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

To study application of multi-ribozyme expression system on expression inhibition of multidrug resistance-associated protein (MRP1) in HEK293 cells, the multi-ribozyme expression system containing 20 cis-acting ribozymes for self-cleavage and 10 trans-acting ribozymes for targeting to MRP1 gene specific site were constructed. HEK293 cells cotransfected multi-ribozyme expression system with MRP1 target gene or full length of MRP1 gene respectively were analyzed by RT-PCR, Western blot analysis and MTT assay. The results showed that multi-ribozyme systems were able to dramatically decrease fluorescent fusion protein expression in HEK293 cells. RT-PCR analysis indicated that the extent of MRP1 target mRNA decrease was correlated with the number of trans-acting ribozyme contained in multi-ribozyme expression system. Similar changes have been observed from Western blot. MTT assay showed that multi-ribozyme systems were able to reverse MDR generated by MRP1 gene in HEK cells. These results suggested that inhibitory effects of multiple copies of ribozymes contained in the system were better than that of single ribozyme contained. Therefore, this strategy could be used in treatment of tumor or other diseases via gene therapy.

Effects of various exogenous hydrogen peroxide (H2O2) concentrations on leaf senescence of tobacco (Nicotiana tabacum L.) were studied. The results showed that 0.1 and 1 mmol/L H2O2 had little effects on the leaf senescence of tobacco, the contents of chlorophyll and soluble protein were decreased in varying degrees by 10, 100 mmol/L H2O2 treatment, the decreased chlorophyll and soluble protein contents were significantly correlated with increased endogenous H2O2 content. SDS-PAGE of soluble protein and localized staining of H2O2 showed consistency with respective content. Chloroplast ultrastructure were observed in the mesophyll cells of tobacco and there was a little change by 10 mmol/L H2O2 treatment, and a remarkable breakdown of granal stacks and disorganized thylakoid membranes were revealed by 100, 200 mmol/L H2O2 treatment. RT-PCR analysis showed the expression level of senescence associated genes SAG12 was low by 10mmol/L H2O2 treatment and increased by 100 mmol/L H2O2 treatment. These results strongly suggested the tobacco leaf senescence was induced by 10 mmol/L H2O2 and accelerated the progression by high concentration (100, 200 mmol/L) H2O2 stress.

Anatomical, histochemical and phytochemical methods were used to investigate the structure, the localization and content changes of total saponin of the root tuber of Pseudostellaria heterophylla on different developmental stages. Results showed that the primary structure and secondary structure of P. heterophylla adventitious root were similar to that of other herbaceous dicotyledons. 80% of mature root tuber were secondary xylem which consisted of most parenchyma cells and less vessels. The secondary phloem were composed of parenchyma cells too. The results of histochemical methods showed that saponin distributed in pericycle and parenchyma cells of primary phloem in the primary structure of root. In secondary structure and mature root tuber, saponin distributed in periderm and secondary vascular structure except cork layer and vessel. Besides, the colors of the secondary phloem were darker. The results of phytochemical test showed that the content of saponin in skin areas was higher than that in xylems in February and July. The result was consistent with that of histochemistry. The content of saponin in the head of the root was higher than that in the end of the root which was in turn higher than that in the middle part of the root. In the developmental process of root tuber, the content of saponin showed a dynamic tendency of high-low-high. This characteristics were related to the development of root system.

By employing a recombination inbreed line (RIL) population of 247 lines derived from an inidca-inidca cross Zhenshan 97Bx Milyang 46, a linkage map consisting of 250 DNA markers was used for QTL mapping of plant height, maximum root length, root dry weight and relative water content under normal and salt stress (0.7% NaCl). Six QTLs for these traits were identified under salt stress, with 6.01%-16.50% phenotypic variations. Six QTLs for these traits were identified under normal conditions, with 4.39%-18.50% phenotypic variations. Meanwhile, root length and relative water content were simultaneously affected by two QTLs located in the interval RM246-RG101 on chromosome 1 and the interval RZ399-RG393 on chromosome 3.

Asymmetric interspecific somatic hybrids between Brassica oleracea var. botrytis and B. nigra were produced by PEG-induced fusion of protoplasts radiated by different doses of UV. B. nigra, genotype St.461, has resistance to black rot, black leg and clubroot diseases which are popular in cabbage production. The regenerated plants were analyzed by several means including morphology observation, relative DNA content measurement by flow cytometry (FCM), chromosome counting, DNA molecular marker. Morphology observation indicated that the regenerated plants varied in morphology from intermediate type to cauliflower type. DNA contents of 20% regenerated plants were less than the sum of donor and recipient. Number of chromosomes in 23% of the regenerated plants were less than the sum of both parents. DNA molecular marker analysis (SRAP) demonstrated that the genetic information from the recipient parent was retained more or less complete and intact in the hybrids, while specific amplification bands of donor genome were lost in the hybrids from 20% to 97.77% . Furthermore, black-rot resistance test was performed in twenty-two regenerated plants, and seventeen of which showed good resistance against Xamthomonas campestris pv campestris. That gave the elementary proof of transferring of alien pathogen resistant genes from wild B. nigra to B. oleracea via UV mediated asymmetrical somatic hybridization. In conclusion, UV irradiation induced production of asymmetric hybrids between cauliflower and black mustard. Chromosome elimination or a limited introduction of donor chromosomes occurred in most of the hybrids, however, the degree of elimination was independent on UV doses.

To explore the effect of cilostazol in the pathophysiology of diabetic retinopathy and its mechanism, we intraperitoneal injection streptozotocin (STZ) to induce rats diabetic model to study the alteration of the thrombospondin-1 (TSP-1) in the retina of diabetic rats, cilostazol treatment diabetic rats and normal rats by immunohistochemistry, real-time quantitative reverse transcription-polymerase chain reaction. The weight, blood sugar and urine sugar were also measured before and after model induction of these three groups. The data of weight, blood sugar and urine sugar indicated no significant difference in these three groups before diabetes induction. Four weeks after the injection of STZ, the weight, blood sugar and urine sugar had significant difference among these three groups (P < 0.01). When compared with the normal retina, TSP-1 expression was increased in the diabetic rat's retina, as shown by increased optical density and immunohistochemistry positive cell number but this was not serious in cilostazol treatment rats (P < 0.01). Our study confirmed that up-regulation of TSP-1 expression in retina of streptozotocin-induced diabetic rats under hyperglycemia condition and cilostazol treatment could prevent TSP-1 overexpression. This indicates a protective way in the pathophysiology of diabetic retinopathy.

Wheat cultivar Jing 411 which is susceptible to powdery mildew, wheat cultivar Brock and near isogenic lines (NILs) of Jing411, which are resistant to powdery mildew were analysized for polymorphisms using 225 pairs of AFLP primers Only two pairs of primers Pst+GAC/Mse+ TCT (P1) and Pst+AGC/Mse+ACC (P2) stably produced polymorphic bands between the resistant and susceptible plants. Two specific fragments were obtained. By cloning and sequencing these two specific fragments, it was showed that the specific fragment amplified by primer P1 had 268bp, and the fragment amplified by P2 had 227bp. They were named AFLP marker P1(268) and P2(227) respectively. Linkage analysis of these two markers revealed that the polymorphism existed in a 106 F2 segregating population. These two markers closely linked to a powdery mildew resistance gene in wheat cultivar Brock, linkage distance were 3.6 and 1.9 cM respectively. These two markers will be useful for marker-assisted selection and gene pyramiding in wheat resistance breeding.

A cytoplasm male sterile pepper (Capsicum annum L.) was examined using cytochemical method to study its pollen abortion. Thick sections of both anthers of male sterility line 8214A and its maintainer 8214B at different stages were stained using Periodic Acid-Schiff's (PAS) reaction to detect starch distribution. Anther structure and starch distribution in both anthers of male sterility and maintainer line were similar before the meiosis of microspore mother cells. After meiosis, the size of tapetal cells of fertile anthers of maintainer line increased and became high vacuolation. Abundant small starches appeared in the connective cells from tetrad stage to early stage of microspore development. At the late stage of microspore, the tapetal cells began to degenerate and the starches in the connective cells became large. Bi-cellular pollen synthesized starches after the large vacuole of vegetative cell disappeared, and abundant starches were stored in the mature pollen. In the anthers of male sterile line, meiosis of microspore mother could occurred and the tetrads could be formed in the locule, but the tetrads were extruded together because the locule could not enlarge its space. Finally, the tetrad microspores degenerated. The development of vascular tissue of the sterile anthers was normal and abundant starches were stored in the connective cells, which suggested that the function of plant transporting polysaccharide into anther was normal but tapetum could not transport the polysaccharide into locule. According to our result, the pollen abortion occurred in the tetrad stage and the abnormal development of tapetal cells might be the reason which induced tetrad microspore abortion in this male sterile pepper.

The cDNA of IgE constant domain of rat was cloned from the spleen of allergy asthma rat by RT-PCR. The IL-1ra segment was obtained from intermediate vector pBV220-IL-1ra. By overlap extension PCR, the fusion gene IL-1ra-Fcepsilon was cloned, then inserted into the eukaryotic expression plasmid pIRES2-EGFP to obtain a recombinant expression plasmid pIRES2-EGFP-IL-1ra-Fcepsilon. The recombinant expression plasmid was transfected into 293T cells using lipofectamin and instillated into the rat lung through trachea. The expression of IL-1ra-Fcepsilon was identified by Western blot, RT-PCR, and this protein could inhibit the activity of IL-1 in vitro. Green fluorescent protein could be detected in the transfected 293T cells and the rat lungs at different times. The research paved the way for the gene therapy of allergy asthma.