Screening for UV-C irradiation-enhanced transcription factors that regulate the metabolism of phenolic compounds in tomato fruit / 生物工程学报

Solanum lycopersicum phenylalanine ammonia-lyase 5 (SlPAL5) gene regulates the metabolism of phenolic compounds. The study of transcription factors that regulate the expression of SlPAL5 gene is of great significance to elucidate the regulatory mechanism underlying the biosynthesis of phenolic compounds in tomato fruit induced by UV-C irradiation. Here, yeast one-hybrid library of tomato fruit was constructed, and the yeast one-hybrid technology was used to screen the transcription factors that regulate the expression of SlPAL5, the key gene related to the synthesis of phenolic compounds in tomato fruit. As a result, a transcription factor, SlERF7, was obtained and sequenced, followed by the blast homology analysis. Further experiments confirmed that SlERF7 interacted with the promoter of SlPAL5 gene. In addition, UV-C irradiation significantly increased the expression level of SlERF7. These results indicate that SlERF7, which is regulated by UV-C irradiation, might be involved in regulating the transcription of SlPAL5, which provided foundations for further studying the regulation mechanism of the biosynthesis of phenolic compounds in tomato fruit induced by UV-C irradiation.

Construction of yeast one-hybrid library and screening of transcription factors regulating FPS expression in Ganoderma lucidum / 中草药

Objective: To screen the upstream regulatory transcription factors of farnesyl diphosphate synthase (FPS) in the triterpenoid synthesis pathway in Ganoderma lucidum. Methods: In this study, the FPS promoter was cloned and connected to the pAbAi plasmid to construct bait vector pAbAi-FPS, which was transformed into Y1H yeast competent cells to construct bait yeast. The yeast one-hybrid cDNA library was constructed by using SMART technology, then the purified ds-cDNA and pGADT7-Rec were co-transformed into bait yeast strain to screen the upstream transcriptional regulatory factors of PFS. Results: The bait vector containing pAbAi-FPS was constructed and the bait strain was screened, the cDNA library was constructed and transformed to the bait strain. A total of 37 positive clones were screened and sequenced. The sequences of conserved domain were predicted and performed blast search against the whole-genome database to identify their function. As a result, a total of 18 upstream regulatory factors were screened out including three transcription factors, five ribosomal proteins, and 10 other transcription regulators. Conclusion: The results indicated that transcription factors GlSNF2, GlMHR, and GlZn2Cys6 were candidate genes for regulating the expression of FPS, and this study offered data for further study on the regulation mechanism of FPS expression.

Construction of yeast one-hybrid library and screening of transcription factors regulating LS expression in Ganoderma lucidum / 中国中药杂志

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.

PDIL1-2 can indirectly and negatively regulate expression of the AGPL1 gene in bread wheat

BACKGROUND: ADP-glucose pyrophosphorylase (AGPase), the key enzyme in plant starch biosynthesis, is a heterotetramer composed of two identical large subunits and two identical small subunits. AGPase has plastidial and cytosolic isoforms in higher plants, whereas it is mainly detected in the cytosol of grain endosperms in cereal crops. Our previous results have shown that the expression of the TaAGPL1 gene, encoding the cytosolic large subunit of wheat AGPase, temporally coincides with the rate of starch accumulation and that its overexpression dramatically increases wheat AGPase activity and the rate of starch accumulation, suggesting an important role. METHODS: In this study, we performed yeast one-hybrid screening using the promoter of the TaAGPL1 gene as bait and a wheat grain cDNA library as prey to screen out the upstream regulators of TaAGPL1 gene. And the barley stripe mosaic virus-induced gene-silencing (BSMV-VIGS) method was used to verify the functional characterization of the identified regulators in starch biosynthesis. RESULTS: Disulfide isomerase 1-2 protein (TaPDIL1-2) was screened out, and its binding to the TaAGPL1-1D promoter was further verified using another yeast one-hybrid screen. Transiently silenced wheat plants of the TaPDIL1-2 gene were obtained by using BSMV-VIGS method under field conditions. In grains of BSMV-VIGS-TaPDIL1-2-silenced wheat plants, the TaAGPL1 gene transcription levels, grain starch contents, and 1000-kernel weight also significantly increased. CONCLUSIONS: As important chaperones involved in oxidative protein folding, PDIL proteins have been reported to form hetero-dimers with some transcription factors, and thus, our results suggested that TaPDIL1-2 protein could indirectly and negatively regulate the expression of the TaAGPL1 gene and function in starch biosynthesis.

The Application of Yeast One Hybrid System in Plant Transcription Factor Gene Resistant to Osmotic Stress / 中国生物工程杂志

The yeast one hybrid system is the new type system deriving from the yeast two hybrid system to study the interaction between DNA and protein.The principle and the technical line of this system was briefly introduced,and the progress of this system on the research of plant transcription factor gene resistant to osmotic stress was particularly summarized,such as cloning the transcription factor genes of osmosis resistance type;confirming the interaction between known DNA and protein;making sure the localization of the confirmed DNA binding domain with the interaction;also validating the transcription activation of the transcription factor gene.Furthermore,the problems existing in this system on the research of plant transcription factor gene resistant to osmotic stress was analyzed,and the development prospect according to the research was forecasted.

Screening out CpG immunostimulatory sequence-specific DNA-binding proteins with Yeast-one-hybrid system / 第二军医大学学报

Objective: To screen out DNA binding proteins specifically recognizing CpG immunostimulatory sequence (ISS) for further investigating the molecular mechanisms of ISS. Methods: Yeast one hybrid system was adapted in screening a human bone marrow cDNA library using 4 copies of ISS as bait. The ISS binding activity of the positive clone was confirmed by electrophoretic mobility shift assay (EMSA). Results: Four dual positive colonies were obtained, two of them encoded proteins with unknown functions. The other 2 encoded light chains of immunoglobulin with amino sequences homology to anti DNA Ab and HBsAb respectively. EMSA showed HBsAb specifically bound to CpG ODN at pH6.4 and pH 5.8. Conclusion: HBsAb may have ISS specific DNA binding activity.