉林粳稻耐盐型吉8945和盐敏感型吉农大808为试验材料,通过双向聚丙烯酰胺凝胶电泳技术(2-DE)以及基质辅助激光解吸附离子化时间飞行质谱(MALDI-TOF-MS/MS)技术,分别对早期幼苗(10d苗龄)在正常和盐胁迫条件(100 mmol/L NaCl)下盐胁迫响应蛋白质组进行分析、鉴定,在叶片中,与对照相比,盐处理后吉8945有21个点明显上调,吉农大808中35个点上调表达,其中12个点在吉8945和吉农大808均上调表达且差异明显。选取其中6个点进行MALDI-TOF-MS/MS质谱分析,鉴定出两种蛋白质,分别为磷酸丙酮酸水合酶和核酮糖1,5-二磷酸羧化酶/加氧酶。     

Structure of the nucleotide activation switch in glycogen phosphorylase a

Adenosine monophosphate is required for the activation of glycogen phosphorylase b and for release of the inhibition of phosphorylase a by glucose. Two molecules of adenosine monophosphate (AMP) bind to symmetry related sites at the subunit interface of the phosphorylase dimer. Adenosine triphosphate (ATP) binds to the same site, but does not promote catalytic activity. The structure of glucose-inhibited phosphorylase a bound to AMP and also of the complex formed with glucose and ATP is described. Crystallographic refinement of these complexes reveals that structural changes are associated with AMP but not ATP binding. The origin of these effects can be traced to different effector binding modes exhibited by AMP and ATP, respectively. The conformational changes associated with AMP binding traverse multiple paths in the enzyme and link the effector and catalytic sites.     

小麦叶片内1,5-二磷酸核酮糖羧化酶/加氧酶大亚基由53 000到50 000裂解反应的定位研究

研究了小麦叶片内1，5-二磷酸核酮糖羧化酶／加氧酶（Rubisco，EC 4．1．1．39）大亚基（LSU）由53000裂解为50000的反应。结果显示，成熟叶片的粗提液在pH5．5的条件下反应后能检测到50000的裂解产物，而暗诱导衰老叶片的粗提液在pH7．5的条件下也能发现LSU的这一降解。分别从成熟叶片和衰老叶片中提取叶绿体，以其裂解液为反应体系研究LSU由53000裂解为50000这步反应的细胞器定位。结果显示，衰老叶片中的叶绿体裂解液在pH7．5时反应1h后能检测到50000降解产物，而成熟叶片叶绿体裂解液在pH5．5和pH7．5的条件下反应后均未检测到LSU的50000裂解产物。上述结果表明：衰老叶片中ISU由53000部分裂解为50000的反应定位于叶绿体内，而成熟叶片中LSU由53000裂解为50000的反应可能定位于叶绿体外。     

Stop-transfer regions do not halt translocation of proteins into chloroplasts

Protein targeting in eukaryotic cells is determined by several topogenic signals. Among these are stop-transfer regions, which halt translocation of proteins across the endoplasmic reticulum membrane. Two different stop-transfer regions were incorporated into precursors for a chloroplast protein, the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Both chimeric proteins were imported into chloroplasts and did not accumulate in the envelope membranes. Thus, the stop-transfer signals did not function during chloroplast protein import. These observations support the hypothesis that the mechanism for translocation of proteins across the chloroplast envelope is significantly different from that for translocation across the endoplasmic reticulum membrane.     

Annotation and validation of genes involved in photosynthesis and starch synthesis from a Manihot full-length cDNA library

A full-length cDNA library from leaf and root tissues of cassava (Manihot esculenta) Arg7 and one accession of its wild ancestor W14 (M. esculenta ssp. flabellifolia) has been constructed. The library is comprised of four sub-libraries, containing 32640 recombinant clones, 6028 cDNA clones from their 5′ ends, and 128 clones from the 3′ ends were sequenced. In total, 5013 high-quality expressed sequence tags (ESTs) and 1259 unigenes were obtained. Of these, 746 unigenes were identified by their sequence homologies to ESTs from model plants, and 323 unigenes were mapped onto 114 different KEGG pathways. From these, 24 differentially expressed genes involved in starch metabolism and photosynthesis were identified and five of them were selected to compare their expression level between Arg7 and W14. Notably, Arg7 has a higher net photosynthesis rate in leaves, higher ribulose-1,5-bisphosphate carboxy-lase oxygenase activities in leaves, and higher AGPase activity in roots. This resource is the first EST collection from wild cassava and should be of value for gene discovery, genome annotation and studies of Manihot evolution.     

6-苄基腺嘌呤和脱落酸对烟草光合功能衰退的影响

研究了6-苄基腺嘌呤（6-BA）和脱落酸（ABA）对2个不同基因型烟草品种（Nicotiana tabacum L．‘NC89’＆‘JYH’）叶片光合功能衰退的影响。结果表明，6-BA处理显著提高两品种烟草叶片的瞬时光合速率（IAPS）、叶绿素含量、叶绿体全电子传递活性和核酮糖-1，5-二磷酸羧化酶（RuBPCase）活性，延长光合功能期，提高烟草叶片光合电子传递和碳同化的衰退比，延缓光合功能衰退；ABA处理，结果相反。品种JYH对这2种激素的反应较NC89敏感。     

巴西橡胶三品系叶片RuBP羧化酶的免疫酶标定位

通过提纯的RuBP羧化酶制备兔抗RuBP羧化酶抗体,以辣根过氧化物酶进行酶联标记,对典型的C4植物甘蔗、C3植物水稻和巴西橡胶三品系叶切片进行RuBP羧化酶的定位.通过显微及超微观察,结果表明:C4和C3植物叶切片中RuBP羧化酶的分布明显不同,C4植物的特异颗粒颜色反应(棕色)存在于维管束鞘细胞;C3植物的特异颗粒颜色反应(棕色)存在于叶肉细胞.巴西橡胶IAN873、RRIM600品系叶片内RuBP羧化酶在鞘细胞和叶肉细胞的叶绿体中均有特异颗粒颜色反应(棕色);而天任31 45品系只有叶肉细胞的叶绿体中有特异颗粒颜色反应(棕色).试验还表明,叶肉细胞的RuBP羧化酶发育可能在前,鞘细胞RuBP羧化酶发育可能在后.     

Photosynthesis-associated gene families: differences in response to tissue-specific and environmental factors

The endogenous small subunit of the ribulose-1,5-bisphosphate carboxylase gene rbcS and the light-harvesting chlorophyll a/b-binding protein gene (LHCP) of pea are expressed in a light-inducible manner and are active mainly in green chloroplast-containing tissue. Chimeric genes under control of the 5'-flanking sequences of the rbcS ss3.6 or LHCP AB80 genes from pea were used to study the factors relating to the issue-specific and lightinducible expression of these nuclear-encoded genes in transgenic tobacco plants. The results show that plastid development plays a crucial role in the activation of expression of these chimeric genes. Particular members of each of the above gene families respond differently to tissue-specific and environmental factors. Furthermore, the light-inducible expression directed by the 5'-flanking sequence of ss3.6 rbcSgene is not exclusively mediated by phytochrome, but probably is controlledin large part by another photoreceptor.     

芸薹属两个亚种间杂种光合作用优势及其机理

 【目的】探讨芸薹属作物杂种是否具有光合作用优势及其机理。【方法】利用气体交换和叶绿素荧光技术系统地研究了‘矮脚黄’白菜和‘白蔓菁’芜菁及其正反交杂种的叶片光合作用的特性，并对卡尔文循环中的关键酶核酮糖1，5-二磷酸羧化/加氧酶（RuBisCO）进行了相对定量研究。【结果】在整个试验期间，杂种F1的净光合速率（Pn）显著高于双亲，具有较强的杂种优势。杂种气孔导度（Gs）也高于亲本，但其胞间CO2浓度（Ci）和气孔限制值（l）与亲本间没有明显的差异。杂种总叶绿素含量介于双亲之间，仅表现出微弱的中亲优势。杂种PSII光合电子传递量子效率（ΦPSII）及光合电子传递速率（ETR）显著高于亲本，其变化趋势和PSII反应中心开放程度（qP）一致。RuBisCO最大羧化速率（Vcmax）、最大电子传递速率（Jmax）和RuBisCO蛋白含量在杂种中均有所增加。【结论】气孔导度、叶绿素含量和光呼吸均不是杂种光合优势产生的主要原因。光合优势的产生很可能是由于碳固定效率的增加反馈上调了光合电子传递速率引起的。     

干旱诱导的甘蓝型油菜SSH文库及抗旱相关基因表达的分析

从前期构建的干旱诱导的甘蓝型油菜SSH文库中随机挑取了646个阳性克隆,对其进行测序及生物信息学分析。结果表明:通过测序,获得639条高质量EST序列,186条单一序列(重叠群89条,单拷贝序列97条);比对分析发现,166条单一序列有同源序列;乙醛酸和二羧酸代谢、碳代谢、光合中的碳固定、氨基酸的生物合成、氮代谢等在植物抵抗干旱胁迫中发挥着重要的作用;MYB、NAC5、锌指蛋白等转录因子以及苹果酸脱氢酶、核酮糖1,5–二磷酸羧化酶/加氧酶、果糖–1,6–二磷酸醛缩酶、磷酸核酮糖激酶等与光合相关的基因以及P5CS基因、F–box蛋白等参与油菜干旱胁迫应答;单一序列涉及的功能中所占比例较大的是蛋白绑定和催化活性。以抗旱性较强的甘蓝型油菜Holiday为材料,在开花初期进行干旱处理,在处理的第1、3、5、7天(土壤含水量分别为60%、40%、30%、20%)进行叶片取样,采用实时荧光定量PCR,分析基因表达量,发现随机选取的文库中的3个基因(P5CSb、BnSOS2和CAM)在干旱胁迫下表达水平均上调,推测这3个基因在抵御干旱胁迫中发挥了重要作用。     

Combining Phytate/Ca2+ Fractionation with Trichloroacetic Acid/Acetone Precipitation Improved Separation of Low-Abundant Proteins of Wheat (Triticum aestivum L.) Leaf for Proteomic Analysis

Proteomic assessment of low-abundance leaf proteins is hindered by the large quantity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) present within plant leaf tissues. In the present study, total proteins were extracted from wheat (Triticum aestivum L.) leaves by a conventional trichloroacetic acid (TCA)/acetone method and a protocol first developed in this work. Phytate/Ca²⁺ fractionation and TCA/acetone precipitation were combined to design an improved TCA/acetone method. The extracted proteins were analysed by two-dimensional gel electrophoresis (2-DE). The resulting 2-DE images were compared to reveal major differences. The results showed that large quantities of Rubisco were deleted from wheat leaf proteins prepared by the improved method. As many as (758±4) protein spots were detected from 2-DE images of protein extracts obtained by the improved method, 130 more than those detected by the TCA/acetone method. Further analysis indicated that more protein spots could be detected at regions of pI 4.00–4.99 and 6.50–7.00 in the improved method-based 2-DE images. Our findings indicated that the improved method is an efficient protein preparation protocol for separating low-abundance proteins in wheat leaf tissues by 2-DE analysis. The proposed protocol is simple, fast, inexpensive and also applicable to protein preparations of other plants.     

Phosphorylase kinase of the liver: deficiency in a girl with increased hepatic glycogen

Studies of a child with glycogenosis revealed an increased concentration of glycogen and low phosphorylase activity in her liver. Using mixtures of homogenates of the patient's liver and of normal liver, we found the low phosphorylase activity to be caused by a deficiency of phosphorylase kinase and not of hepatic phosphorylase. The fact that phosphorylase activity was restored to normal values by the addition of phosphorylase b kinase from rabbit muscle substantiates this conclusion.     

小麦叶片蛋白质组的2D-LC分离及Nano LC-MS/MS分析

 【目的】二维液相色谱（2D-LC）与双向电泳（2-DE）技术具有互补性,本文旨在探讨利用2D-LC和纳升级液相色谱串联质谱（Nano LC-MS/MS）技术分离鉴定小麦叶片蛋白质组的新方法。【方法】小麦叶片蛋白经提取、脱盐后,进行第一维阴离子交换分离;收集洗脱组分进行SDS-PAGE分析,并对非高丰度蛋白组分进行第二维反相液相色谱分离;随机选择含较低吸收峰的部分组分经胰蛋白酶水解后进行Nano LC-MS/MS 分析;将串联质谱数据通过MASCOT搜索NCBInr和EST数据库,并对二级质谱获得的蛋白序列进行MS BLAST分析。【结果】经第一维阴离子交换色谱分离,收集得到15个组分,其中第15组分包含小麦叶片丰度最高的蛋白——1,5-二磷酸核酮糖羧化酶/加氧酶（RuBisCO）;其余14个组分经反相液相色谱分离,共收集1 551个组分,获得1 867个色谱峰;随机对其中6个含较低吸收峰的收集组分酶解和Nano LC-MS/MS 检测,9种蛋白得到鉴定。【结论】结果初步表明,利用2D-LC和Nano LC-MS/MS技术可有效地分离和鉴定小麦叶片蛋白质组,为更深入全面地研究小麦叶片蛋白质组奠定基础。