葡萄SA 和JA 信号转导重要基因克隆及其对外源信号应答分析

 以‘藤稔’葡萄（Vitis vinifera × V. labrusca‘Fujiminori’）的叶片为试材,克隆了水杨酸（SA）和茉莉酸（JA）信号转导途径中重要基因NPR1、PR1、COI1和LOX2,利用定量和半定量PCR法研究其在SA与JA处理后的表达情况,发现PR1特异地受到SA诱导,而施加JA后抑制其表达;LOX2特异地受到JA的诱导,SA处理抑制其表达。上、下游基因的表达情况说明,PR1和LOX2可作为葡萄SA和JA信号转导途径的标记基因,同时研究发现葡萄中NPR1、PR1、COI1和LOX2表达量的快速上升出现在SA和JA处理后6 ~ 24 h;SA与JA信号转导途径存在着协同或拮抗作用,它们之间的相对浓度决定着这种相互关系的变化。     

茉莉酸甲酯诱导的苹果悬浮细胞的防卫响应

 以苹果悬浮细胞为试材，以硝普钠(SNP)为一氧化氮(NO)供体，L-NAA(Nx-nitro-L- arginine)为一氧化氮合酶(NOS)抑制剂，PTIO(2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl- 3-oxide)为NO清除剂，研究了茉莉酸甲酯(MeJA)处理苹果悬浮细胞诱发NO和H₂O₂信号分子及其对PAL酶活性、植保素和丙二醛(MDA)的影响。结果表明，MeJA处理能明显诱发NO生成，在处理6 h时NO生成量达到峰值，而L-NAA和PTIO则能够抑制NO生成。另外，MeJA处理也显著提高了细胞内的H₂O₂水平，而SNP单独处理不能提高H₂O₂水平。在各种处理中，PAL活性以及植保素和MDA含量均表现出先升后降趋势，分别在4 h或6 h时达到峰值。本研究结果揭示了MeJA可以激发NO生成，而细胞内H2O2水平的提高则是MeJA和NO协同作用的结果，植保素和MDA含量以及PAL活性的提高主要受NO的诱导，但也存在其它诱导途径。     

茉莉酸甲酯处理对葡萄果实NO 和H2O2 水平及植保素合成的影响

 以10 μmol · L-1 茉莉酸甲酯（Methyl jasmonate,MeJA）熏蒸处理‘巨峰’葡萄果实6 h,随后转入1 ℃下贮藏28 d。结果表明,MeJA 处理显著抑制了葡萄果实在贮藏期间腐烂率和失重率的上升,促进内源NO 释放量和H2O2 含量在贮藏前期的上升,同时诱导植保素合成相关酶苯丙氨酸解氨酶（PAL）、肉桂酸–4–羟化酶（C4H）、对香豆酰–CoA 连接酶（4-CL）和白藜芦醇合成酶（RS）活性以及植保素白藜芦醇和白藜芦醇脱氢二聚体含量的上升。由此推测,MeJA 在葡萄果实细胞内发挥了信号传导作用,通过调控下游信号分子H2O2 和NO 的水平来提高植保素合成相关酶活性,从而促进了植保素的积累,提高果实的抗病性,降低了其腐烂率。     

圆叶葡萄‘Noble’的芪合成酶STS启动子的功能分析

【目的】分析圆叶葡萄‘Noble’的芪合成酶(stilbene synthetase,STS)基因上游调控序列的顺式作用元件及其功能,为研究白藜芦醇在葡萄抗病机制中的作用提供理论基础。【方法】在前期通过染色体步移法分离得到圆叶葡萄‘Noble’芪合成酶Mr STS基因上游调控序列的基础上,利用Plant CARE在线启动子预测工具对其顺式作用元件进行初步预测,构建该启动子5’端侧翼序列缺失表达载体,启动报告基因绿色荧光蛋白(green fluorescent protein,GFP)表达,并通过农杆菌介导法真空瞬时转化离体葡萄叶片,研究激素和霜霉菌诱导条件下GFP蛋白的活性差异。【结果】PlantCARE在线预测结果表明,Mr STS基因上游调控序列含有启动子的特定结构,如TATA-box、CAAT-box,另外也含有一些与逆境相关的顺式作用元件,如ABA响应元件ABRE、Me JA响应元件CGTCA-motif、赤霉素响应元件P-box、激发子响应元件Box-W1和胁迫响应元件TC-rich repeats等;不同诱导条件下,5’端侧翼序列缺失表达载体启动GFP蛋白的活性存在显著差异。【结论】Mr STS基因上游调控序列含有启动子核心区域和与逆境相关的顺式作用元件,并受ABA、Me JA和霜霉菌的诱导。     

唐菖蒲茉莉素受体基因GhCOI1的克隆与表达分析

通过RACE技术在唐菖蒲(Gladiolus hybridus)品种‘Rose Supreme’的籽球苗叶片中克隆得到茉莉素受体COI1基因的c DNA序列,命名为Gh COI1。该基因全长为2 603 bp,包含一个编码613个氨基酸的开放阅读框,5′utr和3′utr分别为484 bp和277 bp,预测的蛋白质分子量为69.46 k D;其氨基酸序列具有典型的F-box基序和LRRs结构域。同源比对和系统进化树的分析结果表明,Gh COI1与番茄Le COI1氨基酸序列的相似性最高,达到64.3%;与水稻Os COI1的亲缘关系最近。利用实时荧光定量PCR分析基因表达的结果表明,Gh COI1在唐菖蒲叶片中的相对表达量最高,根中其次,匍匐茎、新球、籽球、花瓣、雄蕊、雌蕊里中的相对表达量较低,推测在唐菖蒲中存在器官专一性表达的COI1同源基因。采用0.05、0.1和0.5 mmol·L-1 Me JA喷施处理唐菖蒲叶片,检测到施用0.1 mmol·L-1 Me JA可显著上调Gh COI1的表达。同时伴随此浓度Me JA处理时间的增加,12 h内Gh COI1的表达量呈现先上升再下降后上升的趋势。洋葱表皮细胞瞬时表达结果表明Gh COI1蛋白定位于细胞质和质体中。本研究中初步验证了Gh COI1在茉莉素信号途径中的作用。     

茉莉酸甲酯诱导辣椒抗青枯病与活性氧代谢的关系

为探究茉莉酸甲酯(Methyl Jasmonate,Me JA)诱导辣椒抗青枯病效应与活性氧代谢相关酶的关系,以辣椒易感青枯病品种‘粤红1号’和抗青枯病品种‘辛香8号’幼苗为试验材料,在0.1mmol·L-1 Me JA喷雾处理后12、24、48、72和96 h接种青枯劳尔氏菌(Ralstonia solanacearum),对其进行青枯病病情指数与活性氧代谢相关酶——超氧化酶歧化酶(Superoxide Dismutase,SOD)、过氧化氢酶(Catalase,CAT)、过氧化物酶(Peroxidase,POD)和抗坏血酸过氧化物酶(Ascorbate Peroxidase,APX)活性以及丙二醛(Malondialdehyde,MDA)含量测定及相互关系的分析。结果表明:‘粤红1号’和‘辛香8号’的病情指数随喷Me JA处理时间的推移表现为先降后升,但都低于对照;而诱导效果则相反,Me JA处理对两个辣椒品种幼苗抗性的最适诱导时间均为接种前48 h。接种后0~96 h,Me JA喷雾处理的辣椒幼苗叶片SOD、CAT、POD和APX酶活性均显示先升后降的趋势,且明显高于对照,接种后24~48 h达到最高,而MDA含量则明显低于对照。因此,Me JA可诱导辣椒幼苗抗青枯病,其实现途径可能与提高活性氧代谢相关酶活性和缓解膜脂过氧化有关。     

氟唑活化酯诱导大白菜抗根肿病效果与机理初步研究

为明确新型植物诱导抗病剂氟唑活化酯对大白菜根肿病的诱导抗病效果及抗病机理,研究了氟唑活化酯的诱导浓度、在白菜根部的运输过程以及氟唑活化酯诱导白菜后相关防御基因的表达情况。结果显示以氟唑活化酯25 mg ? L-1对大白菜进行叶面喷雾诱导时根肿病的病情指数最低。通过台盼蓝（Trypan blue）染色观察到氟唑活化酯诱导大白菜叶片后,根部对根肿菌也产生了一定的抗性。利用Real-time PCR技术检测大白菜防御相关基因的表达,发现氟唑活化酯诱导大白菜2 h后JA途径中的COI1和LOX2基因以及SA途径中的PR-1基因都有显著的过量表达,其中JA信号途径相关基因表达变化更明显。结果说明氟唑活化酯可以诱导大白菜产生系统获得抗性,两种信号传导途径都参与了抗病过程,但以JA途径为主,SA途径为辅。     

阿拉伯半乳糖蛋白对香蕉悬浮细胞胚性状态的影响

采用火箭琼脂糖凝胶电泳法测定了培养15 d后的香蕉悬浮细胞液体培养基中的阿拉伯半乳糖蛋白(ara-binogalactan-proteins,AGPs)的含量,发现具有体胚发生能力的贡蕉、大蕉和龙牙蕉过山香品种的胚性悬浮细胞(em-bryogenic suspension cells,ECS)培养基中的AGPs含量分别达到36、48、56 mg.L-1,而在不能进行体胚诱导的香芽蕉威廉斯品种的非胚性悬浮细胞(non-embryogenic suspension cells,NECS)的培养基中检测不到AGPs,说明悬浮细胞分泌到培养基中的AGPs含量与香蕉悬浮细胞的胚性状态有一定的关系。在香蕉ECS的悬浮培养过程中,添加不同浓度的βGlcY试剂(β-glucosyl Yariv)到悬浮培养基中可引起胚性细胞与非胚性细胞(ECS/NECS)的比值下降;在香蕉的ECS体胚诱导过程中,添加不同浓度βGlcY试剂到体胚诱导培养基中,引起体胚发生频率下降。这些结果表明,AGPs在香蕉悬浮细胞的胚性保持过程中起到重要的作用,并且具有剂量效应。     

壳寡糖诱导草莓细胞活性氧代谢的变化

 以草莓悬浮培养的细胞为对象, 研究了壳寡糖处理对活性氧代谢的效应。结果表明, 壳寡糖可诱导草莓悬浮培养细胞的活性氧迸发, 其峰值出现于20～30 min , 同时也可诱导活性氧清除酶活性上升,SOD 和CAT活性的最大值在处理后60～90 min。     

白鹤芋胚性细胞悬浮培养和高效植株再生体系的建立

以白鹤芋（Spathiphyllum cannifolium）试管苗叶柄为外植体诱导获得胚性愈伤组织,建立了胚性细胞悬浮培养系并高频率再生出植株。结果表明,最优的悬浮培养条件为：装有20 mL液体培养基的100 mL三角瓶中接种0.3 g胚性细胞团,悬浮培养基为MS + 0.5 mg ? L-1 TDZ + 1.0 mg ? L-1 2,4-D + 30 g ? L-1蔗糖或麦芽糖,pH 5.8;继代间隔14 d;每个继代周期,胚性细胞团可增殖至接种量的5倍以上;植株再生最优的分化培养基为1/2MS + 0.3 mg ? L-1 6-BA + 30 g ? L-1蔗糖 + 8.0 g ? L-1琼脂粉,pH 5.8,平均每个胚性细胞团可分化再生出25.1株小植株;胚性细胞的快速增殖和高频率植株再生的状态可保持24周。     

In vivo screening and characterization of peptides specifically binding to vasculature endothelial cells of liver in mice with septic shock

AIM: To screen peptides specifically binding to vasculature of liver in mice with septic shock. METHODS: Peptide display libraries were injected into septic shock mice by tail vein injection. After circulating for 10 min, the phages from mouse livers were recovered, amplified and purified following a routine protocol, and then re-injected for the next round screening. After screening for 4 rounds in vivo, the phage libraries were injected into normal mice by tail vein injection and the phages were recovered by serum collection to subtract the peptides binding with endothelial cells of normal mice. Then the phage clones were sequenced and further analyzed by bioinformatics. The interested phage clones were reinfused to the mice with septic shock, and appraised by titering the phage distribution in vivo and immunohistochemical staining of the mouse liver. RESULTS: After 4 rounds of biopanning, the phage recovery rates increased step by step, indicating that the phage library was successfully enriched in the livers of mice with septic shock. Fifty percent (5/10) of the analyzed sequences were identical, and with a sequence of LTTWAPA. In vivo titering and immunohistochemical staining displayed that this sequence selectively targets liver of mice with septic shock. CONCLUSION: The peptide of LTTWAPA specifically binds to vascular endothelial cells of liver in septic shock mice, which may have important significance for the therapy of septic shock.     

Selection of targeted glioblastoma tumor cell-binding and internalizing peptides through phage display vector

AIM: To isolate peptides targeted binding and internalizing into glioblastoma cell line SWO-38. METHODS: Tumor cells were screened five rounds of whole cell screen through the Ph.D.-12 phage display library. The monoclone specific binding efficiency to the tumor cell was analyzed, and the DNA of phages were extracted, sequenced and translated to the sequences of amino acid. RESULTS: In the phage library after five rounds of screen , 10 of 13 monoclones had highly selective binding to SWO-38 cells. We found two repeated peptide sequences. CONCLUSION: Whole cell screening against tumor cells through random phage peptide library can obtain phage peptides with highly specific binding and internalizing ability. The peptides could be used as a therapy vector for tumor targeted delivery.     

Screening of tissue factor targeting peptides alternately from phage display peptide library

AIM: To screen tissue factor (TF) targeting peptides by establishment of a new and effective phage display method to acquire the peptides specifically bound to the transmembrane receptor. METHODS: Five rounds of panning were alternately conducted by targeting TF and HT-29 cell line which showed the detectable TF expression (screen for targeting receptor and cell alternately with phage display, STRCA). The 30 phage clones were assessed by enzyme-linked immunosorbent assay (ELISA). DNA sequencing was performed for the phage clones. The affinity of synthetic peptides was verified with competitive inhibition ELISA. The repeated experiment was conducted to verify the reliability of the results. RESULTS: The phages were effectively enriched after 5 rounds of panning with the improvement of the recovery rate from (2.25×10^-4)% to (1.32×10^-2)%. In 30 individual phages, ELISA positive rate was 76.7%, and the repetition of A, B, C and D peptides showed 23.3% (7/30), 23.3% (7/30), 26.7% (8/30) and 10.0% (3/30),respectively. E peptide constructively consisted of A and B. The five synthetic peptides were verified by ELISA, and the IC₅₀ of each peptide showed 3.25 nmol/L, 6.72 mol/L, 3.24×10³ mol/L, 2.08×10² mol/L and 45.77 mol/L,respectively. The positive phages were selected again in the second experiment to compare the results of the first experiment and the repeatability was 33.3%. CONCLUSION: STRCA can select TF targeting peptides with high affinity, which has the potential to become a therapeutic screening of transmembrane receptor-binding peptides.     

Screening of binding proteins of HMGB1 promoter by phage display technique

AIM: To screen the binding proteins to HMGB1 promoter by phage display technique. METHODS: HMGB1 promoter was incubated with phage display library. Unbound phages were eluted and phages bound to HMGB1 promoter were amplified. Twenty individual clones were randomly selected and identified by enzyme-linked immunosorbent assay (ELISA). Positive clones were characterized by DNA sequencing and the sequences were subjected for computer analysis. RESULTS: Positive phages binding to HMGB1 promoter were enriched after 4 rounds of biopanning. Twenty phage clones were selected and eleven clones of which were identified to bind specifically to HMGB1 promoter. The sequences in full length were obtained and searched for homologous sequences from GenBank. Altogether eight coding sequences were obtained, six of which were known proteins including activator protein-1(AP-1) and two of which were uncharacterized ones. CONCLUSION: Several proteins were obtained that bind specifically with HMGB1 promoter. The results will be useful for further studying the expression and regulation mechanism of HMGB1.     

Advances in research of phage peptide library

Phage peptide libraries are collections of the specific length of short peptides, and they are based on phages as their carriers. They include mimotope libraries and pept ide antibody libraries. Phagedisplayed pept ide libraries have been used to isolate specific ligands for numerous protein targets, and they have been proven useful in defining antigen momitopes, rapid determination of binding energetics at protein-protein interfaces, designing of vaccine and tumor research aspects. This review summarized the research progression of phage peptide library.     

Screening of lung cancer specific peptide ZS-9 from a phage display peptide library and NCI-H1299 cells

〓AIM: To obtain the polypeptides specifically bound to NCI-H1299 cell line from peptide libraries and to identify these polypeptides affinity and specific to lung cancer. METHODS: The lung cancer NCI-H1299 cell line was used as the antigen and MRC-5 was used as control for subtraction biopanning from a phage display peptide library. The positive and specific binding clones were identified by cell enzyme-linked immunosorbent assay (ELISA) and immunochemistry staining. Those DNA sequences of identified clones were sequenced, and the amino acid sequence was deduced and analyzed with bioinformatics. RESULTS: After 3 rounds of panning, 9 phage clones were identified by ELISA, one of them specially bound to the NCI-H1299 and A549. The DNA sequence result of ZS-9 was CAT AAT AAG CAT CTT CCG TCT ACG CAG CCT CTT GCG. Hence, the polypeptide sequence was HNKHLPSTQPLA deduced from its DNA sequence. The amino acid sequence was analyzed in NCBI and Swiss-Prot, the results showed that it has no similarity with the known proteins sequence in the database. All these results showed that we have discovered a novel lung cancer surface associated antigen ligand. CONCLUSION: A peptide which is specific binding to lung cancer cell line NCI-H1299 and A549 has been selected from phage display peptide libraries. Therefore, it provides a potential tool for early diagnosis of lung cancer or targeted drug delivery in chemotherapy.     

Whole cell screening of phage-display peptide library for mimicry peptides of glioma SWO-38

AIM and METHODS:The Ph.D.-7 phage display library was used to isolate peptides specific for glioma SWO-38 cell by whole cell screening.Moreover,binding efficiency analysis was carried out to test the binding specificity of the clones obtained.RESULTS:After three rounds of biopanning,a high concentration of phage clones was obtained and two of them were found to be highly specific to glioma SWO-38.CONCLUSION:Highly specific clones against neurtral glioma cel s can be obtained from a phage display library by simple procedures.     

Investigation of amino acid sequence of specific ouabain conjugated peptides

AIM: The purpose of the study was to investigate the gene and amino acid sequence of specific ouabain conjugated peptides (OCP) in order to get an experimental bases to block or antagonist the actions between endogenous ouabain(EO) and sodium pump in hypertension. METHODS: Screening the phage displayed 12-peptide library by biopaning for OCP. The sequence of each selected peptide was determined and the sequence was analyzed through internet. The bioactivity was determined by erythrocyte [⁸⁶Rb] uptaking. RESULTS: Three kinds of peptides were screened out. Peptide A (12 peptide) was occupied in 66.7%(8/12), peptide B (8 peptide) 16.7% (2/12) and peptide C (12 peptide) 8.3% (1/12). There was only one case without insertron. The analysis of protein showed that there were no homogenous between peptide A, B, C and sodium pump. The amino acid sequence of specific OCP was Leu-Leu-Ala-Asp-Thr-Thr-His-His-Arg -Pro-Trp-Thr. CONCLUSIONS: Determination of the sequence of OCP supplies an important experimental foundation for ouabain research. The results also show that phage display peptide library is an effective, simple and efficient method to select specific steroid receptors.     

Construction of phage random eight-peptide library

AIM: To construct random eight-peptide library for the study on atherosclerosis and restenosis. METHODS and RESULTS: Random oligodeoxynucleotides encoded eight peptides were synthesized and amplified by polymerise chain reaction( PCR).The product was cloned into phage surface display vector fUSE5 in Sfi I site and electroporated into competent MC1061. The library was identified through PCR, hybridization, DNA sequencing and affinity biopanning of streptavidin. Because the upstream primer is complementary to part vector clone site sequences and part exogenous gene sequences, and the other one complementary to pⅢ gene of vector, thus only clones inserted exogenous gene could be amplified easily. Additionally we used the probe oligodeoxynucleotide complementary to vec for clone site sequences to identify clones which were not inserted exogeneous genes. Furthermore, two hybridizing positive clones were sequenced. Their sequences are consistent with two oligodeoxynucleotide probe sequences. As a result, 2.1×10⁸ special clones were obtained. Affinity biopanning proved that the libraries could be amplified steadily.CONCLUSION: The eight-peptide library is reliable.     

Preparation and characterization of the antibodies against human myofibrillogenesis regulator 1 and application in neonatal rat cardiomyocytes

AIM: To prepare and purify the polyclonal antibodies against human myofibrillogenesis regulator 1 (hMR-1), then to characterize the purity, titer, specificity and the availability.METHODS: Two polypeptides named peptide 1 and 2 were synthesized based on the bioinformatics analysis of the sequence of hMR-1 by using software TMHMM and DNAStar, then coupled with keyhole limpet hemocyanin (KLH) for immunization. These peptides for immunization were mixed and injected into New Zealand rabbits to prepare antibodies specifically against hMR-1. ELISA assay was used to detect the titers of the antibodies. After purification by immunoaffinity chromatography, antibodies were identified by Western blotting and immunocytofluorescent assays. Applications of the antibodies on neonatal rat cardiomyocytes were also employed.RESULTS: (1)The titers of antibodies were 1∶105. In WB assay, a specific 17kD band was detected, corresponding to the predicted molecular weight of hMR-1; the positive fluorescent signals were distinct. (2)On the neonatal rat cardiomyocytes model, we observed a peri-nucleus location. The fluorescent signal of hMR-1 overexpression group was much stronger than that in vector control and normal control groups.CONCLUSION: All these results indicate that the antibodies obtained from poly peptides mixture immunization have either human original or rat original antigens. The antibody is available for using in Western blotting or immunofluorescent assays.