Bait Vector Research Articles

Construction of a Membrane Yeast Two-Hybrid Library and Screening of MsPYR1-Like Interacting Proteins in Malus sieversii.

To investigate the biological effects of the ABA receptor pyrabactin resistance 1-like (PYR1-like) in Malus sieversii seeds, the proteins interacting with MsPYR1-like were screened by the membrane yeast two-hybrid library based on the split-ubiquitin system, and to construct the bait vector pBT3-SUC-PYR1 for Malus sieversii cDNA library, which had no self-activating effect on the yeast cells of the pPR3-N membrane yeast two-hybrid library. The library titer assay showed that it could meet the requirements for membrane yeast two-hybrid library screening. After sequencing, GenBank database blast, and yeast rotary validation, 28 candidate proteins interacting with MsPYR1-like were obtained, including ribosomal proteins, late embryogenesis abundant proteins, F-actin-capping proteins, phytochrome-interacting proteins, low-temperature-inducible 65kDa protein-like, senescence-associated, PP2C and SnRK2 family members, and unknown proteins. Gene ontology analysis of the interaction proteins was related to plant hormone response and negative regulation of seed germination, overexpression of MsPYR1-like in Arabidopsis negatively regulates seed germination, and the study of the biological roles of MsPYR1-like interacting proteins lays the foundation for revealing the lifting of seed dormancy in Malus sieversii.

Novel C-type lectin mediated non-specific cytotoxic cells killing activity through NCCRP-1 in nile tilapia (Oreochromis niloticus)

Non-specific cytotoxic cells (NCCs) are vital immune cells involved in teleost's non-specific immunity. As a receptor molecule on the NCCs' surface, the non-specific cytotoxic cell receptor protein 1 (NCCRP-1) is known to play a crucial role in mediating their activity. Nevertheless, there have been limited studies on the signal molecule that transmits signals via NCCRP-1. In this study, a yeast two-hybrid (Y2H) library of tilapia liver and head kidney was constructed and subsequently screened with the bait vector NCCRP-1 of Oreochromis niloticus (On-NCCRP-1) to obtain a C-type lectin (On-CTL) with an interacting protein sequence. Consequently, the full-length sequence of On-CTL was cloned and analyzed. The expression analysis revealed that On-CTL is highly expressed in the liver and is widely distributed in other tissues. Furthermore, On-CTL expression was significantly up-regulated in the brain, intestine, and head kidney following a challenge with Streptococcus agalactiae. A point-to-point Y2H method was also used to confirm the binding between On-NCCRP-1 and On-CTL. The recombinant On-CTL (rOn-CTL) protein was purified. In vitro experiments demonstrated that rOn-CTL can up-regulate the expression of killer effector molecules in NCCs via its interaction with On-NCCRP-1. Moreover, activation of NCCs by rOn-CTL resulted in a remarkable enhancement in their ability to eliminate fathead minnow cells, indicating that rOn-CTL effectively modulates the killing activity of NCCs through the NCC receptor molecule On-NCCRP-1. These findings significantly contribute to our comprehension of the regulatory mechanisms governing NCC activity, paving the way for future research in this field.

Variation of gene expression of fatty acid acyl CoA reductase associated with wax secretion of a scale insect, Ericerus pela, and identification of its regulation factors through the accessible chromatin analyses and yeast one-hybrid.

The Chinese white wax scale insect (CWWSI), Ericerus pela, can secret an amount of wax equivalent to their body weight. Previous studies demonstrated the fatty acyl-CoA reductase (far3) plays a pivotal role in wax secretion of CWWSI. The high expression of far3 is crucial for the massive wax secretion. However, the transcription regulation of far3 was not clear. To identify regulatory factors that control the expression of far3, the assay for transposase-accessible chromatin (ATAC) and yeast one-hybrid (Y1H) were carried out in this study. The ATAC sequencing of the CWWSI at the early wax-secretion stage ATAC-seq resulted in 22.75 GB raw data, generated 75,827,225 clean reads and revealed 142,771 peaks. There was one significant peak in the 3 kb upstream regulation regions. The peak sequence is located between -1000 and -670 bp upstream of the far3 transcription start site, spanning a length of 331 bp. This peak sequence served as bait for creating the pAbAi-peak recombinant vector, used in Y1H screenings to identify proteins interacting with far3 gene. The results indicate a successful CWWSI cDNA library construction with a capacity of 1.2 × 107 colony forming unit, a 95.8% recombination rate, and insert sizes between 1,000 and 2,000 bp. Self-activation tests established that 100 ng/mL of AbA effectively inhibited bait vector self-activation. Finally, a total of 88 positive clones were selected. After sequencing and removal of duplication, 63 unique clones were obtained from these screened colonies. By aligning the clone sequences with full-length transcriptome and genome of CWWSI, the full-length coding sequences of these clones were obtained. BlastX analysis identified a transcription factor, nuclear transcription factor Y beta, and two co-activators, cAMP-response-element-binding-protein-binding protein and WW domain binding protein 2. Reverse transcription quantitative polymerase chain reaction analysis confirmed that their expression patterns were consistent with the developmental stages preceding wax secretion and matched the wax secretion characteristics during ovulation periods. These results are beneficial for further research into the regulatory mechanisms of wax secretion of CWWSI.

Gene Expression and Interaction Analysis of FsWRKY4 and FsMAPK3 in Forsythia suspensa.

Forsythia suspensa is a deciduous shrub that belongs to the family Myrtaceae, and its dried fruits are used as medicine. F. suspensa contains several secondary metabolites, which exert pharmacological effects. One of the main active components is forsythin, which exhibits free radical scavenging, antioxidant, anti-inflammatory, and anti-cancer effects. Mitogen-activated protein kinase (MAPKs) can increase the activity of WRKY family transcription factors in a phosphorylated manner, thereby increasing the content of secondary metabolites. However, the mechanism of interaction between MAPKs and WRKYs in F. suspensa remains unclear. In this study, we cloned the genes of FsWRKY4 and FsMAPK3, and performed a bioinformatics analysis. The expression patterns of FsWRKY4 and FsMAPK3 were analyzed in the different developmental stages of leaf and fruit from F. suspensa using real-time fluorescence quantitative PCR (qRT-PCR). Subcellular localization analysis of FsWRKY4 and FsMAPK3 proteins was performed using a laser scanning confocal microscope. The existence of interactions between FsWRKY4 and FsMPAK3 in vitro was verified by yeast two-hybridization. Results showed that the cDNA of FsWRKY4 (GenBank number: OR566682) and FsMAPK3 (GenBank number: OR566683) were 1587 and 522 bp, respectively. The expression of FsWRKY4 was higher in the leaves than in fruits, and the expression of FsMAPK3 was higher in fruits but lower in leaves. The subcellular localization results indicated that FsWRKY4 was localized in the nucleus and FsMAPK3 in the cytoplasm and nucleus. The prey vector pGADT7-FsWRKY4 and bait vector pGBKT7-FsMAPK3 were constructed and co-transferred into Y2H Glod yeast receptor cells. The results indicated that FsWRKY4 and FsMAPK3 proteins interact with each other in vitro. The preliminary study may provide a basis for more precise elucidation of the synthesis of secondary metabolites in F. suspensa.

Transferrin Mediated NCC Killing Activity through NCCRP-1 in Nile Tilapia (Oreochromis niloticus)

Non-specific cytotoxic cell l (NCC) is a kind of important lymphocyte participating in the non-specific immune response in teleost. Non-specific cytotoxic cell receptor protein 1 (NCCRP-1) is a receptor molecule on the surface of NCC and plays an important role in mediating the activity of NCC. However, there are few reports on which signal molecule could transmit signals through NCCRP-1. In this study, yeast two-hybrid library of tilapia liver and head kidney was constructed, and a transferrin from Oreochromis niloticus (On-TF) with interaction protein sequence was obtained by screening the library with bait vector NCCRP-1 of Oreochromis niloticus (On-NCCRP-1). Then, the open reading frame (ORF) of On-TF was cloned, which had 2088 bp in length, encoding polypeptides of 695 amino acids. The deduced amino acid sequence was highly homologous to teleost and similar to mammalian TF, containing two TR_FER domains (25-343th aa and 344-686th aa) for binding iron ions. Furthermore, a point-to-point yeast two-hybrid method was used to further verify whether On-NCCRP-1 could bind to On-TF. The recombinant On-TF (rOn-TF) protein was purified by prokaryotic expression system. In vitro experiments showed that rOn-TF could up-regulate the expression of killing effector molecule of NCC by On-NCCRP-1, and rOn-TF-activated NCCs showed a significantly improved ability to kill FHM cells, indicating that rOn-TF could regulate the NCC signaling pathway through NCC receptor molecule On-NCCRP-1. The results provide a more theoretical basis for understanding the regulation mechanism of NCC activity.

Screening of genes encoding proteins that interact with ISG15: Probing a cDNA library from a snakehead fish cell line using a yeast two-hybrid system

Interferon-stimulated gene 15 (ISG15) regulates cellular life processes, including defense responses against infection by a variety of viral pathogens, by binding to target proteins. At present, various fish ISG15s have been identified, but the biological function of ISG15 in snakehead fish is still unclear. In this study, total RNA was extracted from snakehead fish cell line E11, ds cDNA was synthesized and purified using SMART technology, and the resulting cDNA library was screened by co-transforming yeast cells. The library titer was 4.28 × 109 CFU/mL. Using snakehead ISG15 as the bait protein, the recombinant bait vector pGBKT7-ISG15 was constructed and transformed into the yeast strain Y2HGold. The toxicity and self-activation activity of the bait vector were detected on the deficient medium, and the prey proteins interacting with ISG15 were screened. In total, 19 interacting proteins of ISG15 were identified, including mitotic checkpoint protein BUB3, hypothetical protein SnRVgp6, elongation factor 1-beta, 60S ribosomal protein L9, dual specificity protein phosphatase 5-like, eukaryotic translation initiation factor 3 subunit I and ferritin. A yeast spotting assay further probed the interaction between ISG15 and DUSP5. These results increase our understanding of the interaction network of snakehead ISG15 and will aid in exploring the underlying mechanisms of snakehead ISG15 functions in the future.

Screening and characterisation of proteins interacting with the mitogen-activated protein kinase Crmapk in the fungus Clonostachys chloroleuca

Clonostachys chloroleuca 67-1 (formerly C. rosea 67-1) is a promising mycoparasite with great potential for controlling various plant fungal diseases. The mitogen-activated protein kinase (MAPK)-encoding gene Crmapk is of great importance to the mycoparasitism and biocontrol activities of C. chloroleuca. To investigate the molecular mechanisms underlying the role of Crmapk in mycoparasitism, a high-quality yeast two hybrid (Y2H) library of C. chloroleuca 67-1 was constructed, and proteins interacting with Crmapk were characterised. The library contained 1.6 × 107 independent clones with a recombination rate of 96%, and most inserted fragments were > 1 kb. The pGBKT7-Crmapk bait vector with no self-activation or toxicity to yeast cells was used to screen interacting proteins from the Y2H library, resulting in 60 candidates, many linked to metabolism, cellular processes and signal transduction. Combined bioinformatics and transcriptome analyses of C. chloroleuca 67-1 and ΔCrmapk mutant mycoparasitising Sclerotinia sclerotiorum sclerotia, 41 differentially expressed genes were identified, which might be the targets of the Fus3/Kss1-MAPK pathway. The results provide a profile of potential protein interactions associated with MAPK enzymes in mycoparasites, and are of great significance for understanding the mechanisms of Crmapk regulating C. chloroleuca mycoparasitism.

Construction of Yeast One-Hybird Library for Screening of Eugenol Synthase Gene Bait vectors in Rosa chinensis

Roses are cultivated as ornamental plants in gardens, cut flowers and potted flowers, but also serviced in the perfume industry. Rose flowers product different volatile compounds including eugenol, being floral attractant for pollinators and used as an important composition of essential oil. Here, the eugenol synthase gene (RcEGS1) promoter was cloned from R. chinensis 'Old Blush'. The sequences of RcEGS1 promoter were 1776bp, contained the conserved elements, and predicted to be with several cis-regulatory elements involved in secondary metabolism of plants. Yeast one-hybrid library of R. chinensis 'Old Blush' at flower opening was constructed with the three-frame reading. The library capacity was more than 1.12×107CFU. The length of fragments inserted was 1 Kb to 3 Kb, which is fully meeting the demand of selecting the yeast library. The bait yeast vectors with cis-transcription elements MBS-box, G-box and one fragment bait yeast vectors were constructed, respectively. Furthermore, the transcript factors R2R3-type MYB and bHLH3 were screened using the yeast one-hybrid system. The results suggested the two TFs might play an important role in the pathway of volatile biosynthesis in roses.

Screening and verification of proteins of Salvia miltiorrhiza polyphenol oxidase interaction

Polyphenol oxidase(PPO) is an important antioxidant enzyme in plants. It has the functions of scavenging active oxygen and synthesizing phenols, lignin, and plant protection factors, and can enhance the plant's resistance to stress and resistance to pests and diseases. Our previous research found that Salvia miltiorrhiza PPO gene can positively regulate salvianolic acid B synthesis. In order to further explore the mechanism, a pGBKT7-PPO bait vector was constructed using the cloned S. miltiorrhiza polyphenol oxidase gene(SmPPO, GenBank accession number: KF712274.1), and verified that it had no self-activation and no toxicity. The titer of S. miltiorrhiza cDNA library constructed by our laboratory was 4.75 × 107 cfu·mL~(-1), which met the requirements for library construction. Through yeast two-hybrid test, 22 proteins that could interact with SmPPO were screened. Only yeast PAL1 and TAT interacted with SmPPO through yeast co-transformation verification. Further verification was performed by bimolecular fluorescence complementary detection(BiFC). Only TAT and SmPPO interacted, so it meant that TAT and SmPPO interacted. TAT and SmPPO were truncated according to the domain, respectively. The first 126 amino acids of SmPPO and tyrosine amino transferase(TAT) were obtained to interact on the cell membrane and chloroplast. SmPPO was obtained by subcellular localization test, which was mainly loca-lized on the nucleus and cell membrane; TAT was localized on the cell membrane. Real-time quantitative PCR results showed that the SmPPO gene was mainly expressed in roots and stems; the TAT gene was expressed in roots, and the expression level in stems and flowers was low. This article lays a solid foundation for the in-depth study of the molecular mechanism of the interaction of S. miltiorrhiza SmPPO and TAT to regulate the synthesis of phenolic substances.

Construction and characterization of a high-quality cDNA library of Cymbidium faberi suitable for yeast one- and two-hybrid assays

BackgroundCymbidium faberi is one of the oldest cultivars of oriental orchids, with an elegant flower fragrance. In order to investigate the molecular mechanism and the functions of related proteins in the methyl jasmonate (MeJA) signaling pathway, one of the main components of flower fragrance in C. faberi, yeast one- and two-hybrid three-frame cDNA libraries were constructed.ResultsIn this study, a modified cDNA library used for yeast one- and two-hybrid screening was successfully constructed, with a recombinant efficiency of 95%. The lengths of inserted fragments ranged from 750~3000 bp, and the library capacity reached 6 × 109 CFU/ μg of cDNA insert, which was suitable for the requirements of subsequent screening. Finally, a homologous protein related with pathogenesis was screened out by the bait vector of CfbHLH36, which may participate in the MeJA signaling pathway.ConclusionThe yeast one- and two-hybrid library of C. faberi provides large amounts of useful information for the functional genomics research in C. faberi, and this method could also be applied to other plants to screen DNA-protein and protein-protein interactions.

Screening and Bioinformatics Analysis of Proteins Interacting with StMAPKK1 in Potato by Yeast Two-Hybrid System

Mitogen-activated protein kinase (MAPK) cascade reaction is one of important and complex signal networks involved in plant growth and development, hormone and stress response. As one of its main members, mitogen-activated protein kinase kinase (MAPKK) is located in the middle of the cascade reaction and plays a key role in signal collection and divergence. There were shown that potato ( Solanum tuberosum L.) StMAPKK1 (PGSC0003DMT400000744) gene responded to drought stress. Therefore, StMAPKK1 gene was firstly selected to screen its interacting protein. In this study, the bait vector pGBKT7-StMAPKK1 was constructed by homologous recombination and used to screen potato cDNA library by yeast ( Saccharomyces cerevisiae ) two-hybrid system. Five StMAPKK1-interacting proteins, hydrolase (hydrolyzing O-glycosyl compound), RING-H2 subgroup RHE protein, cyanate hydratase, ARF GTPase activator, and a C2 domain-containing protein, were obtained through this screening and were identificated by bioinformatics analysis, and the interaction was verified by small-scale hybridization verification. The results provided theoretical basis for further study on the signal pathway and biological function of potato StMAPKK1.

Construction of yeast one-hybrid library and screening of transcription factors regulating LS expression in Ganoderma lucidum

Lanosterol synthase( LS) is a key enzyme involving in the mevalonate pathway( MVA pathway) to produce lanosterol,which is a precursor of ganoderma triterpenoid. And the transcriptional regulation of LS gene directly affects the content of triterpenes in Ganoderma lucidum. In order to study the transcriptional regulation mechanism of LS gene,yeast one-hybrid technique was used to screen the transcription regulators which interact withthe promoter of LS. The bait vector was constructed by LS promoter,then the vector was transformed yeast cells to construct bait yeast strain. One-hybrid c DNA library was constructed via SMART technology. Then the c DNA and p GADT7-Rec vector were co-transformed into the bait yeast strain to screen the upstream regulatory factors of the promoter region of LS by homologous recombination. Total of 23 positive clones were screened. After sequencing,blast was performed against the whole-genome sequence of G. lucidum. As a result,8 regulatory factors were screened out including the transcription initiation TFIIB,the alpha/beta hydrolase super family,ALDH-SF superfamily,60 S ribosomal protein L21,ATP synthase β-subunit,microtubule associated protein Cript,prote asome subunit β-1,and transaldolase. Until now,the regulation effect of these 8 regulatory factors in G.lucidum has not been reported. This study provides candidate proteins for in-depth study on the expression regulation of LS.

The Penaeus stylirostris densovirus capsid protein interacts with the Litopenaeus vannamei BCCIP protein

Viral capsid proteins play an important role in the viral infection process. To identify the cellular proteins in shrimp that interact with the Penaeus stylirostris densovirus capsid protein (PstDNV-CP), we constructed a yeast two-hybrid (Y2H) cDNA library of the muscle tissue of Litopenaeus vannamei, and hybridized the bait vector pGBKT7-CP with this library. Cloning and sequencing showed that the shrimp protein interacting with PstDNV-CP was a homolog of BRCA2 and CDKN1A(p21)-interacting protein (BCCIP). We named this protein L. vannamei BCCIP (LvBCCIP). Further analysis showed that LvBCCIP interacted with L. vannamei calmodulin (LvCaM). We validated the interactions between PstDNV-CP and LvBCCIP, and between LvBCCIP and LvCaM, with GST pulldown assays. The gene expression of LvBCCIP increased significantly after PstDNV challenge. In addition, the PstDNV titer of PstDNV-challenged shrimp was significantly reduced after LvBCCIP expression was inhibited using double-stranded RNA (dsRNA) interference. These results indicated that LvBCCIP is critical to PstDNV pathogenesis in L. vannamei. Interestingly, the growth rate of L. vannamei was significantly reduced when LvBCCIP gene expression was silenced, indicating that LvBCCIP may also be associated with growth regulation in L. vannamei. Thus, the interaction between PstDNV-CP and LvBCCIP might explain why PstDNV infection leads to runt-deformity syndrome in shrimp.

Protein-protein interactions among the structural proteins of Chilo iridescent virus.

Chilo iridescent virus (CIV), officially named invertebrate iridescent virus 6 (IIV6), is a nucleocytoplasmic virus with a ~212-kb linear dsDNA genome that encodes 215 putative open reading frames (ORFs). Proteomic analysis has revealed that the CIV virion consists of 54 virally encoded proteins. In this study, we identified the interactions between the structural proteins using the yeast two-hybrid system. We cloned 47 structural genes into both bait and prey vectors, and then analysed the interactions in Saccharomyces cerevisiae strain AH109. A total of 159 protein-protein interactions were detected between the CIV structural proteins. Only ORF 179R showed a self-association. Four structural proteins that have homologues in iridoviruses (118L, 142R, 274L and 295L) showed indirect interactions with each other. Seven proteins (138R, 142R, 361L, 378R, 395R, 415R and 453R) interacted with the major capsid protein 274L. The putative membrane protein 118L, a homologue of the frog virus 3/Ranagrylio virus 53R protein, showed direct interactions with nine other proteins (117L, 229L, 307L, 355R, 366R, 374R, 378R, 415R and 422L). The interaction between 118L and 415R was confirmed by a GST pull-down assay. These data indicate that 415R is a potential matrix protein connecting the envelope protein 118L with the major capsid protein 274L.

Screening of proteins interacting with ERF transcriptional factor from Populus simonii × P.nigra by yeast two-hybrid method

ABSTRACTTo investigate the biology functions of ERF transcriptional factor in Populus simonii × P.nigra, the proteins interacting with ERF were screened by the yeast two-hybrid method. The bait vector harbouring ERF and cDNA library of Populus simonii × P.nigra were constructed. The bait had no self-activation and toxicity to yeast cells in the yeast two-hybrid system. The library titering experiment indicated that it could meet the requirement for the yeast two-hybrid library screen. Five proteins that interacted with ERF were obtained, including galactinol-sucrose galactosyl transferase 2, phosphoinositide-specific phospholipase C family protein, MLP-like protein, senescence-associated protein and an unknown protein. Real-time polymerase chain reaction showed that the expression levels of ERF and five interacting protein genes increased after salt treatment. Gene ontology analysis associated most of the interacting proteins with stress response. These results showed that ERF might share similar function with these proteins and they act together to regulate the salt response of Populus simonii × P.nigra. This study provided experimental evidence for further revealing the biological function of the ERF transcription factor in poplar.

The protein-protein interactions between Amsacta moorei entomopoxvirus (AMEV) protein kinases (PKs) and all viral proteins

Entomopoxviruses are an important group of viruses infecting only insects. They belong to Poxviridae which infect both invertebrates and vertebrates, including humans. Protein kinases are known to have roles at virus morphogenesis, host selectivity, the regulation of cell division and apoptosis in some vertebrate poxviruses. In this study, 2 protein kinases (PKs) (AMV153 and AMV197) of Amsacta moorei entomopoxvirus (AMEV) were investigated for the interactions among 230 viral proteins using yeast two-hybrid system (Y2H). For this purpose, two protein kinases and 230 viral genes were cloned into the bait and prey vectors, respectively. Bait vectors were introduced into Saccharomyces cerevisiae AH109. Expression of the bait genes were confirmed by western blot analysis. Both yeast strains of bait were transformed individually with each prey clone and grown on a selective medium (minimal synthetic defined) to determine the protein–protein interactions between bait and prey proteins. Transformations identified totally 16 interactions among AMEV protein kinases and all viral proteins of which 5 belong to AMV153 and 11 belong to AMV197. One of the five interactions detected for AMV153 protein kinase is self-association. Its other four interactions are with two virus entry complex proteins (AMV035 and AMV083), a membrane protein (AMV165) and a subunit of RNA polymerase (AMV230). The other protein kinase, AMV197, interacted with two virus entry complex proteins (AMV035 and AMV083) as AMV153, a caspase-2 enzyme (AMV063), a Holliday junction resolvase (AMV162), a membrane protein (AMV165), a subunit of RNA polymerase (AMV230) and five other hypothetical proteins (AMV026, AMV040, AMV062, AMV069, AMV120) encoded by AMEV genome. Glutathione S-transferase (GST) pull-down assay was used to confirm all interactions described by Y2H analysis. In addition, the theoretical structures of the two of 16 interactions were interpreted by docking analysis. Consistent with Y2H and pull down assays, docking analysis also showed the interactions of AMV063 with AMV153 and AMV197. Detected interactions of the AMEV viral proteins with viral protein kinases could lead to the understanding of the regulation of the viral activities of interacted viral proteins.

An OTU deubiquitinating enzyme in Eimeria tenella interacts with Eimeria tenella virus RDRP

BackgroundChicken coccidiosis, a disease caused by seven species of Eimeria (Apicomplexa: Coccidia), inflicts severe economic losses on the poultry industry. Eimeria tenella is the one of the most virulent species pathogenic to chickens. Many parasitic protozoans are parasitised by double-stranded (ds) RNA viruses, and the influence of protozoan viruses on parasitic protozoans has been extensively reported. E. tenella RNA virus 1 (Etv) was identified in E. tenella, and the complete genome sequence of Etv was analysed. Here, we screened Etv-RNA-dependent RNA polymerase (RDRP)-interacting host protein E. tenella ovarian tumour (OTU) protein-like cysteine protease (Et-OTU) using a yeast two-hybrid system with pGBKT7-RDRP plasmid serving as bait. A previous study demonstrated that Et-OTU could regulate the telomerase activity of E. tenella, indicating that Et-OTU affects E. tenella proliferation. However, whether Etv-RDRP affects the molecular biological characteristics of E. tenella by interacting with OTU remains unclear.ResultsWe obtained seven positive clones from the initial screen, and six of the seven preys were identified as false-positives. Finally, we identified an RDRP-associated protein predicted to be an E. tenella OTU protein. A α-galactosidase assay showed that the bait vector did not activate the GAL4 reporter gene, indicating no autoactivation activity from the RDRP bait fusion. Pull-down and co-immunoprecipitation assays verified the interaction between Et-OTU and Etv-RDRP both intracellularly and extracellularly. Additionally, Et-OTU was able to deconjugate K48- and K6-linked di-ubiquitin (di-Ub) chains in vitro but not K63-, K11-, K29-, or K33-linked di-Ub chains. The C239A and H351A mutations eliminated the deubiquitinase (DUB) activity of Et-OTU, whereas the D236A mutation did not. Additionally, when combined with RDRP, the DUB activity of Et-OTU towards K48- and K6-linked chains was significantly enhanced.ConclusionEtv-RDRP interacts with Et-OTU both intracellularly and extracellularly. Etv-RDRP enhances the hydrolysis of Et-OTU to K6- or K48-linked ubiquitin chains. This study lays the foundation for further research on the relationship between E. tenella and Etv.

Construction of Yeast One-hybrid Bait Reporter Vector for Screening the Binding Proteins of CassavaMeCWINV1Promoter

Cellwall invertase (CWIN) hydrolyzes sucrose into glucose and fructose irreversibly, playing key roles in carbohydrate partitioning and plant defence. MeCWINV1 is one of the CWINs in cassava, which contains several light-responsive elements and stress-responsive elements in promoter region. To analyze the regulatory function of MeCWINV1 in the cassava starch accumulation and stress defense response, a 865 bp MeCWINV1 promoter fragment was cloned and inserted into yeast one-hybrid bait vector to construct p CW1 -AbAi vector, then was transformed to Y1HGold yeast strains to screen the binding proteins. It might provide a framework for further investigation on the regulation mechanism of MeCWINV1 gene in cassava.

Straddling the divide: invasive aquatic species in Illinois and movement between the Great Lakes and Mississippi basins

The Laurentian Great Lakes and the Mississippi River are two of the largest freshwater ecosystems in North America, and each contains large numbers of non-native species. In 1900 the Great Lakes and Mississippi were permanently connected with the opening of the Chicago Sanitary and Ship Canal in the US state of Illinois. More recently, movement of trailered boats, bait, and other overland vectors have increased and further enabled movement of non-native aquatic species. To investigate the role of Illinois in continent-wide species spread we assembled a comprehensive database of recorded occurrences of aquatic non-native species in Illinois inland waters. For each species we determined vector, location, current stage in the invasion sequence (introduced or established), and ecological impacts. The arrival of non-native species has accelerated over time. Sixty species are now established, and a further 39 have been recorded. The Great Lakes have been a stronger source of species to Illinois and the Mississippi River basin than the reverse. Over the last two decades the most important vectors delivering non-native aquatic species have been unintentional release and shipping. Through a survey of Illinois aquatic ecologists we found that established species exhibit a continuous range of ecological impacts and that almost a third have high or very high ecological impacts. Local, national, and continental approaches will be required to mitigate the threat of further invasive species spreading into Illinois and between the Great Lakes and Mississippi basins.

利用酵母单杂交文库技术筛选银杏端粒结合蛋白

目的：为了获得与银杏端粒结合序列相结合的端粒结合蛋白，为木本植物端粒结合蛋白的研究提供实验依据，从而丰富对银杏端粒的研究。方法：以银杏叶片为实验材料，利用酵母单杂交文库筛选获得可能为银杏端粒结合蛋白的基因片段，并通过GFP酵母单杂交实验验证获得基因片段与端粒序列的结合特异性。结果：使用端粒特异性结合序列(TTTAGGG)3的诱饵载体没有获得合适大小的扩增片段，而使用端粒特异性结合序列(TTTAGGG)5的诱饵载体成功获得52个扩增片段，这说明端粒DNA序列(TTTAGGG)3过短，不利用蛋白质的结合。比较这些扩增片段序列并去除相同序列，结果显示这52个扩增片段分别属于10个基因片段。对这10个基因片段进行进一步序列分析，并使用酵母单杂交技术验证，结果显示其中一个基因能够与银杏端粒序列特异性结合。结论：本研究通过酵母单杂交文库筛选，GFP酵母单杂交验证等实验方法建立了银杏端粒结合蛋白的筛选方法，并成功获得了一个和银杏端粒重复序列特异性结合的端粒结合蛋白。 Objective: To provide a powerful experimental basis for the research of telomere binding proteins and telomeres in woody plants especially in Ginkgo, we investigated the telomere-binding proteins that bind with telomere sequence in ginkgo. Methods: Using Ginkgo leaves as experimental material, we obtained some gene sequences encoding telomere-binding proteins through yeast one-hybrid library screening technology, then verified their binding specificity by GFP yeast one- hybrid experiments. Results: We did not get any desired DNA sequences using bait vector contain telomere specificity sequence (TTTAGGG)3. However, we obtained 52 DNA sequences using bait carrier contain telomere specificity sequence (TTTAGGG)5 successfully. Our results suggested that the telomere specificity sequence (TTTAGGG)3 might be too short to binding any proteins. After removing repeat sequences, we found that 10 genes were encoded by these 52 DNA sequences through sequences analysis. One gene among them was confirmed that could bind with Ginkgo te-lomere specificity sequence. Through GFP yeast one-hybrid technology. Conclusion: Our study es-tablished a screening method to investigate telomere binding proteins in Ginkgo through yeast one-hybrid library screening and GFP yeast one-hybrid technology, and obtained a telomere binding protein which binding with Ginkgo telomere specificity sequence specifically.