五个水稻类受体激酶的抗体制备及检测

类受体激酶在植物发育、自交不亲和、雄性不育、抗逆和抗病等生命过程中起着重要的调控作用。为了对水稻类受体激酶的功能及生物学特性进行深入研究,本实验克隆表达了水稻中5个类受体激酶抗原表位片段,以纯化的蛋白质为抗原免疫新西兰兔,获得了特异性较高的多克隆抗体。Western blotting检测结果表明,5个类受体激酶均在叶片中表达。     

跨膜逆向转运蛋白NHXFS1多克隆抗体的制备及应用

NHXFS1基因是通过DNA家族改组（DNA family shuffling）技术,以拟南芥、水稻和菊花的液泡膜Na＋/H＋逆向转运蛋白基因（NHX1）为亲本获得的活性显著增强的新基因。为制备该蛋白的多克隆抗体,对该蛋白进行跨膜结构分析,选取跨膜蛋白的C末端为靶标,并将其克隆到原核表达载体pET32a中,成功构建了原核融合蛋白pET32a-NHXFS1-抗原表达载体,转化大肠杆菌BL21（DE3）并诱导表达。通过镍柱亲和层析纯化该融合表达蛋白,获得了纯度约为80%的纯化蛋白,用于免疫新西兰大白兔制备多克隆抗体。ELISA实验表明,该抗体的效价达到1：128 000,提取表达NHXFS1蛋白的酵母液泡经该多克隆抗体Western blot检测,证明该抗体具有较好的NHXFS1蛋白特异性。NHXFS1多克隆抗体的制备为进一步认识NHXFS1新蛋白结构与功能以及植物耐盐分子生物学的研究奠定了基础。     

花生类受体蛋白激酶基因AhBAK1的克隆及原核表达

BAK1是富含亮氨酸重复序列型类受体蛋白激酶,参与调控植物先天免疫反应过程中的程序性细胞死亡过程。实验室已有的花生铝胁迫转录组数据揭示AhBAK1为铝胁迫响应基因。为研究其在花生抗铝机制中的作用,该文分析了铝胁迫下AhBAK1在花生耐铝品种‘99-1507’和铝敏感品种‘中花2号’(‘ZH2’)根尖中的转录变化,采用RT-PCR技术进行扩增AhBAK1的完整CDS序列,并对序列特征进行了分析。结果表明:AhBAK1显著响应铝处理,且在‘99-1507’中有着更强的诱导表达; AhBAK1包括625个氨基酸残基,属富亮氨酸重复区类受体激酶,具跨膜域和蛋白激酶催化结构域,不存在信号肽,预测定位于细胞质膜;我们进一步构建了含有AhBAK1激酶域的pGEX-6p-1-AhBAK1-CD重组质粒,体外诱导表达出约70kD可溶性蛋白,经凝胶亲和层析纯化,最终得到基于蛋白印迹实验(Western Blot)验证正确的重组蛋白。为进一步研究AhBAK1的生物学功能和生化功能奠定了基础。     

拟南芥细胞壁关联蛋白激酶融合表达及多克隆抗体的制备

为了研究拟南芥中细胞壁关联蛋白激酶1(WAK1)基因功能,通过RT-PCR得到WAK1 cDNA片段,将其亚克隆至pET28a表达载体中进行表达,获得融合蛋白.用纯化的融合蛋白pET28aWAK1免疫家兔,得到抗WAK1抗体.Western blot结果显示该抗体能特异识别在原核表达系统内表达的抗原,抗WAK1的抗体对拟南芥中的WAK1蛋白具有高度的特异性.     

水稻中类受体蛋白激酶FERONIA-like Receptor 1的表达纯化及抗体制备

类受体蛋白激酶参与调控植物的器官发育和抗逆性,是一种重要的蛋白激酶。通过对水稻中类受体蛋白激酶家族成员FERONIA-like receptor 1 (FLR1)进行蛋白质结构预测分析,发现FLR1蛋白具有跨膜结构,其N端在细胞膜外(FLR1-BW, 1～450 aa), C端在细胞膜内(FLR1-BN, 474～892 aa)。为了得到FLR1抗体用于其功能分析,将FLR1的N端和C端分别构建到原核表达载体pET-32a、pGEX-4T-1、pET-22b、pTriEx-4上,比较FLR1-BW和FLR1-BN在不同载体和不同条件下的表达效果。结果显示:FLR1-BW在pET-32a及pGEX-4T-1中能够有效表达,最优条件为16℃/16 h、0.5 mmol/L IPTG;而FLR1-BN蛋白表达后会对大肠杆菌产生细胞毒性,但在293细胞中能够表达。进一步利用纯化的FLR1-BW蛋白制备FLR1多克隆抗体,结果显示该抗体特异识别FLR1蛋白。本研究为类受体蛋白激酶的抗体制备提供了良好的思路并为研究FLR1功能奠定了基础。     

水稻YTB中OSVDAC5蛋白的原核表达与抗体制备

目的:克隆水稻YTB osvdac5基因,原核表达后获得纯化的OSVDAC5蛋白,制备相应的抗体.方法:采用Trizol法提取水稻总mRNA,反转录为cDNA,通过PCR扩增得到该基因与原核表达载体连接,构建重组质粒pET-30a-osvdac5,并转入大肠杆菌进行原核表达,SDS-PAGE检测表达产物.通过镍柱纯化获得的单一目的蛋白用于抗体制备,用Western Blot检测抗体的特异性.结果:克隆到原核表达载体中osvdac5基因的ORF为813 bp,编码271个氨基酸.在大肠杆菌中15℃、0.7mmol/L的IPTG浓度诱导17 h是pET-30a-osvdac5融合蛋白表达的优选条件,表达的OSVDAC5蛋白属于包涵体蛋白.镍柱纯化后的OSVDAC5为30 kD左右的单一条带.Western Blot分析表明,抗体能够与30 kD处的OSVDAC5蛋白进行特异性结合.结论:成功克隆了水稻YTB osvdac5基因,原核表达蛋白OSVDAC5制备的多免隆抗体具有一定特异性,能与免疫抗原结合,这为进一步研究OSVDAC5蛋白在植物不同生长发育时期中的表达模式奠定了基础.     

水稻类受体激酶OsCR4的抗体制备及特异性检测

运用生物信息学的方法对水稻类受体激酶OsCR4的抗原性进行分析,选取胞外部分片段与GST融合,在细菌大规模诱导该融合蛋白,利用GST sepharose进行亲和层析纯化,所得蛋白使用SDS-PAGE结合KCl/DTT染色切胶的方法得到收集。以此融合蛋白作为抗原免疫新西兰兔,获得了1：512,000效价的多克隆抗体,该抗体可特异识别水稻叶片微粒体组分中的OsCR4蛋白。     

水稻Qb-SNARE蛋白OsNPSN11多克隆抗体制备、鉴定与应用

在真核生物细胞囊泡运输过程中的膜融合主要是由SNARE蛋白介导的, OsNPSN11是从水稻中克隆的Qb-SNARE家族基因, 文章将OsNPSN11构建到原核表达载体pET-30a中与6个His标签融合, 重组质粒pET-OsNPSN11转化BL21(DE3)0.5 mmol/L IPTG诱导4 h后获得了高效表达。用镍离子亲和树脂(Ni²⁺-NTA His Bind Resin) 纯化融合蛋白, 以纯化后的蛋白为抗原免疫新西兰家兔制备多克隆抗体, Western blotting结果显示, 该抗体能特异识别在原核系统表达的抗原, 以及水稻不同组织质膜组分中的OsNPSN11, 可用于转基因水稻中目标蛋白的表达分析。     

植物凝集素类受体蛋白激酶研究进展

自然界中植物的生长发育受到各种环境变化的影响。为了响应外界各种环境条件,植物演化出一系列识别和传递环境信号的蛋白分子,其中比较典型的是植物细胞质膜上的类受体蛋白激酶(RLKs)。凝集素类受体蛋白激酶(LecRLKs)是类受体蛋白激酶家族中的一个亚族,它主要包含3个结构域:细胞外凝集素结构域、跨膜结构域和细胞内激酶结构域。根据细胞外凝集素结构域的不同,LecRLKs可分为3种不同类型:L、G和C型。近年来,研究表明LecRLKs在植物生物/非生物胁迫和发育调控中发挥非常重要的作用。该文综述了植物凝集素类受体蛋白激酶的研究历史、结构特点、分类以及生物学功能,并重点阐述凝集素类受体蛋白激酶在植物生物/非生物胁迫响应和调控发育方面的功能。对不同类型和不同功能的植物凝集素类受体蛋白激酶进行阐述将有利于对该类蛋白开展功能研究,并为作物改良提供有益借鉴。     

花生铝响应类受体蛋白激酶AhPRK4的原核表达分析

花粉类受体蛋白激酶(pollen receptor-like protein kinase,PRK)是一类富含LRR结构域的类受体蛋白激酶,不仅在花粉发育和植物受精中发挥作用,也在胁迫响应中发挥作用。基于对前期花生根尖铝胁迫转录组数据的分析,我们发现了在转录水平响应铝胁迫的花粉类受体蛋白激酶基因AhPRK4,为探究AhPRK4在花生铝胁迫中的功能,该文进一步分析了铝胁迫处理下AhPRK4在花生耐铝品种‘99-1507''和铝敏感品种‘中花2号''(‘ZH2'')根尖中的转录变化,通过序列分析、进化树构建等分析了AhPRK4蛋白的结构特点和亲缘关系,克隆了AhPRK4的胞内域序列(AhPRK4-CD),并通过原核表达和体外磷酸化体系分析了AhPRK4-CD的自磷酸化活性。结果表明:(1)不同铝处理时间及不同铝浓度处理后,AhPRK4的转录水平上调,显著响应铝处理,是铝诱导基因;(2)AhPRK4含有673个氨基酸,属于LRR-III蛋白激酶家族成员,具跨膜域和信号肽,且预测具有磷酸化活性位点;(3)体外诱导表达出约71 kD的可溶性蛋白(GST-AhPRK4-CD),经凝胶亲和层析纯化,得到基于蛋白印迹实验(Western Blot)验证正确的重组蛋白,重组蛋白可发生磷酸化修饰,但无明显的自磷酸化现象。综上认为,AhPRK4是一个铝胁迫应答基因,参与花生铝胁迫早期应答机制,且能发生磷酸化修饰。     

Astragalar morphology of late Eocene anthropoids from the Fayum Depression (Egypt) and the origin of catarrhine primates

The phylogenetic relationships of the late Eocene anthropoids Catopithecus browni and Proteopithecus sylviae are currently a matter of debate, with opinion divided as to whether these taxa are stem or crown anthropoids. The phylogenetic position of Catopithecus is of particular interest, for, unlike the highly generalized genus Proteopithecus, this taxon shares apomorphic dental and postcranial features with more derived undoubted catarrhines that appear in the same region 1-2 Ma later. If these apomorphies are homologous and Catopithecus is a stem catarrhine, the unique combination of plesiomorphic and apomorphic features preserved in this anthropoid would have important implications for our understanding of the crown anthropoid morphotype and the pattern of morphological character transformations that occurred during the early phases of stem catarrhine evolution.Well-preserved astragali referrable to Proteopithecus, Catopithecus, and the undoubted early Oligocene stem catarrhine Aegyptopithecus have provided additional morphological evidence that allows us to further evaluate competing hypotheses of interrelationships among Eocene-Oligocene Afro-Arabian anthropoids. Qualitative observations and multivariate morphometric analyses reveal that the astragalar morphology of Proteopithecus is very similar to that of early Oligocene parapithecids and living and extinct small-bodied platyrrhines, and strengthens the hypothesis that the morphological pattern shared by these taxa is primitive within crown Anthropoidea. In contrast, Catopithecus departs markedly from the predicted crown anthropoid astragalar morphotype and shares a number of apomorphic features (e.g., deep cotylar fossa, laterally projecting fibular facet, trochlear asymmetry, mediolaterally wide astragalar head) with Aegyptopithecus and Miocene-Recent catarrhines. The evidence from the astragalus complements other independent data from the dentition, humerus and femur of Catopithecus that support this taxon's stem catarrhine status, and we continue to maintain that oligopithecines are stem catarrhines that constitute the sister group of a clade containing propliopithecines and Miocene-Recent catarrhines.     

中国异极衣科地衣二新记录种

该文综合运用形态学、解剖学和化学等方法对山东大型地衣进行分类研究,发现了两个中国新记录种,即德氏蜂窝衣(Heppia despreauxii)和多孢小极衣(Lichinella myriospora)。德氏蜂窝衣隶属于蜂窝衣属(Heppia),生于光线充足且裸露的土壤上,主要识别特征为下皮层缺失、子实层IKI+蓝色;多孢小极衣隶属于小极衣属(Lichinella),生于干燥的钙质岩石上,主要识别特征为其子实层IKI+酒红色变为蓝色。该文对这两个中国新记录种进行了详细描述,与近似物种进行了对比讨论,并且提供了其地衣体、子囊盘及其解剖特征图片。同时,该文还补充报道了白棋盘蜂窝衣(Heppia solorinoides)的有性繁殖结构特征和数据。蜂窝衣属和小极衣属均为山东新记录属。以上研究结果为中国异极衣科(Lichinaceae)地衣研究提供基础资料。     

藏药多刺绿绒蒿种子萌发特性研究

多刺绿绒蒿(Meconopsis horridula)为罂粟科绿绒蒿属一年生草本植物,是一种极具观赏价值和药用价值的高山植物,目前处于濒危状态,因此研究多刺绿绒蒿种子的萌发特性对其种子育苗及人工栽培具有重要意义。为了提高多刺绿绒蒿的种子发芽率,该研究以多刺绿绒蒿的种子为材料,分析了不同消毒剂、浸种时间、温度和外源植物激素对种子萌发特性的影响。结果表明:(1)最适消毒方法为75%乙醇1 min+3%H2O25 min,最适浸种时间为24 h,最适温度和光照条件为20℃/10℃(光照12 h/黑暗12 h),用无菌水浸种后的种子发芽率为49.67%。(2) GA_3100～600 mg·L~(-1)和NAA 5～30 mg·L~(-1)可以提高种子的发芽率、发芽势和发芽指数,缩短发芽启动时间和发芽持续时间,对种子的萌发有促进作用。(3) 6-BA 5 mg·L~(-1)和10 mg·L~(-1)对种子的萌发有一定的促进作用,但不显著,6-BA浓度≥15 mg·L~(-1)则抑制种子的萌发。(4)用GA3500 mg·L~(-1)浸种后的种子发芽指标最好,发芽率、发芽势和发芽指数分别为69.67%、33.00%、4.51,种子的发芽起始时间和发芽持续时间分别为10.67 d、11.67 d。     

scabrous Modifies Epithelial Cell Adhesion and Extends the Range of Lateral Signalling during Development of the Spaced Bristle Pattern in Drosophila

The role of scabrous (sca) in the evenly spaced bristle pattern of Drosophila is explored. Loss-of-function of sca results in development of an excess of bristles. Segregation of alternately spaced bristle precursors and epidermal cells from a group of equipotential cells relies on lateral inhibition mediated by Notch and Delta (Dl). In this process, presumptive bristle precursors inhibit the neural fate of neighbouring cells, causing them to adopt the epidermal fate. We show that Dl, a membrane-bound ligand for Notch, can inhibit adjacent cells, in direct contact with the precursor, in the absence of Sca. In contrast, inhibition of cells not adjacent to the precursor requires, in addition, Sca, a secreted molecule with a fibrinogen-related domain. Over-expression of Sca in a wild-type background, leads to increased spacing between bristles, suggesting that the range of signalling has been increased. scabrous acts nonautonomously, and we present evidence that, during bristle precursor segregation, Sca is required to maintain the normal adhesive properties of epithelial cells. The possible effects of such changes on the range of signalling are discussed. We also show that the sensory organ precursors extend numerous fine cytoplasmic extensions bearing Dl molecules, and speculate on a possible role for these structures during signalling.     

中国兰科二新记录种

兰科植物资源调查是掌握地区兰科本底资料的基础,对研究兰科植物的地理分布和资源多样性具有重要意义。该文报道了分别产自中国西藏自治区墨脱县和巴宜区的石豆兰属(Bulbophyllum Thou.)中国分布新记录种——尼泊尔大苞兰(B.raskotii J.J.Verm.,Schuit.&de Vogel)和曲唇兰属[Panisea(Lindl.)Steud.]中国分布新记录种——林芝曲唇兰(P.panchaseensis Subedi)。此二新记录种均生长在海拔约2000 m的常绿阔叶林中的树干或岩壁上。此外,还提供了二新记录种的形态特征描述和彩色图片等信息,并附有国产曲唇兰属的分种检索表。该研究结果扩充了我国兰科植物的记录,为我国兰科植物多样性和保护研究提供了新资料。     

不同光质对白及组培苗生长及光合特性的影响

白及的自然繁殖率极低,组培育苗是其种苗繁殖的主要方式之一。为探索提高白及组培育苗质量及缩短育苗周期的高效人工光环境,该文以紫花白及(Bletilla striata)为试验材料,研究LED光质对白及组培苗的生长和光合特性的影响。结果表明:提高红蓝光组合中的蓝光占比,有利于促进白及组培苗的生长和生物量的积累,而白及的球茎大小与红光的占比呈正相关;在红蓝组光合中增加25%的绿光(2R1B1G),可显著提高白及叶片的叶绿素含量和净光合速率,促进组培苗根系和叶片的生长。综上结果表明,2R1B1G处理下的白及组培苗株高、茎粗、叶绿素含量、净光合速率和根系发育均表现出最佳优势,2R1B1G处理的LED光谱可推荐作为白及组培育苗的光质配方。     

Essential amino acids of starch synthase IIa differentiate amylopectin structure and starch quality between <Emphasis Type="Italic">japonica</Emphasis> and <Emphasis Type="Italic">indica</Emphasis> rice varieties

Four amino acids were variable between the ‘active’ indica-type and ‘inactive’ japonica-type soluble starch synthase IIa (SSIIa) of rice plants; Glu-88 and Gly-604 in SSIIa of indica-cultivars IR36 and Kasalath were replaced by Asp-88 and Ser-604, respectively, in both japonica cultivars Nipponbare and Kinmaze SSIIa, whereas Val-737 and Leu-781 in indica SSIIa were replaced by Met-737 in cv. Nipponbare and Phe-781 in cv. Kinmaze SSIIa, respectively. The SSIIa gene fragments shuffling experiments revealed that Val-737 and Leu-781 are essential not only for the optimal SSIIa activity, but also for the capacity to synthesize indica-type amylopectin. Surprisingly, however, a combination of Phe-781 and Gly-604 could restore about 44% of the SSIIa activity provided that Val-737 was conserved. The introduction of the ‘active’ indica-type SSIIa gene enabled the japonica-type cv. Kinmaze to synthesize indica-type amylopectin. The starch in the transformed japonica rice plants exhibited gelatinization-resistant properties that are characteristic of indica-rice starch. Transformed lines expressing different levels of the IR36 SSIIa protein produced a variety of starches with amylopectin chain-length distribution patterns that correlated well with their onset temperatures of gelatinization. The present study confirmed that the SSIIa activity determines the type of amylopectin structure of rice starch to be either the typical indica-type or japonica-type, by playing a specific role in the synthesis of the long B₁ chains by elongating short A and B₁ chains, notwithstanding the presence of functional two additional SSII genes, a single SSI gene, two SSIII genes, and two SSIV genes in rice plants.     

Stratigraphy, artefact industries and hominid associations for Sterkfontein, member 5

A revised stratigraphy for the early hominid site of Sterkfontein (Gauteng Province, South Africa) reveals a complex distribution of infills in the main excavation area between 2.8 and 1.4 m.y.a, as well as deposits dating to the mid to late Pleistocene. New research now shows that the Member 4 australopithecine breccia (2.8-2.6 Ma) extends further west than was previously thought, while a late phase of Member 4 is recognized in a southern area. The artefact-bearing breccias were defined sedimentologically as Member 5, but one supposed part of these younger breccias, the StW 53 infill, lacks in situ stone tools, although it does appear to post-date 2.6 Ma when artefacts first appear in the archaeological record. The StW 53 hominid, previously referred to Homo habilis, is here argued to be Australopithecus. The first artefact-bearing breccia of Member 5 is the Oldowan Infill, estimated at 2-1.7 Ma. It occupies a restricted distribution in Member 5 east and contains an expedient, flake-based tool industry associated with a few fossils of Paranthropos robustus. An enlarged cave opening subsequently admitted one or more Early Acheulean infills associated in Member 5 west with Homo ergaster. The artefacts attest to a larger site accumulation between ca. 1.7 and 1.4 Ma, with more intensive use of quartzite over quartz and a subtle but important shift to large flakes and heavier-duty tools. The available information on palaeoenvironments is summarized, showing an overall change from tropical to sub-tropical gallery forest, forest fringe and woodland conditions in Member 4 to more open woodland and grassland habitats in the later units, but with suggestions of a wet localized topography in the Paranthropus -bearing Oldowan Infill.     

甜荞FeFUL2基因的克隆与表达分析

为了探索甜荞FUL同源基因参与花与籽粒发育调控的分子机制,该文采用同源克隆的方法从甜荞(Fagopyrum esculentum)长花柱和长雄蕊突变体(lpls)中克隆到1个长837 bp的FeFUL2基因(GenBank登录号为MG779493.1),其包含长690 bp的完整开放阅读框,编码1个由229个氨基酸残基组成的MADS-box转录因子。通过对FeFUL2进行分子系统发生、同源蛋白比对与转录因子结构分析,结果显示FeFUL2与核心真双子叶植物AP1/FUL亚家族转录因子中的euFUL进化系聚于1个进化分支,属甜荞euFUL型MADS-box转录因子,且包含1个57个氨基酸残基长的高度保守的MADS结构域、1个69个氨基酸残基长的次级保守的K结构域,其C末端转录激活区在序列长度和氨基酸残基组成上与其他euFUL型转录因子差异较大,但仍含有2个euFUL型转录因子特有的保守基元:FUL motif和paleo AP1 motif。用qPCR检测基因表达的组织特异性显示:FeFUL2基因在甜荞lpls突变体的根、茎、叶、花被片、雄蕊、雌蕊和发育4 d的幼果中均有表达,但其在花被片中表达量极显著高于该基因在其他器官中的表达量(LSD,P0.01)。综合转录因子的结构与基因的表达模式推测,FeFUL2基因与其他euFUL型基因的功能可能存在一定差异,其在花发育过程中可能主要参与甜荞花被片的发育调控。     

四川卧龙国家级自然保护区马先蒿属一新种——熊猫马先蒿

熊猫马先蒿(Pedicularis pandania)是在四川卧龙国家级自然保护区发现的马先蒿属一新种。该新种属于互生叶类群,其基生叶成丛,花冠属于短管有喙型,且下唇将喙包裹住,花管在近萼端发生约180度的扭旋使得花冠下唇在侧上方,这一特征明显区别于其他国产马先蒿物种。分子系统发育分析结果表明,熊猫马先蒿是Clade 7 成员,虽然与火焰系、假欧氏系、喙齿系、拟蕨系和长喙系等成员构成姐妹关系,但熊猫马先蒿的花冠形态特征明显区别于近缘物种。