Transferring Translucent Endosperm Mutant Gene Wx-mq and Rice Stripe Disease Resistance Gene Stv-bi by Marker-Assisted Selection in Rice (Oryza sativa)

A high-yielding japonica rice variety, Wuyunjing 7, bred in Jiangsu Province, China as a female parent was crossed with a Japanese rice variety Kantou 194, which carries a rice stripe disease resistance gene Stv-b＇ and a translucent endosperm mutant gene Wx-mq. From F2 generations, a sequence characterized amplified region （SCAR） marker tightly linked with Stv-b＇ and a cleaved amplified polymorphic sequence （CAPS） marker for Wx-mq were used for marker-assisted selection. Finally, a new japonica rice line, Ning 9108, with excellent agronomic traits was obtained by multi-generational selection on stripe disease resistance and endosperm appearance. The utilization of the markers from genes related to rice quality and disease resistance was helpful not only for establishing a marker-assisted selection system of high-quality and disease resistance for rice but also for providing important intermediate materials and rapid selection method for good quality, disease resistance and high yield in rice breeding.

A Mutant Gene Found to Be the Pathogenic Origin of Infantile Cataract

According to a report in the June 24 issue of Nature Genetics, mutations in a gene named heat-shock transcription factor 4 (HSF4) have been discovered to be responsible for lamellar and Marner cataract. Experts believe that this will open new horizons for revealing the pathogenic origin of congenital cataract. ……

Genetic Analysis and Molecular Mapping of a Novel Chlorophyll-Deficit Mutant Gene in Rice

A rice etiolation mutant 824ys featured with chlorophyll deficiency was identified from a normal green rice variety 824B. It showed whole green-yellow plant from the seedling stage, reduced number of tillers and longer growth duration. The contents of chlorophyll, chlorophyll a, chlorophyll b and net photosynthetic rate in leaves of the mutant obviously decreased, as well as the number of spikelets per panicle, seed setting rate and 1000-grain weight compared with its wild-type parent. Genetic analyses on F1 and F2 generations of 824ys crossed with three normal green varieties showed that the chlorophyll-deficit mutant character was controlled by a pair of recessive nuclear gene. Genetic mapping of the mutant gene was conducted by using microsatellite markers and F2 mapping population of 495R/824ys, and the mutant gene of 824ys was mapped on the short arm of rice chromosome 3. The genetic distances from the target gene to the markers RM218, RM282 and RM6959 were 25.6 cM, 5.2 cM and 21.8 cM, respectively. It was considered to be a new chlorophyll-deficit mutant gene and tentatively named as chill（t）.

Genetic Analysis and Molecular Mapping of Novel White Striped Leaf Mutant Gene in Rice

A new white striped leaf mutant wsll was discovered from Nipponbare mutated by ethyl methanesulfonate. The mutant showed white striped leaves at the seedling stage and the leaves gradually turned green after the tillering stage. The chlorophyll content of wsll was significantly lower than that of wild-type during the fourth leaf stage, tillering stage and booting stage. The numbers of chloroplast, grana and grana lamella were reduced and the thylakoids were degenerated in wsll compared with wild type. Genetic analysis showed that the wsll was controlled by a single recessive gene. Molecular mapping of the wsll was performed using an F2 population derived from wsll/Nanjing 11. The wsll was finally mapped on the telomere region of chromosome 9 and positioned between simple sequence repeat markers RM23742 and RM23759 which are separated by approximately 486.5 kb. The results may facilitate map-based cloning of wsll and understanding of the molecular mechanism of the regulation of leaf-color by WSL1 in rice.

Morphological Structure and Genetic Mapping of New Leaf-Color Mutant Gene in Rice (Oryza sativa)

Leaf-color mutations are a widely-observed class of mutations, playing an important role in the study of chlorophyll biosynthesis and plant chloroplast structure, function, genetics and development. A naturally-occurring leaf-color rice mutant, Baihuaidao 7, was analyzed. Mutant plants typically exhibited a green-white-green leaf-color progression, but this phenotype was only expressed in the presence of a stress signal induced by mechanical scarification such as transplantation. Prior to the appearance of white ~eaves, mutant plant growth, leaf color, chlorophyll content, and chloroplast ultrastructure appeared to be identical to those of the wild type. After the changeover to white leaf color, an examination of the mutated leaves revealed a decrease in total chlorophyll, chlorophyll a, chlorophyll b, and carotenoid content, a reduction in the number of chloroplast grana lamella and grana, and a gradual degradation of the thylakoid lamellas. At maturity, the mutant plant was etiolated and dwarfed compared with wild-type plants. Genetic analysis indicated that the leaf mutant character is controlled by a recessive nuclear gene. Genetic mapping of the mutant gene was performed using an F2 population derived from a Baihuaidao 7 ~ Jiangxi 1587 cross. The mutant gene was mapped to rice chromosome 11, positioned between InDel markers L59.2-7 and L64.8-11, which are separated by approximately 740.5 kb. The mutant gene is believed to be a new leaf-color mutant gene in rice, and is tentatively designated as gwgl.

Fine Mapping and Cloning of Leafy Head Mutant Gene pla1-5 in Rice

We identified a leafy head mutant plal-5 （plastochron 1-5） from the progeny of japonica rice cultivar Taipei 309 treated with 60Co-γ ray irradiation. The plal-5 mutant has a dwarf phenotype and small leaves. Compared with its wild type, plal-5 has more leaves and fewer tillers, and it fails to produce normal panicles at the maturity stage. Genetic analysis showed that the plal-5 phenotype is controlled by a single recessive nuclear gene. Using the map-based cloning strategy, we narrowed down the location of the target gene to a 58-kb region between simple sequence repeat markers CHR1027 and CHR1030 on the long arm of chromosome 10. The target gene cosegregated with molecular markers CHR1028 and CHR1029. There were five predicted genes in the mapped region. The results from sequencing analysis revealed that there was one base deletion in the first exon of LOC_Os10g26340 encoding cytochrome P450 CYP78A11 in the plal-5 mutant, which might result in a downstream frame shift and premature termination. These results suggest that the P450 CYP78A11 gene is the candidate gene of PLA1-5.

Genetic Analysis and Gene Mapping of a Rice Tiller Angle Mutant tac2

Tiller angle, a very essential agronomic trait, is significant in rice breeding, especially in plant type breeding. A tiller anglo controlling 2 （tac2） mutant was obtained from a restorer line Jinhui 10 by ethyl methane sulphonate mutagenesis. The tac2 mutant displayed normal phenotype at the seedling stage and the tiller angle significantly increased at the tillering stage, A preliminary physiological research indicated that the mutant was sensitive to GA. Thus, it is speculated that TAC2 and TAC1 might control the tiller angle in the same way. Genetic analysis showed that the mutant trait was controlled by a major recessive gene and was located on chromosome 9 using SSR markers. The genetic distances between TAC2 and its nearest markers RM3320 and RM201 were 19.2 cM and 16,7 cM, respectively.

Identification and Genetic Mapping of a Lesion Mimic Mutant in Rice

A lesion mimic stripe mutant, designated as Ims1 （lesion mimic stripe 1）, was obtained from the M2 progeny of a ^60Co y-radiation treated japonica rice variety Jiahua 1. The Ims1 mutant displayed propagation type lesions across the whole growth and developmental stages. Physiology and histochemistry analysis showed that the mutant exhibited a phenotype of white stripe when grown under high temperature （30 ℃）, and the lesion mimic caused by programmed cell death under low temperature （20 ℃）. The genetic analysis indicated that this lesion-mimic phenotype is controlled by a single locus recessive nuclear gene. Furthermore, by using simple sequence repeat markers and an F2 segregating population derived from two crosses of Ims1 ×93-11 and Ims1 ×Pei＇ai 64S, the Imsl gene was mapped between markers Indel1 and MM0112-4 with a physical distance of 400 kb on chromosome 6 in rice.

In Vitro Anti-tumor Immune Response Induced by Dendritic Cells Transfected with Recombinant Adenovirus Carrying Mutant K-ras Genes

Summary： The specific anti-tumor immune response induced by mouse bone marrow dendritic cells （DCs） lransfected with recombinant adenovirus carrying mutant k-ras genes was investighted. DCs were generated from mouse bone marrow in the presence of rmGM-CSF （3.3 ng/mL） and rmIL-4 （1.3 ng/mL） and detected by FACS, and then transfecled with the recombinant adenovirus encoding mutant k ras gene. The efficacy of transfection and T cell stimulating activity of DCs were detected. CTL activity of the mice vaccinated with DCs was observed. The resuhs showed thai DCs had dendritic veiled morphology. BmDCs highly expressed B7-1（80%）, B7-2（77%）, MHC Ⅱ （70%）, CDllc （65%）, CD40 （70%） and CD54 （96%） with FACS, and no significant difference in the expression was observed before and after the transfection （P〈0.05）. The DCs transfeeled by mutant k-ras gene could significantly stimulate lymphoeytes proliferation as compared with those transfeeted by Ad e or non-modified DCs （P〈0.05）. DC vaccine transfected by mutant k-ras gene could induce CTL activity against Lewis lung cancer, but not against B16. The specific eytotoxicity against Lewis lung cancer in Ad-k-ras/12-transdueed DC group was signifieantly higher than those in the control, vector and non transfeeted DCs groups （P〈0.05）. It was concluded that special antitumor response could be induced by DCs transfected with recombinant adenovirus carrying mutant k-ras genes.

Genetic Analysis and Mapping of TWH Gene in Rice Twisted Hull Mutant

A mutant with twisted hulls was found in a breeding population of rice （Oryza sativa L.）. The mutant shows less grain weight and inferior grain quality in addition to twisted hulls. Genetic analysis indicated that the phenotype of mutant was controlled by a single recessive gene （temporarily designated as TW（H）. To map the TWH gene, an F2 population was generated by crossing the twh mutant to R725, an indica rice variety with normal hulls. For bulked segregant analysis, the bulk of mutant plants was prepared by mixing equal amount of plant tissue from 10 twisted-hull plants and the bulk of normal plants was obtained by pooling equal amount tissue of 10 normal-hull plants. Two hundred and seven pairs of simple sequence repeat （SSR） primers, which are distributed on 12 rice chromosomes, were used for polymorphism analysis of the parents and the two bulks. The TWH locus was initially mapped close to the SSR marker RM526 on chromosome 2. Therefore, further mapping was performed using 50 pairs of SSR primers around the marker RM526. The TWH was delimited between the SSR markers RM14128 and RM208 on the long arm of chromosome 2 at the genetic distances of 1.4 cM and 2.7 cM, respectively. These results provide the foundation for further fine mapping, cloning and functional analysis of the TWH gene.

Genetic Analysis and Mapping of a Thermo-sensitive White Stripe-Leaf Mutant at Seedling Stage in Rice(Oryza sativa)

A thermo-sensitive white stripe-leaf mutant （tws） was selected from the M2 progeny of a japonica variety, Jiahua 1, treated by ^60 Co γ-radiation. In comparison with the wild type parent, the mutant displayed a phenotype of white stripe on the 3rd and 4th leaves, but began to turn normal green on the 5th leaf when grown at low temperatures （20℃ and 24℃）. Furthermore, the content of total chlorophyll showed an obvious decrease in the leaves with white stripe. These results suggest that the expression of the mutant trait was thermo-sensitive and correlated with the leaf age of seedlings. The genetic analysis indicated that the mutant trait was controlled by a single recessive nuclear gene, designated as tws. In addition, by using SSR markers and an F2 segregating population derived from the cross between the tws mutant and 9311, tws was mapped between the markers MM3907 and MM3928 with a physical distance of 86 kb on dce chromosome 4.

Genetic Analysis and Molecular Mapping of Light-Sensitive Red-Root Mutant in Rice

The light-sensitive red-root mutant, designated as HG1, was newly observed from an indica rice variety, Nankinkodo, when seedlings were grown with roots exposed to natural light. The root color of the mutant began to turn slight-red when the roots were exposed to the light at the intensity of 29 ）Jmol/（m^2·s）, then turned dark-red at the light intensity of 180 pmol/（m^2·s）, suggesting that the root color of the mutant was evidently sensitive to light. Furthermore, genetic analysis showed that the character of light-sensitive red-root of the HG1 mutant was controlled by a single dominant gene, tentatively designated as Lsr. With simple sequence repeat markers, Lsrgene was located between the markers RM252 and RM303 on chromosome 4 with the genetic distances of 9.8 cM and 6.4 cM, respectively. These results could be useful for fine mapping and cloning of Lsrgene in rice.

Identification and Gene Mapping of a multi-floret spikelet 1 (mfs1) Mutant Associated with Spikelet Development in Rice

In this study, a rice spikelet mutant, multi-floret spikelet 1 （mfsl）, which was derived from ethylmethane sulfonate （EMS）- treated Jinhui 10 （Oryza sativa L. ssp. indica） exhibited pleiotropic defects in spikelet development. The mfsl spikelet displayed degenerated the empty glume, elongated the rachilla, the extra lemma-like organ and degraded the palea. Additionally, mfsl flowers produced varied numbers of inner floral organs. The genetic analysis revealed that the mutational trait was controlled by a single recessive gene. With 401 recessive individuals from the F2 segregation population, the MFS1 gene was finally mapped on chromosome 5, an approximate 350 kb region. The present study will be useful for cloning and functional analysis of MFS1, which would facilitate understanding of the molecular mechanism involved in spikelet development in rice.

An Arabidopsis embryonic lethal mutant with reducedexpression of alanyl-tRNA synthetase gene

In present paper, one of the T-DNA insertional embryonic lethal mutant of Arabidopsis is identifled and designated as acd mutant. The embryo development of this mutant is arrested in globular stage. The cell division pattern is abnormal during early embryogenesis and results in disturbed cellular differentiation. Most of mutant embryos are finally degenerated and aborted in globular stage. However, a few of them still can germinate in agar plate and produce seedlings with shorter hypocotyl and distorted shoot meristem. To understand the molecular basis of the phenotype of this mutant, the joint fragment of T-DNA/plant DNA is isolated by plasmid rescue and Dig-labeled as probe for cDNA library screening. According to the sequence analysis and similarity searching, a 936bp cDNA sequence (EMBL accession#: Y12555) from selected positive clone shows a 99.8 % (923/925bp) sequence homology with Alanyl-tRNA Synthetase (A1aRS) gene of Arabidopsis thaliana. Furthermore, the data of in sitll hybridization experiment indicate that the expression of AlaRS gene is weak in early embryogenesis and declines along with globular embryo ’development’ in this mutant.Accordingly, the reduced expression of AlaRS gene maybe closely related to the morphological changes in early embryogenesis of this lethal mutant.

Genetic Analysis and Gene Mapping of Multi-tiller and Dwarf Mutant d63 in Rice

A spontaneous mutation, tentatively named d63, was derived from the twin-seedling progenies of rice crossed by diploid SARIII and Minghui 63. Compared with wild-type plants, the d63 mutant showed multiple abnormal phenotypes, such as dwarfism, more tillers, smaller flag leaf and reduced seed-setting rate and 1000-grain weight. In this study, two F2 populations were developed by crossing between d63 and Nipponbare, d63 and 93-11. Genetic analysis indicated that d63 was controlled by a single recessive gene, which was located on the short arm of chromosome 8, within the genetic distance of 0.40 cM from RM22195. Hence, D63 might be a new gene as there are no dwarf genes reported on the short arm of chromosome 8.

Responses of Drought-Resistant Mutant vem1 to Stress and Cloning of VEM1 Gene in Arabidopsis

[ Objective] This study aimed to investigate the responses of drought-resistant mutant veml to stress and clone VEM1 gene in Arabidopsis. [ Method] A drought-resistant mutant veml was isolated from the Arabidops/s mutant pool. The germination rates of wild-type （WT） and mutant veml were detected to investigate the responses of mutant veml to mannitol, NaCl and ABA stress. [ Result] The mutant veml was resistant to mannitol and NaC1 stress but sensitive to ABA stress. VEM1 gene was cloned by Tail-PCR technology and sequenced. The sequencing result was submitted to NCBI for sequence alignment and gene mapping using BLAST. Database analysis suggested that VEM1 gene was a transposable clement gene. [ Conclusion] This study laid the foundation for functional analysis of drought-resistant gene VEM1.

Influence of the Interaction of the Mutant Inl Gene and the Type of Fruiting Branches ss on the Anatomical Features of the Stem in the Indeterminate and Determinant Forms of <i>G. hirsutum</i>L.

For the first time, a comparative analysis of the structure of the apical meristem and the node of the main shoot of two forms of the indeterminate kind Namangan-77 and the determinant line of Determinant-2 and Determinant-3 of G. hirsutum in the kidney ding phase was carried out. In the apical meristem of the indeterminate form Namangan-77 is characterized by the recessive homozygous state of the mutant gene (inlinl) and the dominant homozygous, heterozygous state of the fruiting branch gene S-s: inlinlSS, inlinlSs, inlinlss—more pronounced vegetative, vegetative, with this, this form is predominated by the continuation of first-order shoots and the monopodial branching type with unlimited apical growth, accompanied by a uniform elongation of the internodes. In the apical meristem, in the forms of the determinant lines, Determinant-2 and Determinant-3 are characterized by the dominant homozygous state of the mutant gene (InlInl) and the recessive homozygous state of the allelic gene (ss)—InlInlss—there is a generative collateral kidney in the axillary leaf axial sinus, which is why these forms of the vegetative apex of the shoot when they transit to the reproductive state turn into a floral apical meristem and a sympodial branching type what happens through the development of inflorescences. Obviously, this is a consequence of a change in the phytohormonal status in the apical part of the stem as a result of the interaction of the mutant gene Inl and gene S. The node of the main stem in all forms was also studied, and a three-beam-three-lacuna type of structure was revealed, which is a fairly persistent characterizing feature of large taxa and can be used in their taxonomy.

Genetic Analysis of Selected Mutants of Cowpea (<i>Vigna unguiculata</i>[L.] Walp) Using Simple Sequence Repeat and <i>rcb</i>L Markers

Genetic diversity evaluation of mutant lines is essential to facilitate their conservation and utility in breeding programs. Characterization of plant genotypes using morphological markers has limitations which make the procedure inefficient. Application of molecular tools for characterization and diversity assessment has been found useful to complement phenotypic evaluation of plant population. Therefore genetic diversity of some cowpea mutant lines was studied using simple sequence repeats (SSR) markers. DNA barcoding marker, ribulose-1,5-bisphosphate carboxylase(rbcL) of the chloroplast DNA (cpDNA) was also used for characterization and identification of the mutants to species level. The mean polymorphic information content (0.51) obtained from the microsatellites showed high polymorphism in accessing wide genetic diversity among the mutants and their parents. Dendrogram generated revealed 8 groups with most mutants clustered separately from their parents. Sequence analysis revealed insertions/deletions (InDels) and base substitutions as the two main classes of mutations induced in the plastid DNA of the mutants studied. The nucleotide frequencies were 26.95% (A), 34.43% (T), 24.09% (C) and 14.53% (G). A total of 61.38% AT rich region was identified, while GC rich region was found to be 38.62%. Highest rate of mutations were observed in region 3 - 4 indicating that the region is less conserved in cowpea rbcL gene. The present study proved that SSR markers are useful for the genetic diversity assessment of cowpea mutants. It also proved the efficiency of rbcL markers in mutants’ identification. The results indicate that the mutants are valuable genetic resources that have been developed to widen cowpea genetic base.

Isolation of Gene Mutation from a Pathogenicity-Enhanced Mutant of Magnaporthe oryzae

To find new genes involved in fungal pathogenicity, a mutant （B11 ） exhibiting enhanced pathogenicity was isolated from an Agrobacterium-mediated transformed Magnaporthe oryzae mutant library. Southern blotting analysis showed that T-DNA insertion in the B11 genome was a single copy. TAIL-PCR and sequence alignment analyses revealed that a putative gene locus MG01679 was interrupted by the T-DNA fragment. By using the PCR-based method, the DNA and cDNA of the mutant gene MG01679 was cloned and sequenced. The open reading frame of MG01679 includes one intron and two exons, and the coding sequence is 696 bp in length and encodes a 231 amino acid peptide. Protein similarity analysis indicated that the gene belongs to the ThiJ/Pfp I protein family, and the gene was thus designated MgThiJ1. MgThiJ1 showed 57% similarity to FOXG_09029 from Fusarium oxysporum and 54% similarity to FGSG_08979 from F. graminearum in protein sequence. MgThiJ1 gene might act as a negative regulator in vegetative growth and pathogenesis in filamentous fungi, and its specific mechanism needs to be studied further.

Antitumor activity of mutant bacterial cytosine deaminase gene for colon cancer

AIM:To evaluate bacterial cytosine deaminase(bCD) mutant D314A and 5-fluorocytosine(5-FC) for treatment of colon cancer in a mouse model.METHODS:Recombinant lentivirus vectors that contained wild-type bCD gene(bCDwt),and bCD mutant D314A gene(bCD-D314A) with green fluorescence protein gene were constructed and used to infect human colon carcinoma LoVo cells,to generate stable transfected cells,LoVo/null,LoVo/bCDwt or LoVo/bCDD314A.These were injected subcutaneously into Balb/c nude mice to establish xenograft models.Two weeks post-LoVo cell inoculation,PBS or 5-FC(500 mg/kg) was administered by intraperitoneal(i.p.) injection once daily for 14 d.On the day after LoVo cell injection,mice were monitored daily for tumor volume and survival.RESULTS:Sequence analyses confirmed the construction of recombinant lentiviral plasmids that contained bCDwt or bCD-D314A.The lentiviral vector had high ef-ficacy for gene delivery,and RT-PCR showed that bCDwt or bCD-D314A gene was transferred to LoVo cells.Among these treatment groups,gene delivery or 5-FC administration alone had no effect on tumor growth.However,bCDwt/5-FC or bCD-D314A/5-FC treatment inhibited tumor growth and prolonged survival of mice significantly(P < 0.05).Importantly,the tumor volume in the bCD-D314A/5-FC-treated group was lower than that in the bCDwt/5-FC group(P < 0.05),and bCDD314A plus 5-FC significantly prolonged survival of mice in comparison with bCDwt plus 5-FC(P < 0.05).CONCLUSION:The bCD mutant D314A enhanced significantly antitumor activity in human colon cancer xenograft models,which provides a promising approach for human colon carcinoma therapy.