Proteomic analysis of differentially expressed proteins in longan flowering reversion buds

A proteomic approach was taken to compare the proteomes of normal flowering buds and flowering reversion buds in longan (Dimocarpus longan Lour. cv. Longyou). Two-dimensional gel electrophoresis (2-DE), coupled with mass spectroscopy and protein database searching, recognized 18 proteins that were differentially expressed in flowering reversion buds. Eleven of these were down-regulated, whereas seven were up-regulated. A subset of 13 proteins was identified by MALDI-TOF-TOF/MS and classified into regulatory proteins (kinase, 20S proteasome alpha 6 subunit, putative alpha 7 proteasome subunit, auxin-induced protein, and abscisic stress ripening-like protein), antioxidant-related proteins (Chain A, GDP-mannose-3′,5′-epimerase, putative lactoylglutathione lyase, and Chain A, ascorbate peroxidase), pollen fertility-related proteins (putative leucoanthocyanidin reductase 2, and putative isoflavone reductase), photosynthesis-related proteins (large subunit, ribulose-bisphosphate carboxylase–oxygenase), and molecular chaperones (disulfide isomerase). Among them, regulatory and antioxidant-related proteins accounted for almost two-thirds of these proteins, suggesting that they may play a more important role in bud differentiation. Identification of these proteins provides insights that may lead to a better understanding of the molecular basis for flowering reversion in longan.     

乙烯利诱导菠萝成花过程中FT基因的克隆与表达分析

【目的】克隆菠萝AcFT基因并研究其表达模式,为深入研究该基因在乙烯利诱导菠萝成花中的功能奠定基础。【方法】从菠萝基因组数据库中获得AcFT基因全长序列,设计全长引物,克隆两个AcFT基因,分别命名为AcFT1和AcFT2,对基因序列进行生物信息学分析;通过qRT-PCR探究乙烯利处理后菠萝AcFT基因在不同组织、时间下的表达模式。【结果】AcFT1和AcFT2分别编码178和177个氨基酸,两者均含有PBP结构域,具有PEBP家族典型结构特征。实时荧光定量PCR分析结果显示,AcFT1和AcFT2都具有组织表达特异性,在茎和叶中相对表达量较高;乙烯利处理后,AcFT1和AcFT2在茎和叶中的表达具有相反的趋势,其中AcFT2明显受到乙烯利上调,呈现先上升后下降趋势,AcFT1则显示为先下降后上升。乙烯利处理后1 d,茎尖组织AcFT2的表达水平显著上升,相对表达量约为对照的178倍,而AcFT1则明显下调;乙烯利处理后31 d,AcFT2在茎尖中的表达水平达到最大值,约为对照的408倍,但AcFT1却处于极低水平。【结论】克隆得到2个AcFT基因,AcFT2基因在乙烯利处理后1 d及随后的花芽分化时期高度表达,表明AcFT2在响应外源乙烯利信号诱导菠萝成花过程中发挥重要作用。     

Identification of differentially expressed genes during flower opening by suppression subtractive hybridization and cDNA microarray analysis in Eustoma grandiflorum

The forward and reverse suppression subtractive cDNA libraries were constructed in petals of Eustoma grandiflorum at bud stage (stage 1) and anthesis (stage 7). Approximately 1000 clones were isolated from stage 1- (S1) and stage 7-specific (S7) libraries. The clones were sequenced and assembled, which yielded 98 contigs and 444 singletons. BLAST search was conducted on these assembled sequences. Generally, probes isolated from the S7 library exhibited higher expression at stage 7 by microarray analysis, as did those of the S1 library at stage 1. A clone set from the S7 library contained genes from later steps of anthocyanin biosynthesis pathway, terpene synthases, GAST (gibberellic acid-stimulated) family proteins, xyloglucan endotransglucosylase/hydrolase, glycosidases, and stress- and senescence-related proteins. In contrast, the S1 library contained genes associated with flavonol biosynthesis, phenylpropanoid metabolism, terpenoid metabolism, and floral organ development. Gene expression profiling for flavonoid biosynthesis was in accordance with preferential accumulation of flavonols at bud stages and anthocyanins at anthesis.     

用于多基因表达检测的猕猴桃macroarray阵列

基因阵列是开展高通量功能基因组学研究的有效手段之一。对适用于多年生果树猕猴桃的macroarray阵列进行了探索,构建了含有42个基因的macroarray阵列,与α-32P标记的cDNA探针杂交产生清晰信号,有效分辨了不同基因或基因家族不同成员的表达差异。DNase处理可去除基因组DNA污染,提高结果准确性。     

Studies on the induction of flowering in juvenile Prunus avium L.

Juvenile Prunus avium shoot apices produced flowering shoots after grafting to mature trees irrespective of treatment of the apices in the season before grafting with G A or G A + cytokinin. Scions grown from mature shoot apices grafted to seedling stocks failed to flower, again irrespective of prior hormone treatment. Similar treatment of mature shoot apices with these hormones, or with zeatin, inhibited concurrent floral initiation, but G A or GA + zeatin treatments increased flowering of scions grown from the treated apices after grafting to untreated mature trees. Localized shoot tip or root drench treatment of one or two year old seedlings with (2RS, 3RS)-paclobutrazol failed to induce flowering, but treatment of three or four year old plants did stimulate flowering. Branch or stem girdling or root flooding applied alone or in combination to three year old plants did not affect flowering. Floral initiation by three or four year old plants was inhibited by treatment with GAs. The results were consistent with the presence in seedlings of a root- produced, xylem transported, graft-transmissible inhibitor(s), which control the initiation of otherwise competent meristems, and which could include factors other than GAs. Juvenile meristem competence to flower was not affected by prior GA treatment.     

华东葡萄抗白粉病杂种后代cDNA文库的构建及EST序列分析

以抗白粉病的华东葡萄杂种后代6-12-4株系为试材,利用SMARTTM技术构建了白粉病病原菌诱导的全长cDNA文库。经检测,原始文库滴度为2.50×106pfu·mL-1,重组率达到99%,扩增文库滴度为3.0×109pfu·mL-1,重组率达到95%,插入片段长度在500～2000bp。随机挑取300个克隆,测序获得260条质量较高的ESTs,聚类及拼接后,共得到183个一致序列(所获序列已经提交GenBank,登录号为FG579859-FG580039)。经蛋白功能预测分析,获得含有TPR结构域的小的富含谷氨酰胺的蛋白、MYB类转录因子、水通道蛋白、富含脯氨酸蛋白、亲环素蛋白、ACI14蛋白、转脂蛋白等抗病相关基因,涉及到植物的信号转导,胁迫诱导,防卫反应等方面。这些EST序列为利用中国野生华东葡萄进行抗白粉病育种研究和利用提供了有价值的抗病基因片段,为丰富世界葡萄属植物抗白粉病基因提供序列数据。     

Isolation and expression analysis of differentially expressed genes in stem tissue of the Greek lemon cv. Adamopoulou

Lemon (Citrus limon (L.) Burm. f.) is susceptible to mal secco, a serious vascular disease caused by the fungus Phoma tracheiphila (Petri) Kant. and Gik., as well as low temperatures. The greek lemon cultivar Adamopoulou, thought to be derived from the Portuguese cultivar Lisbon, exhibits enhanced resistance to mal secco and cold as opposed to cv. Lisbon. Suppression subtractive hybridization (SSH) was employed for the isolation of differentially expressed genes in lemon stem tissue. A subtractive cDNA library was constructed and a total of 296 clones were sequenced. The obtained sequences were edited, resulting in 56 non-redundant ESTs. Sequence analysis revealed homology to previously identified genes involved in defense mechanisms against biotic and abiotic stresses, as well as sequences with no significant similarity in the GenBank. Selected ESTs were analyzed by real-time PCR for confirmation purposes. This analysis revealed significant expression differences between the two cultivars for genes expressing allantoinase, ultraviolet-B-repressible protein, 4-coumarate:CoA ligase and other proteins that are known to be upregulated under biotic and abiotic stress conditions.     

Effect of pH and urea on the efficacy of ethephon for induction of flowering in pineapple

An experiment was carried out to reduce the effective concentration of ethephon for flower induction in pineapple by adding urea to the ethephon solution and adjusting the pH to 9.0 with sodium carbonate. It was found that a 2 % urea solution adjusted to a pH of 9.0 and containing 10 p.p.m. ethephon induced over 90 % of plants to flower within 50–60 days of treatment. This treatment was as good as, or better than, that obtained with several α-naphthalene acetic acid-based products. At a pH of 9.0 and with the addition of urea, the effectiveness of ethephon at a concentration of 10 p.p.m. will lead to economic use of this chemical.     

Effects of lovastatin on differentially expressed genes in HepG2 cells

AIM: To analyze the lovastatin-induced differential gene expression in HepG2 cells using a cDNA microarray assay. METHODS: Total RNA was extracted from the lovastatin-treated HepG2 cells and control group. cDNA was synthesized from RNA with Cy3/Cy5-labelled dCTP. Then the hybridization was conducted. The result was analyzed using Imagene and Genespring software. RT-PCR was carried to confirm the hybridization results. RESULTS: 30 genes were up-regulated while 11 genes were down-regulated in lovastatin-treated HepG2 cells, involved in some major functional areas including signal transduction, cell cycle regulation, tumor immunity, and so on. CONCLUSION: The analysis of differentially expressed genes in lovastatin-treated HepG2 cells is helpful to explore the mechanism of the anti-tumor activity of statins.     

Genome-wide identification and involvement of litchi SPL genes in flowering in response to cold and leaf maturity

Litchi (Litchi chinensis Sonn.) flowers from shoot apical meristems after exposure to prolonged cold differently depending on maturity. The molecular mechanism defining that interaction of cold with maturity is not fully understood. In the present study, potted litchi plants with terminal flushes at mature and turning stage were treated with 60-days of cold. The mature plants, but not the turning plants, were competent to flower, indicating the importance of terminal flush maturity on litchi flowering. This result could be explained by the difference in accumulation of soluble sugars in the apical leaves on terminal flushes. To explore this link, the miR156/SPL (SQUAMOSA-Promoters Binding Protein-like) module, a dominant sugar-mediated flowering regulator, was investigated. Eighteen SPL homologues were identified, 11 of which were up-regulated during the transition from juvenile to adult. Ten LcSPLs were highly expressed in response to cold, but only LcSPL1 and LcSPL2 correlated with the age-dependent flowering in response to cold. Further, 12 LcSPLs, putative targets of LcmiR156, were subjected to transient co-expression assays in tobacco leaves. LcSPL3 and LcSPL10 showed direct binding to the LcFT1 promoter in vitro and in vivo. This trans-activation was verified by yeast one-hybrid (Y1H) assays.     

Identification of differentially expressed genes in fragrant rose Jinyindao with suppressive subtraction hybridization

To investigate the floral fragrance new genes, scent mutant of rose was used here. The suppressive subtraction hybridization (SSH) technique and micoarray analysis of the clones were used to isolate the cDNA fragments, which showed differential expression between the rose scent mutant ‘Wangriqinghuai’ and wild type ‘Jinyindao’ (Rosa × hybrida), and RT-PCR was used to identify up-regulated expressed genes. 16 positive contigs of JSSH were obtained. Some ESTs such as RcOMT1, RcOMT2, RhMYB92 and RhGP1 were known to regulate scent metabolism, and 5 ESTs with no homology in NCBI may represent new genes involved in rose flower fragrance metabolism. SSH technique combined with cDNA micoarray would be useful for analysis and isolation of the genes related to rose floral scent.     

甜樱桃成花相关MADS-box基因的克隆及表达分析

【目的】克隆甜樱桃中可能参与成花途径的MADS基因,分析其基本信息,研究其在不同组织中的表达情况。【方法】以甜樱桃‘萨米特’(‘Summit’)为试材,结合桃基因组分析,克隆得到14个可能参与成花的MADS基因,分别命名为PaSOC1、PaAG、PaAP1、PaAP1-2、PaAP3、PaPI、PaSVP、PaAGL24、PaSEP1、PaSEP2、PaSEP3、PaSEP4、PaSEP5、PaFLC,通过DNAMAN、SMART、protein BLAST和MEGA5等软件分析14个甜樱桃的MADS基因结构、氨基酸结构域及其进化关系,RT-PCR检测其在樱桃根、叶芽、叶、花芽、花、韧皮部中的表达模式。【结果】14个甜樱桃MADS成员大小为612~765 bp,均含有典型的MADS结构域和K-box结构域,含有6~8个内含子。分属7个亚组,PaSEP1、PaSEP2、PaSEP3、PaSEP4、PaSEP5属于SEP亚组,PaAP1、PaAP1-2属于AP1亚组,PaAG属于AG亚组,PaSOC1属于SOC1亚组,PaAP3和PaPI属于AP3/PI亚组,PaSVP属于SVP亚组,PaAGL24属于AGL24亚组,PaFLC并未聚到FLC亚组中。RT-PCR分析显示,14个MADS基因在花芽或花中均有不同程度的表达,此外,除PaAP3、PaSEP1、PaSEP4及PaSEP5之外,其他几个基因在韧皮部中也有不同程度的表达。【结论】获得的甜樱桃MADS-box基因结构高度保守,参与调控成花及花发育过程。     

香蕉果实果胶裂解酶基因cDNA克隆及序列分析

利用已报道果胶裂解酶基因的保守序列设计简并引物,进行RT-PCR,得到1个大约1300bp的香蕉果胶裂解酶基因cDNA片段,命名为MA-pl。DNA序列分析表明:MA-pl片段全长1277bp,包含1个882bp的开放读码框(ORF),编码294个氨基酸;其具有所有果胶裂解酶共有的保守区域:钙协调部位(Asp72,Asp74,Asp96,andAsp100)、酶活性位点(Arg152,Pro154,Arg157)及3个重要的结构域(motifI:WVDH,motifII:DGLVDAVMGSTAITVSNNYF,motifIII:LYQRMPRCRHGYFHVVWNDY);MA-pl氨基酸序列与草莓-1、葡萄、草莓-2、拟南芥、苹果、香蕉(banana-1)的相似性分别为85.8%、74.2%、79.7%、78.6%、72.4%和71.4%。     

Screening and identification of differentially expressed genes in hippocampus of mice during hypobaric hypoxic delayed preconditioning

AIM: This study was designed to explore the differentially expressed genes between hypobaric hypoxic delayed preconditioning (HHDP) and normal mouse hippocampus. METHODS: HHDP was produced by treating the animals at a 7 000 m high altitude for 2.5 h/d for 3 d. At 36 h after last time decompression, total RNA was isolated from hippocampus. cDNA was synthesized and amplified by SMART PCR. cDNA libraries of differentially expressed gene between HHDP and control hippocampus were constructed. 452 clones from forward (subtracted control from preconditioning) cDNA library and 74 clones from backward (subtracted preconditioning from control) one were screened by reverse Northern hybridization. RESULTS: Screening with subtracted probes, hybridization signal of 85 gene fractions decreased and that of 217 gene fractions increased by more than 2 times in HHDP hippocampus compared with control. Screening with unsubtracted probe, hybridization signal of 44 gene fractions decreased and that of 135 gene fractions increased by more than 2 times in HHDP hippocampus compared with control. Some of the clones had been sequenced. Analysis and comparison with the data of GenBank were performed. The results showed that mouse cytochrome C oxidase subunit 1, NADH dehydrogenase subunit 1 and 6, deleted in split-hand/split-foot 1 region (DSS1) and cDNA corresponding to clone IMAGE: 5251089 of mice cDNA library were increased in hippocampus of HHDP mice. cDNA corresponding to clone IMAGE: 3593193, mus musculus adult male olfactory brain cDNA and mus musculus bladder RCB-0544 MBT-2 cDAN were decreased in hippocampus of HHDP mice. CONCLUSION: Many genes expresses differentially in hippocampus of mice during HHDP. This may be one of the molecular mechanisms of HHDP.     

Effect of nicotine in vitro on expressions of apoptosis-related genes in human lung adenocarcinoma cells by cDNA microarray

AIM: To investigate effect of nicotine on growth of human lung adenocarnoma cells and expressions of apoptosis-related gene. METHODS: Lung adenocarcinoma cell line, SPC-A-1, was cultured in the presence of various concentrations (1-1 000 μg/L) of nicotine for 48 hours. MTT was applied to evaluate effect of nicotine in vitro on growth of SPC-A-1 cell line. After SPC-A-1 cells were treated with 100 μg/L for 48 hours, cDNA expression profile microarray was used to detect the expressions of 451 apoptosis-related genes in SPC-A-1 cell line. RESULTS: Significant proliferation in SPC-A-1 cells treated with nicotine (1-10 μg/L) was observed, but this effect decreased with increase in concentration of nicotine in culture. Growth inhibition rate of 1, 10, 100, 1 000 μg/L of nicotine was 27%, -40%, -40% and -93%. Microarray detection showed that significantly different expressions appeared in 80 of 451 apoptosis-related genes. 29 apoptosis-promoted genes and 26 apoptosis-inhibited genes were up-regulated significantly (CY3/CY5>2.0), and 25 genes were significantly down-regulated (CY3/CY5<0.5). CONCLUSION: Nicotine may promote growth of human lung adenocarcinoma cell through regulating many apoptosis-related gene expressions.     

Searching and cloning of differentially expressed genes in liver tumor by restriction fragment differential display PCR

AIM:The aim of this study was to isolate and clone the liver tumor-associated genes by restriction fragment differential display PCR (RFDD-PCR) methods that developed recently. METHODS:Total RNA was extracted from primary carcinoma of the liver and adjacent noncancerous tissue separately. The method of RFDD-PCR was used to search for the differentially expressed genes. Each gene segment was sequenced after cloning. The sequence homogeneity were analyzed and compared with BLAST through Internet. The data were then transported into the Translated BLAST Searches to acquire the related information such as the corresponding protein function. RESULTS:Eighteen cDNA bands were found significantly different at their expression levels with 16 higher and 2 lower in primary carcinoma of the liver cells. Three of them were selected to reamplication by PCR, and then were cloned, identified and sequenced. One of them had no homology as compared with known genes and was likely to be a new one. One had relatively high homology with human ribosomal protein genes (86%). The other one has relatively high homology with selenoprotein P genes (98%). CONCLUSIONS:Compared with DDRT-PCR method, RFDD-PCR is better for rapid screening of the differentially expressed genes. It is simple, sensitive, reproducible and less false positiveness. Three differentially expressed genes are found, their functions and another 15 genes need further analysis.     

Deferring flowering of nobile dendrobium hybrids by holding plants under low temperature after vernalization

Orchids are currently the most valuable potted crop in the United States. To date, no studies focused on making possible the year-round greenhouse production of flowering nobile dendrobium orchids. This experiment was aimed at developing a strategy to defer flowering of nobile dendrobium orchids by holding them under low temperature. Mature Den. Red Emperor ‘Prince’ and Den. Sea Mary ‘Snow King’ were held at 10 °C for various durations (0, 4, 8, 12 or 16 weeks) after vernalization (4 weeks at 10 °C). Plants were forced in a greenhouse after holding. Time to flower, flower differentiation (flowering node percentage, number of aerial shoot and aborted bud) and flower quality (total flower number, flower diameter, flower number per flowering node and flower longevity) were determined. Increase of low temperature holding duration from 0 to 16 weeks extended time to flower up to 3 months and did not affect parameters of flower except producing larger flowers and reducing flower number per flowering node for Den. Red Emperor ‘Prince’. Notably, the flower longevity was not adversely affected. Defoliation was aggravated in Den. Red Emperor ‘Prince’ by longer duration of cooling and was considered a detrimental effect of low temperature holding.