In order to obtain high yield of the HrpNEcc protein with a lower total cost,fermentation and lactose induction conditions for recombinant E.coli BL21(DE3)/pET30a(+)hrpN Ecc were optimized in flasks and the recombinant E.coli was fermentated in 7L fermenter.The optimized incoulum concentration was 5% and the optimized nutrient medium was TB medium.The HrpNEcc protein yield reached 417.60mg/L by adding 3g/L exogenous inducer lactose in the growth prophase of log-phase for the recombinant E.coli.The HrpNEcc protein yield was higher 36.73% than that of the case of no any inducer,and was higher 16.85% than that of the case of adding IPTG.The wet weight of cell pellet of the recombinant E.coli reached 57.24g/L after fermentation in 7L fermenter,the HrpNEcc protein reached 3.29g/L,about 50.2% of total cellular protein.

Cold acclimation can improve freezing tolerance. Here cDNA amplified fragment length polymorphism (cDNA-AFLP) was used to isolate differentially expressed cDNAs and a Pp-LIM only A cDNA was isolated and identified in the cold acclimation of Physcomitrella patens. Real-time RT-PCR indicated it is obviously up-regulated at 6 h, 12 h, 24 h, 48 h and 72 h after cold acclimation. After comparing the cDNA with the gene sequence, seven introns and eight exons were identified in the cDNA. The cDNA putatively encodes a protein of 345 amino acid residues and only contains one LIM domain which has highly similarity with the PDZ/LIM domains of the protein family in animals. We proposed that Pp-LIM only A be a new gene coding for the LIM-domain containing protein and enhance stability of cell membrane via their effects on cytoskeleton during cold acclimation in P. patens.
Acclimatization ; genetics ; physiology ; Amino Acid Sequence ; Amplified Fragment Length Polymorphism Analysis ; Base Sequence ; Bryopsida ; genetics ; physiology ; Cloning, Molecular ; Cold Temperature ; DNA, Complementary ; genetics ; DNA, Plant ; genetics ; Molecular Sequence Data ; Transcription Factors ; genetics

FRUITFULL (FUL) is an MADS box gene that functions early in controlling flowering time, meristem identity and cauline leaf morphology and later in carpel and fruit development in Arabidopsis thaliana. In order to clarify the regulation of FUL expression the upstream regulatory region, -2148 bp - +96 bp and the first intron of the FUL gene were cloned, and vectors with a series of deletion of FUL promoter, and the ones fused with the first intron were constructed. Vectors harboring the fusion of cis-acting elements with the constitutive promoters of TUBULIN and ACTIN were also constructed. Beta-Glucuronidase activity assays of the transgenic Arabidopsis plants showed that two cis-elements were involved in the repression of FUL expression, with one of the two being probably the binding site of the transcriptional factor AP1. And the two CArG boxes played a important role in FUL initiation particularly. Furthermore, the first intron of FUL was shown to participate in the development of carpel and stamen as an enhancer.
Actins ; genetics ; Arabidopsis ; genetics ; metabolism ; Arabidopsis Proteins ; genetics ; Base Sequence ; Enhancer Elements, Genetic ; Flowers ; genetics ; metabolism ; Gene Expression Regulation, Plant ; Introns ; genetics ; MADS Domain Proteins ; genetics ; Molecular Sequence Data ; Promoter Regions, Genetic ; genetics