Fen zi xi bao sheng wu xue bao = Journal of molecular cell biology最新文献

Pollen grains of Allium tuberosum Roxb broke and released their content including generative cells using osmotic shock method. In a medium containing 0.05% CaCl2, 0.01% Boric acid, 0.01%KH2PO4, 15%PEG 10% sucrose (710 mOsmol/kg H2O) 86% pollen grains germinated and grew out pollen tubes, which broke after transferred into 6% mannitol solution, and released tube content including generative cell. When pollen grains were cultured in the same medium but adding 0.1% casein, a few generative cells divided into two sperm cells. Stigmas of Allium tuberosum Roxb were pollinated at second day after anthesis and the styles grow 3 h in vivo. Then the styles were cut and cultured in a medium for about 6-8 h, some pollen tubes grew out of the cut end of the style. The cut end of the style was transferred into a solution containing 6% mannitol to burst pollen tubes. Pairs of sperm cells of Allium tuberosum Roxb were released and collected using a micromanipulator.

H2O2 burst in the defense responses of Wheat (Triticum aestivum L.) infected with compatible and incompatible leaf rust fungus races (Puccinia recondita f.sp. tritici) was investigated firstly. Cytofluorimetric analysis using the fluorescent probe DCFHDA scrutinized the generation of H2O2 in wheat primary leaves. The results indicated that H2O2 burst could be induced by either compatible or incompatible leaf rust fungus. However, the kinetics of H2O2 burst in incompatible interaction were biphasic, and the peak of phase II (20h after inoculation) was much higher than that of phase I (12h after inoculation), whereas, there only existed the weak and transient phase I H2O2 accumulation at 12h after inoculation in compatible interactions. In addition, pharmacological experiments were carried out. Antioxidants (AsA, DTT) and inhibitors of the mammalian neutrophil NADPH oxidase (DPI, imidazole) were separately injected into wheat primary leaves before inoculation to study their effects on hypersensitive reaction (HR) and H2O2 production in incompatible interaction. It was observed that treatments of wheat leaves with AsA, DTT, DPI or imidazole obviously suppressed the two peaks of intracellular H2O2 burst, whereas the second peak was inhibited much more. Moreover, the area of hypersensitively dead cells was decreased in the four treatments. These results suggested that NADPH oxidase may play an important role in H2O2 generation during wheat-leaf rust fungus interaction, and H2O2 burst may be involved in HR caused by leaf rust invading.

The sequences of rbcL from four species of Boehmeria were analyzed in this study by PCR amplification and directly sequencing. The sequences of PCR products were about 1,370 bp. RbcL sequences of nine generas were chosen from GenBank, then the rbcL of 13 species were sequenced with clustal X1.83. After sequencing, the length of rbcL was 1,428 bp and has 1,263 conserved sites, 165 variable sites and 71 information sites. By using MEGA3.1, MP tree and NJ tree of Urticaceae were constructed. Based on the difference in rbcL squence, the genetic distances among species were calculated. With these informations, we can further analyze the genetic relationships of Urticaceae. In result, Pellionia, Poikilospermum, Procris, Elatostema and Pilea formed a monophyletic group; Parietaria and Boehmeria formed another monophyletic group. However, Urtica formed a single group, suggesting it had the most distant relationship to the other genus of Urticaceae.

The developmental potential of reconstructed embryos varied according to the source of donor cells, it was thought that the donor cells capabilities to be reprogrammed were different. We established the method of culturing porcine bone marrow mesenchymal stem cells (pMSCs), identified and observed the growth characteristics of pMSCs, and determined pMSCs reprogramming potential as donor cells for nuclear transfer (SCNT). We found that the method of gradient centrifugation to isolate pMSCs from porcine bone marrow was better than the method of anchoring culture; the number of pMSCs achieved peak at day 6, the adhesive rate of cultured cells was 78.50% at 10h and the division index of cultured cells was 24.00 per thousand at day 4. The developmental competence were compared among three kinds of embryos, reconstructed embryos with PF and pMSCs, Parthenogenetic. The blastocysts rate and total cell number of blastocysts were 15.07%, 14.63% vs 30.91% and 24.1 +/- 6.5, 30.67 +/- 17.7 vs 25.8 +/- 11.4. These results indicated that pMSCs could be high proliferation and stable growth characters in vitro, and were suitable donor cells type for nuclear transfer.

Apoptosis in detached corn root cap cells under fluorescent microscope was studied. Homokaryon maize B37 inbred lines was tested and the detached corn root cap cells with cell wall might be stained using Hoechst33342. Apoptosis was found under fluorescence microscope treated by culture filtrate of HMC toxin. The sensitivity of HMC toxin to C cytoplasm was greater than N cytoplasm and there was more resistance response in detached corn root cap cells of N cytoplasm than C cytoplasm. Besides, apoptosis did not coincide at different processing times. These indicated that special disease response occurred in the course of apoptosis in detached corn root cap cells of C cytoplasm maize led by HMC which may be collective result of nucleus and cytoplasm.

Using reverse transcriptase-polymerase chain reaction (RT-PCR), REG 1A mRNA expression was investigated in 235 primary gastric carcinoma samples. And the relation between these results with the clinicopathologic parameters of primary gastric carcinoma was discussed in experiment. The positive REG 1A mRNA is 78% (183/235) of primary gastric carcinoma which revealed by RT-PCR analysis. Moreover, patients with REG 1A-positive differentiated adenocarcinoma were found to have a significantly poorer prognosis compared with REG 1A-negative tumor patients. REG IA mRNA is closely linked to the infiltrative growth pattern and with signet ring cell carcinoma and poorly differentitated adencarcinoma. And the incidence of venous invasion of REG 1A-positive tumors was significantly higher than that of REG 1A-negative tumors. So the results of the experiment demonstrate that the expression of the REG 1A gene is closely related to the infiltrating property of primary gastric carcinoma, and may be as a prognostic indicator of differentiated adenocarcinoma of the stomach in clinic diagnosis.

Using RT-PCR method, the open reading frame (ORF) of AtNHX1-cDNA, encoding the vacuolar Na+/H+ antiportor, was cloned from Arabidopsis thaliana seedlings pretreated with 100 mmol/L NaCl for 24h. This ORF was inserted between CaMV35S promoter, a Omega fragment of TMV RNA 5'UTR and NOS polyA terminator in the T-DNA region of a binary expression vector pNT (Fig1). The recombinant plasmid, designated as pNT-AtNHX1, was then transformed into Agrobacterium tumefaciens LBA4404. Mediated by this engineering Agrobacterium, the AtNHX1 gene was transferred into T0 generation transgenic plant strains of A. melilotoides and 103 regenerated plants resistant to Kanamycin (Kan) were obtained. Some factors influencing the transformation efficiency, such as the concentration and infection duration of Agrobacterium, the concentration of Acetosyringone (AS), were optimized to establish a stable Agrobacterium mediated gene transformation protocol of A. melilotoides. PCR analysis, Southern blot and RT-PCR detection of some T0 transgenic plants showed that the AtNHX1 gene was evidently integrated into the genome of transgenic plants and couldl be transcripted properly. Under the same salt stress conditions, the detection of NaCl resistance revealed the difference between the wild-type calli and the transgenic calli that induced from the transgenic plants, i.e, the relative growth rates of the transgenic calli were remarkably higher than that of the wild-type calli. The K+ and Na+ contents and relative conductivity in the leaves of the transgenic plants and wild-type plants were estimated. It suggested that under the stress of different concentration of NaCl, K+/Na+ ratio in the transgenic plant cells were always higher than that in wild-type, however the situation of relative conductivity was on the opposite. From the facts above mentioned, the transformation of AtNHX1 gene not only enhanced the salt tolerance of transgenic A. melilotoides, but also reduced the cell membrane damage induced by salinity.

Elastin-like polypeptides (ELPs) are biological macromolecules designed on the elastic structure and composition. ELPs are thermally responsive polypeptides that undergo reversible inverse phase transition. Below their inverse transition temperature (Tt), ELPs are soluble in water, but when the temperature is raised above Tt, phase transition occurs, leading to aggregation of the polypeptide. Based on this property of ELPs, we investigated the ELPs as a fusion partner for the expression and purification of the antimicrobial polypeptide halocidin18 (18-amino-acid subunit, Hal18). According to the amino acid sequence of hal18 and the bias for preferred codons of E. coli, the hal18 gene was synthesized by reverse PCR method and fused with the ELPs gene; the fusion gene was cloned into the prokaryotic expression vector pET23a to construct a recombinant expression vector pETH9E and then transformed into E. coli BL21 (DE3) to overexpress the recombinant fusion protein H9E. H9E was purified by inverse transition cycling (ITC) from the soluble fraction of the lysed E. coli. SDS-PAGE analysis results demonstrated that one-round of ITC purification could remove most of the contaminated proteins, and second-round could increase the purity of the fusion protein up to 95%. After cleavage and dialysis, Hal18 showed strong antimicrobial activities against E. coli and M. luteus which were used as representative gram-negative and gram-positive bacteria, respectively. The results demonstrate that ELPs is an ideal fusion protein to express and purify the antimicrobial polypeptides.