EaDREB2B互作蛋白的筛选及分析

甘蔗(Saccharum officinarum)是我国重要的糖料作物之一,90％以上的食用糖是由甘蔗产生的,我国有85％以上的甘蔗种植区是分布在旱地,干旱是限制甘蔗生产重要的因素之一.因此,改良和培育抗旱甘蔗新品种对甘蔗产业的发展具有重要意义.AP2/ERF转录因子是植物中特有的基因家族之一,该基因家族成员通过调控逆...     

橡胶树PR107和CATAS8-79中胶乳蛋白的差异分析及磷酸化蛋白的鉴定

橡胶树是合成天然橡胶的重要植物。PR107和CATAS8-79是具有不同产排胶性状的2个无性系。在以往的研究中,胶乳中蛋白表达差异被认为是影响天然橡胶合成的关键因子之一。然而,这2个无性系之间与胶乳产量相关的蛋白质图谱尚未明确。本研究采用蛋白质组学方法对这些蛋白质进行鉴定,有助于探讨巴西橡胶树胶乳合成机理。经双向凝胶电泳和质谱鉴定分析,共获得65个差异表达蛋白信息。通过磷酸化蛋白质组分析,在PR107胶乳中鉴定出31个磷酸化蛋白质,含有74个磷酸化氨基酸残基,在CATAS8-79胶乳中鉴定出80个磷酸化蛋白质,含有166个磷酸化氨基酸残基。橡胶延伸因子/小橡胶粒子蛋白（REF/SRPP）家族成员被鉴定为差异表达蛋白和磷酸化修饰蛋白,这些蛋白在调节天然橡胶合成中起着重要作用。pro-hevein和hevamine也表现出不同的磷酸化修饰水平,它们主要在天然橡胶排胶过程中起作用。一种丝氨酸-苏氨酸蛋白磷酸酶激酶的磷酸化和去磷酸化修饰可能在天然橡胶合成中起调节作用。这些结果为天然橡胶生物合成调控机制的研究提供了新的理论依据。     

巴西橡胶树HbMET的克隆与表达分析

通过同源克隆和RACE方法获得了巴西橡胶树DNA甲基转移酶(DNA Methyltransferase,MET)基因(Hb MET)。Hb MET长4 896 bp,含有4 635 bp的阅读框架,编码1 545个氨基酸。蛋白分子量174.36 ku,等电点为6.36。氨基酸序列与可可、毛果杨、黄瓜、拟南芥、碧桃、葡萄和烟草等物种的MET家族成员的同源性分别为73%、77%、74%、56%、72%和68%。进化树分析表明,Hb MET氨基酸序列与毛果杨的MET亲缘关系最近。Hb MET在巴西橡胶树的根、树皮、叶、胶乳中均有表达,其中在胶乳中表达量最低;Hb MET在橡胶树自根幼态无性系的胶乳中表达比老态无性系胶乳中的表达低。     

Members of the Kunitz-type protease inhibitor gene family of potato inhibit soluble tuber invertase in vitro

Summary A soluble proteinaceous invertase inhibitor was purified from tubers of cv. Provita. N-terminal amino acid sequencing of the purified protein indicated that the invertase inhibitor was a member of a family of small tuber proteins known as Kunitz-type protease inhibitors. The purified protein was completely inhibitory to soluble tuber invertase at quantities that did not inhibit trypsin. Based on amino acid sequence information of invertase inhibitor protein, seven candidate cDNA clones were identified in libraries of Provita and Saturna tubers. The candidate cDNA sequences differed from each other between 2% and 5%, having several non-conservative amino acid substitutions when compared with sequence related protease inhibitors. The results suggest invertase as an alternative target of tuber “protease” inhibitors.     

Isolation and Characterisation of cDNAs Encoding Protein Disulphide Isomerases and Cyclophilins in Wheat

The enzyme families of protein disulphide isomerases (PDI) and cyclophilins catalyse the isomerisation of disulphide bonds and the rotation of Xaa-Pro peptide bonds in nascent proteins, respectively, making them excellent candidates for regulating the deposition of storage proteins in wheat. This study aimed to isolate and characterise clones encoding cyclophilins and PDIs from a wheat endosperm cDNA library. A number of cDNA clones were isolated, of which three different cDNAs each of PDI and cyclophilin were selected for complete sequencing. The three PDI cDNAs encoded putative protein products of 513 or 516 amino acids, all exhibiting conserved sequences for the N-terminal signal peptide, the two thioredoxin-like domains at the catalytic sites and the C-terminal ER retention signal. The three cyclophilin cDNAs exhibited 68–87% identity to other reported cyclophilins and encoded putative protein products of 171 amino acids containing the conserved tryptophan residue and a 7 amino acid sequence unique to certain plant cyclophilins. The sequence variations within and outside the coding regions of all the cDNAs suggest that both enzymes are encoded by multiple genes. This information will allow further investigations into the roles of these enzymes in storage protein deposition in the developing endosperm and thus in wheat quality.     

The location of disulphide bonds in α-type gliadins

Gliadin prepared from gluten of the cultivar Rektor by extraction with 70% (v/v) aqueous ethanol adjusted to pH 5.5 was separated by RP-HPLC. Amongst 23 components obtained, two α-type gliadins (α3- and α8-gliadin) were selected for the determination of disulphide bonds. After both proteins were digested with thermolysin, differential RP-HPLC (chromatography prior to and after reduction of disulphide bonds) was used for the detection of cystine peptides. Two cystine peptides from α3-gliadin and three cystine peptides from α8-gliadin were isolated by RP-HPLC. The resulting peptides were reduced and alkylated with 4-vinylpyridine, separated by RP-HPLC and their amino acid sequences determined. The cystine peptides from both α-type gliadins had similar structures, and the corresponding fragments had homologous sequences. One cystine peptide of each gliadin was composed of three fragments linked by two disulphide bonds. The second cystine peptide consisted of two fragments linked by one disulphide bond. The third cystine peptide derived from α8-gliadin was different from the second peptide in one position of the sequences (glutamic acid instead of glutamine). Comparing complete sequences of α-type gliadins described in the literature, the cystine peptides from α3- and α8-gliadins were identical with corresponding sequences of clones A1235 and A212, respectively¹¹. The structures of the cystine peptides analysed indicate one intramolecular disulphide bond within domain III of α-type gliadins and two disulphide bonds between domains III and V. The linkages found correspond to homologous linkages determined for low M_r subunits of glutenin and glutenin-bound γ-type gliadins⁶. Obviously, these intramolecular disulphide bonds are not linked randomly, but are strongly directed.     

水稻不育系系谱抗稻瘟病基因同源序列的克隆与序列分析

根据已知NBS-LRR类抗病基因结构中氨基酸的保守区域设计简并引物,对水稻不育系系谱NBS-LRR类抗病基因同源序列进行克隆、测序和聚类。同源序列克隆与分析表明,7个水稻不育系系谱亲本中共获得14个阳性克隆,其中11个含有NBS-LRR类抗病基因所特有的保守氨基酸结构域KinaseⅠ、KinaseⅡ、KinaseⅢ及跨膜区域。这些片段间同源性最高达98.3%,最低仅为29.7%;11个氨基酸片段与抗病基因Xal（AB002266）的同源性较高,42%-45%的氨基酸序列相同,59%-64%的氨基酸序列相似,且与水稻其他NBS-LRR类抗性蛋白相似性很高。通过聚类分析,可以将其分为3类,分别记为G1、G2、G3,并发现抗源谷农13所含有的G3类基因,通过天谷、福伊传给后代谷丰、全丰。     

Potato virus X isolates from potato collected in eastern Canada with different symptoms in tobacco differ in their coat proteins

Three Canadian isolates of potato virus X (PVX) that produced distinctly different symptoms in tobacco (Nicotiana tabacum cv. Samsun) were analyzed for differences in their coat proteins using SDS-PAGE, denaturing isoelectric focusing, peptide analysis and nucleotide sequencing. The amino acid sequence of the coat protein in a mild isolate differed from that of isolates that produced severe or intermediate symptoms at two locations. However, the amino acid sequences of the Canadian isolates that produced severe or intermediate symptoms were identical and were the same as the coat proteins of PVX isolates from Argentina, China, The Netherlands, and United Kingdom. These results suggest that regions of the PVX genome in addition to the coat protein gene may be involved in viral-host interactions.     

Influence of Amino Acid Compositions and Peptide Profiles on Antioxidant Capacities of Two Protein Hydrolysates from Skipjack Tuna (Katsuwonus pelamis) Dark Muscle

Influence of amino acid compositions and peptide profiles on antioxidant capacities of two protein hydrolysates from skipjack tuna (Katsuwonus pelamis) dark muscle was investigated. Dark muscles from skipjack tuna were hydrolyzed using five separate proteases, including pepsin, trypsin, Neutrase, papain and Alcalase. Two hydrolysates, ATH and NTH, prepared using Alcalase and Neutrase, respectively, showed the strongest antioxidant capacities and were further fractionated using ultrafiltration and gel filtration chromatography. Two fractions, Fr.A3 and Fr.B2, isolated from ATH and NTH, respectively, showed strong radical scavenging activities toward 2,2-diphenyl-1-picrylhydrazyl radicals (EC₅₀ 1.08% ± 0.08% and 0.98% ± 0.07%), hydroxyl radicals (EC₅₀ 0.22% ± 0.03% and 0.48% ± 0.05%), and superoxide anion radicals (EC₅₀ 1.31% ± 0.11% and 1.56% ± 1.03%) and effectively inhibited lipid peroxidation. Eighteen peptides from Fr.A3 and 13 peptides from Fr.B2 were isolated by reversed-phase high performance liquid chromatography, and their amino acid sequences were determined. The elevated antioxidant activity of Fr.A3 might be due to its high content of hydrophobic and aromatic amino acid residues (181.1 and 469.9 residues/1000 residues, respectively), small molecular sizes (3–6 peptides), low molecular weights (524.78 kDa), and amino acid sequences (antioxidant score 6.11). This study confirmed that a smaller molecular size, the presence of hydrophobic and aromatic amino acid residues, and the amino acid sequences were the key factors that determined the antioxidant activities of the proteins, hydrolysates and peptides. The results also demonstrated that the derived hydrolysates and fractions from skipjack tuna (K. pelamis) dark muscles could prevent oxidative reactions and might be useful for food preservation and medicinal purposes.     

玉米苗期水分胁迫下相关基因差异表达研究

采用cDNA-AFLP技术对玉米幼苗在PEG胁迫和土壤干旱胁迫下的基因表达进行了研究分析,获得转录本衍生片段(TDFs)488条,经测序和同源比对获得42个cDNAs。同源性BLAST搜索结果表明:18个cDNAs的同源序列均为功能未知的玉米克隆,7个cDNAs序列与功能已知的玉米基因同源,5个TDFs序列与功能未知的水稻克隆同源,7个TDFs序列与功能已知的水稻基因同源,剩余的cDNAs序列分别与西红柿、高粱等同源。在获得的cDNAs片段中,既有与干旱胁迫相关的重要功能蛋白,也有一些重要的转录因子。     

辣椒酵母单杂交系统的建立及调控CaWRKY40表达的转录因子分离

辣椒CaWRKY40的转录表达受到病原菌和高温的诱导并在辣椒抗青枯病以及耐高温中起着重要的作用,但其应答青枯菌侵染和高温胁迫表达的调节机制还不清楚。为了进一步分离调节CaWRKY40表达的转录因子,建立辣椒酵母单杂交cDNA文库,以CaWRKY40启动子的青枯病原菌及高温应答区域CaWRKY40p(W40p)为诱饵,筛选cDNA文库,共获得56个阳性克隆。序列分析结果表明,有27个克隆为非重复序列,对其推定氨基酸序列进行同源比对和功能结构域分析,发现有1个推定转录因子,即ARF（Auxin response factor）,以及其他蛋白如eIF（Eukaryotic initiation factor,真核起始因子）,EF（延伸因子,Elongation factor）等。进一步通过染色质免疫共沉淀验证这些转录因子与W40p的结合。剖析CaWRKY40表达的分子机制有利于深入解析CaWRKY40介导的辣椒抗病及耐高温分子机制。     

酵母双杂交系统筛选与水稻条纹病毒Pc2互作的寄主蛋白基因片段

水稻条纹病毒编码的Pc2蛋白在病毒侵染及宿主症状发生中起作用。研究其与寄主间的分子互作有助于揭示病毒侵染与致病的分子机制。分别将编码Pc2 N端（Pc2-N）与Pc2 C端（Pc2-C）的基因片段克隆到酵母双杂交诱饵载体pGBKT7上，并构建水稻叶片cDNA文库，然后分别用Pc2-N和Pc2-C为诱饵筛选文库寻找与其互作的寄主因子。结果表明，诱饵载体中插入的Pc2-N和Pc2-C编码框和氨基酸序列均正确。Pc2-N对酵母菌株AH109无毒性和自主激活能力，但Pc2-C对酵母具有细胞毒性。文库滴度为4×107 CFU/mL，且大多数插入片段在500~2 000 bp之间。利用Pc2-N为诱饵筛选水稻cDNA文库，获得有8个候选阳性克隆。经序列测定和BLAST分析结果表明，这些克隆共编码5种蛋白，主要为胁迫相关蛋白与光系统相关蛋白。     

Characterisation of Friabilin Polypeptides

Analysis by two dimensional electrophoresis (NEPHGE × SDS-PAGE) of proteins extracted with cold SDS solution from starch granules isolated from flour of the soft wheat cv. Galahad showed the presence of two very basic, approx.M_r15 k polypeptides with mobilities very similar to those of the surface-active, lipid-binding proteins, puroindoline-a and puroindoline-b. Whereas the puroindoline-a component predominated in the flour extract, the two analogous polypeptides extracted from isolated starch granules appeared to be present in similar amounts. When the starch proteins were fractionated by Triton X-114 extraction and phase partioning, the puroindoline-like polypeptides were found in the detergent-rich phase, as would be expected for puroindoline proteins. Purification of the Triton X-114-fractionated proteins from cvs. Galahad and Norin 61 by gel filtration and cation exchange chromatography allowed the separation of three polypeptides, the first having a slightly lower mobility on SDS-PAGE than the other two. The N-terminal sequence of the first 15 amino acids of the first of those proteins isolated form cv. Norin 61 was identical to those of 0·19 and 0·53alpha-amylase inhibitors, whereas the N-terminal sequences of the second and third were identical to those of puroindolines-a and -b, respectively, apart from the eighth amino acid of the puroindoline-b homologue, which was glycine in the starch protein, whereas it is reported to be serine in puroindoline-b. The similarities between the electrophoretic, detergent fractionation and N-terminal sequence properties of two of the cold SDS-extractable starch friabilin polypeptides and those of puroindolines-a and -b strongly suggest that friabilin comprises a mixture of proteins in which the puroindoline polypeptides are important components. The amino acid sequence data indicate that somealpha-amylase inhibitor proteins are also significant components of friabilin. The reason for the differential binding of these proteins to the surfaces of hard and soft wheat starch granules remains to be found, but elucidation of that reason may be important in defining the molecular mechanisms by which endosperm texture variation is controlled.     

茶树两个Dof转录因子的分离及其在温度胁迫中的响应分析

茶树是一种重要的经济作物,温度胁迫对茶叶生产影响较大。本研究以茶树品种迎霜为实验材料,克隆获得2个编码Dof转录因子的基因CsDof1和CsDof2。序列分析显示,CsDof1含有1 389 bp,编码463个氨基酸;CsDof2含有1 458 bp,编码486个氨基酸。CsDof1和CsDof2转录因子都具有典型的锌指结构域,分别位于第133~195和124~186个氨基酸位点之间。对CsDof1和CsDof2转录因子理化性质、亲/疏水性、二级和三级结构分析显示,两者都是亲水性蛋白且显偏碱性。空间结构分析显示,CsDof1和CsDof2均有1个α-螺旋,且Dof结构域的位点完全相同,均有4个半胱氨酸残基。实时定量PCR表明,CsDof1和CsDof2在不同茶树品种、不同温度胁迫下均能诱导表达,且表达呈现差异。     

甜菜Actin基因片段的克隆及序列分析

根据其它植物Actin基因设计一对简并性引物,以甜菜根总RNA为模板,采用RT-PCR的方法扩增出Actin基因片段并克隆到pUCm-T载体上,阳性克隆经PCR鉴定测序。序列分析表明,克隆得到Actin基因家族的两个成员,其片段长度均为598 bp,编码198个氨基酸,它们间的同源性为87%。将其分别命名为BvACT1和BvACT2,已在GenBank中注册,登录号为KF214784和KF214785。多重序列比较表明,Bv A CT1氨基酸序列与其他植物Actin基因的同源性在92%以上,而Bv A CT2则在93%以上。     

瓠瓜Ty1-copia型逆转座子RT基因的分离与序列分析

以3个瓠瓜栽培种为供试材料,利用Ty1-copia逆转座子逆转录酶保守序列设计简并引物,PCR扩增获得260bp左右序列条带,回收产物克隆至T载体进行测序,利用生物信息学软件分析获得的25条逆转录酶序列。结果表明,这些序列长度变化范围为218~267bp,经DNAStar软件比对同源性在26.1%~99.6%之间,存在高度异质性,主要表现为缺失突变,核苷酸序列聚类分析分为6个家族,家族1与家族3分别占总序列数的24.0%与36.0%。翻译成氨基酸序列后,分别有2条与11条序列发生移码与终止密码子突变。与已发表物种的相应序列构建系统发育进化树,发现瓠瓜Ty1-copia型逆转座子RT序列具有一定保守性,同时也与杨树、草莓、马铃薯等有很近的亲缘关系。本研究为后续利用逆转座子开发分子标记研究瓠瓜遗传变异及进化途径奠定基础。     

基于转录组的芒果MYB家族基因的鉴定及分析

MYB家族作为植物中较大的转录因子家族之一,参与调控植物的多种生理活动。本研究基于芒果果实转录组测序结果,鉴定出71个MYB家族蛋白,其中包含1个4R-MYB蛋白、3个R1R2R3-MYB蛋白、60个R2R3-MYB蛋白和7个MYB相关蛋白。进化树及基序分析表明:除个别蛋白外,相同类型的MYB蛋白均聚在一起,且相近分支的MYB蛋白具有相同或相似的基序。60个全长MYB家族蛋白中,大部分为不稳定蛋白,且均为不含跨膜结构和信号肽的亲水性蛋白,亚细胞定位分析均定位于细胞核。进化树分析发现芒果R2R3-MYB蛋白与拟南芥有较高的保守性。R2R3-MYB蛋白保守域分析发现,R2和R3结构域均有多个氨基酸保守不变。GO分析发现芒果R2R3-MYB蛋白共注释到生物学过程、细胞组分和分子功能3大类功能的15个亚类。     

油菜叶片衰老相关基因的分离：ATP硫化酶cDNA的克隆和序列分析

通过筛选缩减ｃＤＮＡ文库中在油菜叶片衰老阶段表达而在绿叶中不表达的ｃＤＮＡ克隆，分离了与油菜叶片衰老有关的基因ＬＳＣ６６。ＤＮＡ序列测定和分析结果表明，ＬＳＣ６６的全长可读框架由１３８０个碱基组成，编码４６０个氨基酸，它的核昔酸序列与拟南芥菜ＡＴＰ硫化酶基因有８６．６７％的同源性，其推导的氨基酸序列与拟南芥菜ＡＴＰ硫化酶的氨基酸序列有９１．７４％的同源性。对ＬＳＣ６６基因产物在植物叶片衰老过程中的作用进行了讨论。     

Identification and Characterization of a New Type III Polyketide Synthase from a Marine Yeast,Naganishia uzbekistanensis

A putative Type III Polyketide synthase (PKSIII) encoding gene was identified from a marine yeast, Naganishia uzbekistanensis strain Mo29 (UBOCC-A-208024) (formerly named as Cryptococcus sp.) isolated from deep-sea hydrothermal vents. This gene is part of a distinct phylogenetic branch compared to all known terrestrial fungal sequences. This new gene encodes a C-terminus extension of 74 amino acids compared to other known PKSIII proteins like Neurospora crassa. Full-length and reduced versions of this PKSIII were successfully cloned and overexpressed in a bacterial host, Escherichia coli BL21 (DE3). Both proteins showed the same activity, suggesting that additional amino acid residues at the C-terminus are probably not required for biochemical functions. We demonstrated by LC-ESI-MS/MS that these two recombinant PKSIII proteins could only produce tri- and tetraketide pyrones and alkylresorcinols using only long fatty acid chain from C8 to C16 acyl-CoAs as starter units, in presence of malonyl-CoA. In addition, we showed that some of these molecules exhibit cytotoxic activities against several cancer cell lines.