Electrostatic effect on electron transfer between cytochrome b5 and cytochrome c

The binding and electron transfer between wild type, E44A, E56A, E44/56A, E44/48/56A/D60A and F35Y variants of cytochrome b5 and cytochrome c were studied. When mixed with cytochrome c, the cytochrome b, E44/48/56A/D60A did not show the typical UV-vis difference spectrum of absorption, indicating that the alteration of the surface electrostatic potential obviously influenced the spectrum. The electron transfer rates of wild type cytochrome bj, its variants and cytochrome c at different temperature and ionic strength exhibited an order of F35Y > wild type > E56A > E44A > E44/48/56A/D60A. The enthalpy and entropy of the reaction did not change obviously, suggesting that the mutation did not significantly disturb the electron transfer conformation. The investigation of electron transfer rate constants at different ionic strength demonstrated that electrostatic interaction obviously affected the electron transfer process. The significant difference of Cyt b, F35Y and E44/48/56A/D60A from the wild type protein further confirmed the great importance of the electrostatic interaction in the protein electron transfer.     

Mutagenesis of Ser24 of Cytochrome b559 α Subunit Affects PSII Acitivies in Chlamydomonas reinhardtii

In order to study the functions of cytochrome b559 (Cyt b559) in photosystem two (PSII) activity, mutant S24F of Chlamydomonas reinhardtii was constructed using site directed mutagenesis, in which Serine24 (Ser24) locating downstream of Histidine23 (His23) in α subunit of Cyt b559 was replaced by Phenylalanine (Phe). Physiological and biochemical analysis showed that mutant S24F could be grown photoautotrophically or photoheterotrophically. However, their growth rate was slower either on HSM or TAP medium than that of the control; Analysis of PSII activity revealed that its oxygen evolution was about 71% of wild type (WT); The Photochemical efficiency of PSII (Fv/Fm) of S24F was reduced 0.23 compared with WT; S24F was more sensitive to strong light irradiance than the wild type; Furthermore, SDS-PAGE and Western-blotting analysis indicated that the expression levels of α subunit of Cyt b559, LHCII and PsbO of S24F were a little less than those of the wild type. Overall, these data suggests that Ser24 plays a significant role in making Cyt b559 structure maintain PSII complex activity of oxygen evolution although it is not directly bound to heme group.     

Characterization and expression of a cDNA, AmphiSDHD,encoding the amphioxus cytochrome b small subunit in mitochondrial succinate-ubiquinone oxidoreductase

In this study, an amphioxus cDNA, AmphiSDHD, encoding the cytochrome b small subunit in mitochondrial succinate-ubiquinone oxidoreductase, was isolated from the gut cDNA library of amphioxus Branchiostoma belcheri tsingtauense. It is 1429 bp in length, with an open reading frame of 465 bp coding for a protein of 154 amino acids. The deduced protein contains a mitochondrial targeting presequence of 65 amino acids rich in basic residues like arginine and hydroxy residues such as serine and threonine. Alignment of the amino acid sequences of AmphiSDHD and other eukaryotic SDHD proteins showed that AmphiSDHD has three transmembrane segments, and includes two histidine residues in the second transmembrane segment that are the putative binding sites for the heme b molecule. The phylogenetic tree constructed suggests that AmphiSDHD appears more closely related to vertebrate SDHD proteins than invertebrate ones. Northern blotting demonstrated that AmphiSDHD is ubiquitously expressed in amphioxus, being in line with the fact that SDHD is a house-keeping protein.     

Improving photosynthesis of microalgae by changing the ratio of light-harvesting pigments

Changing the ratio of light-harvesting pigments was regarded as an efficient way to improve the photosynthesis rate in microalgae, but the underlying mechanism is still unclear. In the present study, a mutant of Anabeana simensis （called SP） was selected from retrieved satellite cultures. Several parameters related with photosynthesis, such as the growth, photosynthesis rate, the content of photosynthetic pigment, low temperature fluorescence spectrum （77K） and electron transport rate, were compared with those of the wild type. It was found that the change in the ratio of light-harvesting pigments in the mutant led to more efficient light energy transfer and usage in mutant than in the wild type. This may be the reason why the mutant had higher photosynthesis and growth rates.     

DnaJ-like protein gene sll1384 is involved in phototaxis in Synechocystis sp. PCC 6803

In Synechocystis sp. PCC 6803, gene sll1384 encodes a protein with a DnaJ domain at its N-terminal portion and a TPR domain at the C-terminal portion. An sll1384 mutant shows no difference from the wild type in adaptation to different temperatures, but almost completely loses its capability of phototactic movement. After complementation with sll1384, the mutant regains the phototaxis. As shown with electron microscopy, on the cell surface, mutant cells have pill that appear to be the same as that of the wild type. Also, the transformation efficiency remains unchanged in the mutant. It is postulated that Sll1384 regulates phototaxis of Synechocystis through protein-protein interaction. It is the first DnaJ-like protein gene identified in a cyanobacterium for a role in phototaxis.     

Effects of point mutation C→T at - 64 of human δ globin gene promoter on DNA binding proteins

By electrophoretic mobility shift assay (EMSA), the effect of point mutation C→T at - 64 of human δ-globin gene on its binding proteins has been studied. Two segments of 36 bp from - 83- - 48 bp of the 6 globin gene promoter, named WOG and MOG, were synthesized. WOG includes wild type CAAT-like box (CCAAC), while MOG includes the mutant CAAT-like box (CCAAT, -64 C→T). Results indicate that: ( i ) in erythroid cell lines MEL, K562 and Hemin induced K562, the affinity of MOG with CCAAT binding protein (CBF) and GATA-1 was greatly increased; ( ii ) in Hemin induced K562 cell line, there were another two novel specific DNA binding proteins, named C and D temporarily, besides the above two factors. The former was combined with WOG and MOG, likely indicating its relation with the increased gene expression after induction. The latter was only combined with MOG, which had possible relationship with the point mutation of - 64 C→T; ( iii ) EMSA also indicates that the suppression mechanisms of the expression of 6 globin gene is different in various periods of human developments. The result evidently supports the hypothesis that the defect CAAT-like box in human 6 globin gene contributes the main reason of its low level expression. The defect c/s-acting element CAAT-like box affects gene expression by its combination with the frans-acting element CBF and GATA-1.     

Protective effect of sodium butyrate on the cell culture model of Huntington disease

This study aimed to develop a cell culture model of Huntington disease and observe the effect of sodium butyrate on this cell culture model.Exon 1 of both a wild type and a mutant IT15 gene from the genomic DNA of a healthy adult and a patient with Hunt- ington disease was amplified and cloned into the eukaryotic expression vector pEGFP-C1.Human neuroblastoma SH-SY5Y cells were tran- siently transfected with these recombinant plasmids in the absence and presence of sodium butyrate(0.1,0.2,0.5,1.0 mmol/L).The MTT assay was used to measure cell viability.The results indicated that the N-terminal fragment of mutant huntingtin formed perinuclear and intranuclear aggregates and caused a decrease of SH-SY5Y cell viability.Sodium butyrate inhibited the decrease of SH-SY5Y cell via- bility caused by the N-terminal fragment of mutant huntingtin.This suggests that sodium butyrate has a protective effect on this cell cul- ture model of Huntington disease.     

Genetic analysis and gene cloning of a triangular hull 1 (tri1) mutant in rice (Oryza sativa L.)

Grain shape and size are two key factors that determine rice yield and quality. In the present study, a rice triangular hull mutant (tri1) was obtained from the progeny of japonica rice variety Taipei 309 treated with 60Co γ-rays. Compared to the wild type, the tri1 mutant presents a triangular hull, and exhibits an increase in grain thickness and protein content, but with a slight decrease in plant height and grain weight. Genetic analysis indicated that the mutant phenotype was controlled by a recessive nuclear gene which is stably inherited. Using a map-based cloning strategy, we fine-mapped tri1 to a 47-kb region between the molecular markers CHR0122 and CHR0127 on the long arm of chromosome 1, and showed that it co-segregates with the molecular marker CHR0119. According to the rice genome sequence annotation there are six predicated genes within the mapped region. Sequencing analysis of the mutant and the wild type indicated that there was a deletion of an A nucleotide in exon 3 of the OsMADS32 gene, which could result in a downstream frameshift mutation and premature termination of the predicted polypeptide. Both semi-quantitative and real-time RT-PCR analyses showed that this gene expressed highly in young inflorescences, while expressed at very low levels in other tissues. These results implied that the OsMADS32 gene could be a candidate of TRI1. Taken together, the results of this study lay the foundation for further investigation into the molecular mechanisms regulating rice caryopsis development.     

Sinorhizobium meliloti nifA gene exerts a pleiotropic effect on nodulation through the enhanced plant defense response

Sinorhizobium meliloti nifA gene is required for the expression of a bunch of nif and fix genes. Here, we report its pleiotropic effects on the nodule formation. Compared with wild type strain, nifA mutant sig- nificantly reduced nodule suppression rate in split-root system. The plants inoculated with mutant strain produced lower amount of daidzein and less necrotic cells on their roots. In addition, the defense genes failed to be evoked by nifA mutant at the early nodulation stage. These findings indicated that host defense response was one of the mechanisms mediated by nifA gene to regulate nodule formation during symbiosis. Even though nifA mutant could increase the number of nodules in host plant, it synthesized lower Nod factors than wild type. This suggested that nifA gene mediated multiple and diverse instances in nodulation formation.     

Magnaporthe oryzae MTP1 gene encodes a type III transmembrane protein involved in conidiation and conidial germination

In this study the MTP1 gene, encoding a type III integral transmembrane protein, was isolated from the rice blast fungus Magnaporthe oryzae. The Mtp 1 protein is 520 amino acids long and is comparable to the Ytp 1 protein of Saccharomyces cerevisiae with 46% sequence similarity. Prediction programs and MTP1-GFP （green fluorescent protein） fusion expression results indicate that Mtp 1 is a protein located at several membranes in the cytoplasm. The functions of the MTP1 gene in the growth and development of the fungus were studied using an MTP1 gene knockout mutant. The MTP1 gene was primarily expressed at the hyphal and conidial stages and is necessary for conidiation and conidial germination, but is not required for pathogenicity. The Amtpl mutant grew more efficiently than the wild type strain on non-fermentable carbon sources, implying that the MTP1 gene has a unique role in respiratory growth and carbon source use.     

Observation of fiber ultrastructure of Ligon lintless mutant in upland cotton during fiber elongation

Lintless mutant is a super-short fiber mutanl in upland cotton only 4-8 mm in fiber length and also named Ligon cotton controlled by one dominant gene Li1. Fiber ultrastructure of the mutant (Li1) and its its wild (li1) in siti and in vitro was observed under an electron microscope to understand its cytological characteristics during the fiber cell elongation. The resulls showed that the mutant fiber in situ had thinner cytoplasm, more small vacuoles, less mitochondria, Golgi apparatus and endoplasmic reticula, and there were more starch granules which were free or packed in the amyloplast beside the cell wall than thai of wild type. It was indicated that scarcity of functional organelles and disability of transformation from starch to sugar might be associated with the tact that the mutant fiber cell was aborted too early to elongate into normal length. Mutant ovule in some media containing GA3 could produce a kind of huge callus that grew faster than normal ovules. The callus was covered with many white, loose, and semitransparent fiber-like cells that apt lo get off from ovule. These fiber-like cells were multicellular fibers generated by cell division and had black dots just like pigment glands in the stem and leaf of cotton. There were lots of micro-tubes beside cytoplasm membrane uf the multiecllular fiber, which were thought to be primary preparation for second wall deposition of multicellular fiber. It was indicated that GA3 might induce the expression of gene(s) that kept inactive in the field condition and then stimulate the original fiber cell in vitro to undergo divisionagain.     

Preliminary study on a gravity－insensitive rice mutant

A gravity-insensitive mutant was isolated from rice (Oryza sativa L. cv. Zhonghua 11) transformed by Agrobacterium tumefaciens. The mutant‘s shoot growth (prostrate growth) was insensitive to gravity; whereas root growth displayed a normal positive gravitropism. Histological observation of root caps and leaf sheaths indicated that there was no significant difference in the number and size of amyloplasts in cells of the mutant and cells of the wild type.     

Morphological and physiological analysis of narrow and striped leaf 1 （nsl1） mutant of rice （Oryza sativa L.） and the gene mapping

The shape and color of rice leaves are impor- tant agronomic traits that directly influence the proportion of sunlight energy utilization and ultimately affect the yield and quality. A new mutant exhibiting stable inheritance was identified as derived from ethyl methane sulfonate （EMS）-treated restorer Jinhui 10, tentatively named as narrow and striped leaf 1 （nsll）. The nsll displayed pale white leaves at the seeding stage and then white striped leaves in parallel to the main vein at the jointing stage. Meanwhile, its leaf blades are significantly narrower than the control group of Jinhui 10. The chloroplast structures of cells in the white striped area of the nsll mutant break down, and the photosynthetic pigments are significantly lower than that of the wild type. Moreover, fluorescence parameters, such as Fo, Fv/Fm, ФpsⅡ, qP, and ETR, in the nsll mutant are significantly lower than those of the wild type, and the photosynthetic efficiency is also significantly decreased. These changes in leaf color and shape, together with physiological changes in the nsll, result in smaller plant height and a decrease in the most important agro- nomic traits, such as the number of grains per panicle, grain weight, etc. Genetic analysis shows that the narrow and striped traits of the nsll mutant are controlled by a single recessive nuclear gene, which is located between InDel 16 and InDel 12 in chromosome 3. The physical distance is 204 kb. So far, no similar genes of such leaf color and shape in this area have been reported, This study has laid asolid foundation for the gene cloning and function analysis of NSL 1.     

Clustered spikelets 4, encoding a putative cytochrome P450 protein CYP724B1, is essential for rice panicle development

Rice （Oryza sativa L.） inflorescence （panicle） architecture is an important agronomic trait, serving as one of the determinants of rice yield. A number of genes related to panicle development have been cloned and functionally characterized so far. However, more information is needed for fully understanding of the mechanism underlying the panicle development. In the present study, we identified a clustered spikelets 4 （cl4） mutant in the 93-11 genetic background. Compared to its wild-type 93-11, cl4 mutant has a typical clustered spikelets phenotype with all primary branches clustered on the base of the main rachis and 2-3 abnormal spikelets clustered on the primary branches. Moreover, cl4 mutant also shows shorter plant height than that of the wild type. Map-based cloning strategy is per- formed to clone the CL4 gene. As a result, CL4 is demonstrated to encode a putative cytochrome P450 protein CYP724B1, which is involved in brassinosteroid biosynthesis. To confirm our mapping result, the CL4 RNAi transgenic plants are generated. And the transgenic plants also show similar phenotype as the cl4 mutant. These results provide strong evidence that CL4 plays an important role in rice panicle development as well as plant height regulation.     

Analysis of Type Ⅱ Toxin-Antitoxin Genes all 3211-asl3212 in Anabaena PCC 7120

The type Ⅱ toxin-antitoxin genes are responsible for the phenotypic switch to a quasi-dormant state that enables cell survival under stresses,a similar function to heterocyst of cyanobacteria. In this paper,we particularly study the role of gene pair all3211-asl3212 under Spectinomycin stress to reveal how the type Ⅱ toxin-antitoxin involved in environmental stress responses. Bioinformatics prediction shows that toxin protein gene All3211 is homologous to Maz F,a member of maz EF family that encoding nucleases. We clone gene all3211-asl3212 into expression vectors to identify its molecular characteristics. Deletion mutant strains of all3211-asl3212 are selected in a tri-parental mating screen. Phenotype comparisons of mutant and wild type reveals no difference of single-deletion-mutants in pigment integrity,the sensitivity to antibiotics,and heterocyst formation. The results show that deletion mutation of single TAS gene pair all3211-asl3212 results in limited effects on the cellular growth of PCC 7120. Thus,we suggest that dosage compensating might be provided from redundant genes or bypass pathways to offset obvious phenotypic differences.