Identification of KASP markers and putative genes for pre-harvest sprouting resistance in common wheat(Triticum aestivum L.)

Common wheat(Triticum aestivum L.)is the most important crop in the world and a typical allopolyploid with a large and complex genome.Pre-harvest sprouting(PHS)leads to a significant reduction in grain quality worldwide.PHS is a complex trait with related QTL located on different chromosomes.However,the study of markers and genes related to PHS resistance is limited especially for whitegrained wheat.Four pairs of near isogenic lines(NILs)from a white-grained wheat cross of Chara
DM5637B*8 targeting a major QTL for PHS resistance(Qphs.ccsu-3A.1)on wheat chromosme 3AL were genotyped using the 90K SNP Illumina iSelect array.Ten SNPs were identified,with a 75%-100%consistency between genotype and phenotype in the resistant or susceptible isolines.The 10 SNPs were converted to cost-effective kompetitive allele-specific PCR(KASP)markers.Screening of 48 wheat cultivars with different phenotypes of PHS identified four KASP markers with 81.3%-85.4%conformity between genotype and phenotype.Further investigation revealed that the four SNPs(BS00022245_51,Kukri_c49927_151,BS00022884_51 and BS00110550_51)corresponding to the four validated KASP markers are residing in three independent genes(TraesCS3A03G1072800,TraesCS3A03G1072400,TraesCS3A03G1071800)close to each other with a distance of 4.28-4.48 Mb to the targeted QTL.These three annotated genes have potential functions related to PHS resistance.Our study revealed that combined use of NILs and the 90K SNP chip is a powerful approach for developing KASP markers and mining functional genes in wheat.The KASP markers for PHS resistance on chromosome 3AL are useful for high-throughput evaluation and marker-assisted selection,and the three identified genes could lead to a better understanding of the genetic pathways controlling PHS.

The relationship of Imp2 and DR3 genes with susceptibility to type Ⅰ diabetes mellitus in south China Han population

AIN To study the relationship of Imp2 and DR3genes with type Ⅰ diabetes mellitus.NETHODS Imp2 genotypes and DR3 wereidentified in 68 patients with type Ⅰ diabetesmellitus(Ⅰ-DM)and 71 healthy controls.Then,Ⅰ-DM patients and controls were respectivelyallocated into DR3-positive and DR3-negativegroups.The frequencies of Imp2 and DR3 genein random subjects,and Imp2 genotypes in DR3-matched subjects were compared between Ⅰ-DMpatients and controls.At the same time,Ⅰ-DMpatients were divided into 3 groups based on theonset age of diabetics:group A≤14 years,group B 15-30 years and group C≥31 years.RESULTS The frequency of DR3 in Ⅰ-DMpatients was significantly higher than that incontrols(47% vs 21%,P【0.005),and it wassignificantly higher in group A than that in groupB+C(70% vs 36%,x^2=7.07,P【0.01).Therewas a significant difference among groups withdifferent onset age of diabetics(x^2=8.19,rp=0.33,P【0.05).In random subjects,thefrequency of Imp2.R/R in Ⅰ-DM patients waslower(43% vs 61%,P【0.05)and Imp2.R/Hhigher(53% vs 28%,P【0.05)than that incontrols,and there was no significant differenceamong groups with different onset age ofdiabetics.In DR3-positive subjects,thefrequency of Imp2.R/R in Ⅰ-DM patients waslower(47% vs 87%,P【0.05)and Imp2-R/H higher(47% vs 13%,P【0.05)than that incontrols.In DR3-negative subjects,thefrequency of Imp2.R/H in Ⅰ-DM patients washigher than that in controls(58% vs 32%,P【0.01),but the frequency of Imp2-R/R and Imp2H/H was not significantly different betweenthese two groups.CONCLUSION DR3 gene may be one of thesusceptible genes of Ⅰ-DM,and significantlyrelated to the onset age of diabetics,and thepersons with DR3 may have an younger onsetage of diabeteS.The Imp2-R/R may be theprotective genotype of Ⅰ-DM,and Imp2-R/H thesusceptible genotype.These were not affectedby DR3 gene.Imp-2 genotypes were not relatedwith the onset age of diabetics.

Specific changes in the expression of imprinted genes in prostate cancer--implications for cancer progression and epigenetic regulation

Epigenetic dysregulation comprising DNA hypermethylation and hypomethylation, enhancer of zeste homologue 2 （EZH2） overexpression and altered patterns of histone modifications is associated with the progression of prostate cancer. DNA methylation, EZH2 and histone modifications also ensure the parental-specific monoallelic expression of at least 62 imprinted genes. Although it is therefore tempting to speculate that epigenetic dysregulation may extend to imprinted genes, expression changes in cancerous prostates are only well documented for insulin-like growth factor 2 （IGF2）. A literature and database survey on imprinted genes in prostate cancer suggests that the expression of most imprinted genes remains unchanged despite global disturbances in epigenetic mechanisms. Instead, selective genetic and epigenetic changes appear to lead to the inactivation of a sub-network of imprinted genes, which might function in the prostate to limit cell growth induced via the PI3K/Akt pathway, modulate androgen responses and regulate differentiation. Whereas dysregulation of IGF2 may constitute an early change in prostate carcinogenesis, inactivation of this imprinted gene network is rather associated with cancer progression.

An improved protein expression system for T3SS genes regulation analysis in Xanthomonas oryzae pv. oryzae

Xanthomonas oryzea pv.oryzae(Xoo)is the causal agent of bacterial blight of rice,which is a significant threat to many of rice-growing regions.The type Ⅲ secretion system(T3SS)is an essential virulence factor in Xoo.Expression of the T3SS is often induced in the host environment or in hrp-inducing medium but is repressed in nutrient-rich medium.The elucidation of molecular mechanism underlying induction of T3SS genes expression is a very important step to lift the veil on global virulence regulation network in Xoo.Thus,an efficient and reliable genetic tool system is required for detection of the T3SS proteins.In this study,we constructed a protein expression vector pH3-flag based on the backbone of pHM1,a most widely used vector in Xoo strains,especially a model strain PXO99A.This vector contains a synthesized MCS-FLAG cassette that consists of a multiple cloning site(MCS),containing a modified pUC18 polylinker,and Flag as a C-terminal tag.The cassette is flanked by transcriptional terminators to eliminate interference of external transcription enabling detection of accurate protein expression.We evaluated the potential of this expression vector as T3SS proteins detection system and demonstrated it is applicable in the study of T3SS genes expression regulation in Xoo.This improved expression system could be very effectively used as a molecular tool in understanding some virulence genes expression and regulation in Xoo and other Xanthomonas spp.

EMP-1 Promotes Tumorigenesis of NSCLC through PI3K/AKT Pathway

This study examined the role of EMP-1 in tumorigenesis of non-small cell lung carcinoma (NSCLC) and the possible mechanism. Specimens were collected from 28 patients with benign lung diseases and 28 with NSCLC, and immunohis to chemically detected to evaluate the correlation of EMP-1 expression to the clinical features of NSCLC. Recombinant adenovirus was constructed to over-express EMP-1 and then infect PC9 cells. Cell proliferation was measured by Ki67 staining. Western blotting was performed to examine the effect of EMP-1 on the PI3K/AKT signaling. Moreover, tumor xeno-grafts were established by subcutaneous injection of PC9 cell suspension (about 5×107/mL in 100 μL of PBS) into the right hind limbs of athymic nude mice. The results showed EMP-1 was significantly up-regulated in NSCLC patients as compared with those with benign lung diseases. Over-expression of EMP-1 promoted proliferation of PC9 cells, which coincided with the activation of the PI3K/AKT pathway. EMP-1 promoted the growth of xenografts of PC9 cells in athymic nude mice. It was concluded that EMP-1 expression may contribute to the development and progress of NSCLC by activating PI3K/AKT pathway.

Mutagenesis reveals that the rice OsMPT3 gene is an important osmotic regulatory factor

Plant mitochondrial phosphate transporters regulate phosphate transport and ATP synthesis. Determining whether they function in abiotic stress response process would shed light on their response to salt stress. We used the CRISPR/Cas9 gene-editing system to mutagenize two mitochondrial phosphate transporters, OsMPT3;1 and OsMPT3;2, to investigate their regulatory roles under salt stress. Two cas9(CRISPR-associated protein9)-free homozygous mutants, mpt33 and mpt30, were confirmed to be stable. Both OsMPT3;1 and OsMPT3;2 were markedly induced by salt stress, and their mutagenesis strongly inhibited growth and development, especially under salt stress. Mutagenesis sharply reduced the accumulation of ATP, phosphate, calcium, soluble sugar, and proline and increased osmotic potential, malondialdehyde, and Na^+ /K^+ ratio under salt stress. Both mutants demonstrate normal growth and development in the presence of ATP, revealing high sensitivity to exogenous ATP under salt stress. The mutants showed lowered rates of Na^+ efflux but also of K^+ and Ca^(2+) influx under salt stress. Mutagenesis of OsMPT3;2 altered the enrichment profiles of differentially expressed genes involved mainly in synthesis of secondary metabolites, metabolism of glycolysis, pyruvate, tricarboxylic acid cycle, in response to salt stress. The mutant displayed significant accumulation differences in 14 metabolites involved in 17 metabolic pathways, and strongly up-regulated the accumulation of glutamine, a precursor in proline synthesis, under salt stress. These findings suggest that the OsMPT3 gene modulates phosphate transport and energy supply for ATP synthesis and triggers changes in accumulation of ions and metabolites participating in osmotic regulation in rice under salt stress, thus increasing rice salt tolerance. This study demonstrates the effective application of CRISPR/Cas9 gene-editing to the investigation of plant functional genes.

Induction of Chondrogenesis of Adipose-derived Stem Cells by Novel Recombinant TGF-β3 Fusion Protein

Summary： A new type of TGF-β3 fusion protein with targeted therapy function was constructed, and its feasibility and target specificity of inducing chondrogenesis were investigated by transfecting LAP-MMP-mTGF-β3 gene into adipose-derived stem cells （ADSCs）. The recombinant pIRES- EGFP-MMP was constructed by inserting the sense and antisense DNA of encoding the amino acid of the synthetic MMP enzyme cutting site into the eukaryotic expression vector pIRES-EGFE LAP and mTGF-β3 fragments were obtained by using RT-PCR and inserted into the upstream and downstream of MMP from pIRES-EGFP-MMP respectively, and the recombinant plasmid of pIRES-EGFP- LAP-MMP-mTGF-β3 was constructed, which was transferred to ADSCs. The ADSCs were cultured and divided in three groups： experimental group （MMP group）, negative control group （no MMP） and non-transfection group. The morphological changes were observed microscopically, and the expression of proteoglycan and type II collagen （Col II） was detected by using Alcian blue staining and immuno- histochemistry staining at 7th, 14th and 21st day after culture. The recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3 was correctly constructed by methods of enzyme cutting and se- quencing analysis. The mTGF-β3 fusion protein was successfully expressed after transfection, and in the presence of the MMP, active protein mTGF-β3 was generated, which significantly promoted differ- entiation of ADSCs into chondrocytes and the expression of cartilage matrix. The novel fusion protein LAP-MMP-mTGF-β3 can targetedly induce differentiation of ADSCs into chondrocytes, which would open up prospects for target therapy of cartilage damage repair in future.

Are Drug Efflux Genes Present among <i>Mycobacterium tuberculosis</i>Isolates from Patients in Lagos, Nigeria?

A major challenge in the treatment of Tuberculosis (TB) is emergence of Multi-Drug Resistant Mycobacterium tuberculosis (MDRTB) strains. Efflux genes have been established to be among factors for drug resistance in Mycobacterium tuberculosis (M. tuberculosis) pulmonary infections by conferring bacterial ability to pump-out drugs from intracellular compartment, making it impossible for drugs to attain intracellular concentration lethal to the organism. There is paucity of data on the role of efflux pump in MDRTB in Nigerian strains of M. tuberculosis. Hence, the aim of this study was to detect the carriage, distribution and frequency of efflux pump genes among MDRTB and non-MDRTB isolates from participants with pulmonary tuberculosis in Lagos, Nigeria. This study was carried out on M. tuberculosis isolated from 1020 participants suspected of pulmonary tuberculosis in Lagos State, Nigeria. A total of 78 M. tuberculosis isolates were obtained from the participants suspected of pulmonary tuberculosis. Forty Eight isolates were confirmed as MDRTB and 30 non-MDRTB. Efflux pump genes were investigated in the isolates using the conventional polymerase chain reaction. Statistical analysis was carried out using the Statistical Package for Social Science (SPSS version 20) to compare the efflux pump gene results between MDRTB and non- MDRTB isolates. Different efflux genes types and frequency were detected in MDRTB and non-MDRTB isolates. Carriage of 2 or more alleles of efflux gene types Rv2486c (efpA), Rv2459c (jefA), Rv1877, Rv1002c, Rv0342, Rv2686c and drrC associated with MDR were detected. Additionally, the frequency of efflux genes alleles in MDRTB was significantly different from those in non- MDRTB isolates.

Molecular regulation mechanisms of lymphangiogenesis

Lymphangiogenesis, the growth of new lymphatic vessels, has long been regarded as a putative efficient pathway to neoplastic metastization. Recent results have shown the necessity of lymphatic molecular markers and growth factors for lymphangiogenesis. Importantly, lymphatic endothelial receptor tyrosine kinase VEGFR-3 and its ligands VEGF-C and VEGF-D play crucial roles in promoting lymphatic vascular growth both during development and in pathological conditions. Isolation of pure cultures of lymphatic and blood vascular endothelial cells and systematic characterization of their transcriptomes provide useful cell culture models and novel potential vascular markers and offer further insights into the lymphatic vascular biology. Ectopic expression of the lymphatic endothelial specific homeobox transcription factor Prox1 in blood endothelial cells results in a shift in the gene expression profile towards the lymphatic endothelial phenotype. It demonstrates the plasticity of endothelial cells and offers the possibility of transcriptional reprogramming of vascular endothelial cells as future putative therapeutic applications.

Correlation of Runt-related transcription factor gene 3 expression in osteosarcoma tissue with cell proliferation and angiogenesis

Objective:To study the correlation of Runt-related transcription factor gene 3 (RUNX3) expression in osteosarcoma tissue with cell proliferation and angiogenesis.Methods: A total of 80 patients with osteosarcoma who were treated in our hospital between February 2014 and February 2017 were collected, and the RUNX3 expression in osteosarcoma tissue and adjacent tissue were detected. According to the RUNX3 expression in tumor tissue, the patients were further divided into high RUNX3 expression group and low RUNX3 expression group, and the proliferation gene and angiogenesis gene expression were compared.Results:RUNX3, KISS-1 and RanBP9 mRNA expression in osteosarcoma tissue were significantly lower than those in adjacent tissue while VCP, Six1, S100A6, IF-1α, MMP-14, bFGF and Ang-2 mRNA expression were significantly higher than those in adjacent tissue;KISS-1 and RanBP9 mRNA expression in osteosarcoma tissue of high RUNX3 expression group were significantly higher than those of low RUNX3 expression group while VCP, Six1, S100A6, IF-1 , MMP-14, bFGF and Ang-2 mRNA expression were significantly lower than those of low RUNX3 expression group.Conclusions:The desease of RUNX3 expression in osteosarcoma tissue is one of the direct causes of increased tumor proliferation activity and strong angiogenesis.

Changes of chlorogenic acid content and its synthesis-associated genes expression in Xuehua pear fruit during development

According to synthetic pathway of plant chlorogenic acid （CGA）, the expression patterns of genes encoding enzymes that are associated with CGA synthesis were studied in normally developed Xuehua pear fruit. The study demonstrated that CGA content in peel and flesh of Xuehua pear decreased as fruit development progressed, with a higher level in peel. The expression levels of PbPAL 1, PbPAL2, PbC3H, PbC4H, Pb4CL 1, Pb4CL2, Pb4CL6, PbHC T1 and PbHC T3 genes decreased in fruit, which was consistent with the pattern of variation in CGA content. That indicated that these genes might be key genes for influencing fruit CGA synthesis in Xuehua pear. However, Pb4CL7 gene expression profile is not consistent with variation of CGA content, hence, it may not be a key gene involved in CGA synthesis.

^(103)Pd-induced apoptosis of proliferative smooth muscle cells in bile ducts of dogs:significance and effects on related genes

BACKGROUND:With the objective of developing a locally- produced radioactive stent,the present study used in vivo animal experiments to explore apoptosis of proliferative smooth muscle cells resulting from facilitation of the expression of genes caused byγ-radiation in order to prevent bile duct restenosis.We therefore explored the effects and significance ofγ-radiation on the activity of caspase-3,Fas and Bcl-2 genes in apoptosis of proliferative smooth muscle cells in the bile duct walls of dogs. METHODS:Twelve dogs were randomly divided into 2 groups(6 in each group).A postinjury bile duct stenosis model was established and radioactive 103 Pd( 103 palladium) or ordinary bile duct stents were implanted into the bile ducts.HE staining,RT-PCR and immunohistochemistry were used to detect the proliferation and apoptosis of bile duct smooth muscle cells in proliferative endomembrane and the expression of related caspase-3,Bcl-2 and Fas genes. RESULTS:The expression of caspase-3 and Fas genes in the bile duct tissues of dogs with radioactive stents was higher than that of dogs with ordinary stents.There was significant apoptosis of proliferative smooth muscle cells in the bile ducts.The expression of the Bcl-2 gene in the bile duct tissues of dogs with radioactive stents was lower than that in those with ordinary stents.There was significant apoptosis of proliferative smooth muscle cells in the dogs with low Bcl-2 gene expression. CONCLUSIONS:Radiation increases the activity of caspase-3 and Fas genes and is associated with apoptosis. The radioactive 103 Pd stent may facilitate apoptosis of proliferative smooth muscle cells in the bile ducts of dogs by activating these genes.The Bcl-2 gene expression level is correlated with the occurrence of apoptosis and the radiosusceptibility of cells.

Sequence Variations in the Bovine IGF-I and IGFBP3 Genes and Their Association with Growth and Development Traits in Chinese Beef Cattle

The objective of this study was to determine the genotype effects of the bovine insulin-like growth factor Ⅰ （IGF-Ⅰ） and its binding protein 3 （IGFBP3） genes on growth and development traits in beef cows, including 130 Chinese Simmental, 42 Nanyang, and 47 Luxi Yellow cattle. Sequence variations in the bovine IGF-Ⅰ and IGFBP3 genes were investigated by single strand conformation polymorphism （SSCP）. SSCPs were detected in 6 fragments, which is the 5＇-flanking region, the 2nd exon, the 5th exon, and the 5th intron of the IGF-1 gene, and the 2nd exon, the 3rd exon of the 1GFBP3 gene. Two polymorphisms, an A-to-G transition in the 2rid exon of the IGF-Ⅰ gene and a T-to-C transition in the 2rid exon of IGFBP3 gene were detected in 3 breeds. The allele frequencies of 2 polymorphisms were 0.0411 （A）, 0.9589 （B）, and 0.7237 （A）, 0.2763 （B）, respectively. These 2 loci were analyzed to associate with body weight, height at withers, body length, heart girth, rump width, and beef production index （BPI） at 0, 6, 12, 24, and 36-month old. The IGFBP3 locus was shown to be associated with rump width, heart girth at 24-month and 36-month. Animals with BB genotype had higher rump width （24.86 ± 0.47） cm at 24-month and （27.50 ± 0.63） em at 36-month. The heart girth was highest for the individuals with BB genotype （171.33 ± 1.84） cm and higher than those with AB genotype （166.68 ± 1.13） cm （P〈 0.05） at 36-month.

Quantitative detection of peripheral MDR genes in breast cancer before chemotherapy and their clinical significances

Objective： To explore expressions of multidrug resistance gene （MDR） product P170 and peripheral blood MDR1 mRNA in breast cancer tissue and their clinical significances. Methods： Fluorescent quantitative RT-PCR and immunohistochemistry were used to detect peripheral blood MDR1 mRNA before chemotherapy in breast cancer tissue samples from 32 cases with P170 positive （trial group） and 11 cases with P170 negative （control group）. Results： There were 14 cases （43.75%） peripheral blood MDR1 mRNA positive and 18 cases （56.25%） negative in 32 cases P170 positive breast cancer patients, while there were 6 cases （54.55%） peripheral blood MDR1 mRNA positive and 5 cases （45.45%） negative in 11 cases P170 negative breast cancer patients; there wasn＇t a significance difference with peripheral blood MDR1 mRNA express rate in P170 positive （43.75%） group and P170 negative （54.55%） group （x2 = 0.383, P 〉 0.05）. There were 12 cases （37.50%） Her-2 positive and 20 cases （62.50%） negative in P170 positive group; there were 2 cases （18.18%） Her-2 positive and 9 cases （81.82%） negative in P170 negative group; there wasn＇t a significance difference with Her-2 positive rate in P170 positive group （37.50%） and P170 negative （18.18%） group （χ^2= 1.391, P 〉 0.05）. There were 13 cases （40.63%） ER positive and 19 cases （59.37%） negative in P170 positive group; there were 7 cases （63.64%） ER positive and 4 cases （36.36%） negative in P170 negative group; there wasn＇t a significance difference with ER positive rate in P170 positive group （40.63%） and P170 negative （63.64） group （χ^2 = 1.742, P 〉 0.05）. There were 20 cases （62.50%） PR positive and 12 cases （37.50%） negative in P170 positive group; there were 4 cases （36.36%） PR positive and 7 cases （63.64%） negative in P170 negative group; there wasn＇t a significance difference with PR positive rate in P170 positive group （62.50%） and P170 negative （36.36%） group （χ^2 = 2.267, P 〉 0.05）. There were 9 cases （28.12%） with lymphaden metastasis and 23 cases （71.88%） without lymphaden metastasis in P170 positive group; there were 5 cases （45.45%） with lymphaden metastasis and 6 cases （54.55%） without lymphaden metastasis in P170 negative group; there wasn＇t a significance difference with lymphaden metastasis rate in P170 positive group （28.12%） and P170 negative （45.45%） group （χ^2 = 1.120, P 〉 0.05）. Conclusion： 1） There were possibly MDR expressing infrequently in carcinoma tissue and peripheral blood before chemotherapy. 2） FQ-RT-PCR for detecting of peripheral blood MDR1 mRNA is special, sensitive and reliable. It can be used as new molecular biology diagnostic maker dynamic detecting peripheral blood MDR1 mRNA for regulating chemotherapy proposal and elevating chemotherapy effect.

Transformation of Two VP1 Genes of O-and Asia 1-Type Foot-and-Mouth Disease Virus into Maize

The expression of antigens in transgenic plants has increasingly been used as an alternative to the classical methodologies for the development of experimental vaccines.This paper reports here the development of a novel oral immunization system for foot-and-mouth disease （FMD） in transgenic maize with two serotypes of the structural protein VP1 of the foot-and-mouth disease virus （FMDV） viz.,O-and Asia 1-type,respectively.The transgenic plantlets were identified and investigated by polymerase chain reaction （PCR）,Southern blot,and real-time PCR.Moreover,it was found that the VP1 genes in transgenic plants could be transmitted stably to the next generation through PCR detection.To our knowledge,this is the first report in an attempt to induce a protective systemic antibody response in animals by feeding the transgenic plants in which two serotypes antigen protein of FMDV expressed together.Results of the experiment provide the possibility of using plant-based vaccines as feedstuff or feedstuff additives.

Imprinting Analysis of RTL1 and DIO3 Genes and Their Association with Carcass Traits in Pigs (Sus scrofa)

Imprinted genes play significant roles in the regulation of fetal growth, development, function of the placenta and postnatal behavior in mammals, but little is known in pigs. In order to investigate the imprinting status of porcine retro-transposon like 1 （RTL1） and type 3 iodothyronine deiodinase （DIO3） genes, DNA or RNA samples of the parents and F1 animals, generated with reciprocal crosses between Large White and Meishan breeds, were isolated, and analyzed by reverse transcription polymerase chain reaction restriction fragment length polymorphism （RT-PCR-RFLP）. The results demonstrated that the RTL1 gene was paternally expressed in 10 tissues, such as the skeletal muscle, heart, spleen, liver, kidney, lung, stomach, fat, small intestine and brain, and D103 gene exhibited paternal expression in the skeletal muscle, heart, spleen, lung, stomach, and brain, in 2-month-old pigs. The association of RTL1 and DI03 with carcass traits was further analyzed in the F2 population of Large White×Meishan pigs. The statistical results showed that the R TL1 A1101G polymorphism （EU781029） was significantly associated with lean meat percentage （LMP） and fat meat percentage （FMP） （P〈0.05）, while the D103 A744C polymorphism （AY533208） was not significantly associated with any carcass traits. These results indicate that the imprinting status of RTL1 and DIO3 is well kept across the mammalian species, and porcine RTL1 may have important roles in muscle growth and fat deposition.

Genome-wide identification and expression analysis of Gossypium RING-H2 finger E3 ligase genes revealed their roles in fiber development,and phytohormone and abiotic stress responses

Background： RING H2 finger E3 ligase （RH2FE3） genes encode cysteine rich proteins that mediate E3 ubiquitin ligase activity and degrade target substrates. The roles of these genes in plant responses to phytohormones and abiotic stresses are well documented in various species, but their roles in cotton fiber development are poorly understood. To date, genome wide identification and expression analyses of Gossypium hirsutum RH2FE3 genes have not been reported. Methods： We performed computational identification, structural and phylogenetic analyses, chromosomal distribution analysis and estimated KJKs values of G hirsutum RH2FE3 genes. Orthologous and paralogous gene pairs were identified by all versus all BLASTP searches. We predicted cis regulatory elements and analyzed microarray data sets to generate heatmaps at different development stages. Tissue specific expression in cotton fiber, and hormonal and abiotic stress responses were determined by quantitative real time polymerase chain reaction （qRT PCR） analysis. Results： We investigated 140 G hirsutum, 80 G. orboreum, and evolutionary mechanisms and compared them with orthologs 89 G. roimondii putative RH2FB genes and their in Arobidopsis and rice. A domain based analysis of the G hirsutum RH2FE3 genes predicted conserved signature motifs and gene structures. Chromosomal localization showed the genes were distributed across all G hirsutum chromosomes, and 60 duplication events （4 tandem and 56 segmental duplications） and 98 orthologs were detected, cis elements were detected in the promoter regions of G hirsutum RH2FE3 genes. Microarray data and qRT PCR analyses showed that G hirsutum RH2FE3 genes were strongly correlated with cotton fiber development. Additionally, almost all the （brassinolide, gibberellic acid （GA）, indole 3-acetic acid drought, and salt）. dentified genes were up regulated in response to phytohormones （IAA）, and salicylic acid （SA）） and abiotic stresses （cold, heat, Conclusions： The genome wide identification, comprehensive analysis, and characterization of conserved domains and gene structures, as well as phylogenetic analysis, cis element prediction, and expression profile analysis of G hirsutum RH2FE3 genes and their roles in cotton fiber development and responses to plant hormones and abiotic stresses are reported here for the first time. Our findings will contribute to the genome wide analysis of putative RH2FE3 genes in other species and lay a foundation for future physiological and functional research on G hirsutum RH2FE3 genes.

CHROMOSOME 3 MAY HARBOR MULTIPLE TUMOR SUPPRESSOR GENES ASSOCIATED WITH PRIMARY GLIOBLASTOMA MULTIFORME

Objective: To investigate whether deletion of chromosome 3 is involved in the carcinogenesis of primary glioblastoma multiforme (GBM) and to localize the possible common deletion region in the aforementioned chromosome. Methods: PCR based microsatellite polymorphism analyses were performed to detect loss of heterozygosity (LOH). Twenty-three loci on chromosome 3 were examined in 20 cases of GBM. Fluorescence-labeled primers and Perkin Elmer 377 DNA Sequencer were applied. Results: 50% informative cases of GBM displayed LOH on chromosome 3. 50% of informative cases displayed LOH on 3q and 35% on 3p. 25.6% of informative loci showed LOH in our series, in which frequent LOH were observed in the chromosomal region from loci D3S1614 (42.9%) to D3S1565 (35.3%) on 3q24–27 and at loci D3S1569 (35.3%) on 3q22–23 and D3S1289 (33.3%) on 3p14.1–14.3. Conclusion: Loss of genetic material on chromosome 3 may play an important part in the tumorigenesis of GBM. The chromosomal regions from loci D3S1614 to D3S1565 on 3q24–27 and at loci D3S1569 on 3q22–23 and D3S1289 on 3p14.1–14.3 are potential sites for novel tumor suppressor genes associated with GBM.

Characterization of TaCOMT genes associated with stem lignin content in common wheat and development of a gene-specific marker

Stem lignin content(SLC) in common wheat(Triticum aestivum L.) contributes to lodging resistance. Caffeic acid 3-O-methyltransferase(COMT) is a key enzyme involved in lignin biosynthesis. Characterization of TaCOMT genes and development of gene-specific markers could enable marker-assisted selection in wheat breeding. In the present study, the full-length genomic DNA(gDNA) sequences of TaCOMT genes located on chromosomes 3 A, 3 B, and 3 D were cloned by homologous cloning. Two allelic variants, TaCOMT-3 Ba and TaCOMT-3 Bb, were identified and differed by a 222-bp insertion/deletion(InDel) in the 3′-untranslated region(3′-UTR). A co-dominant gene-specific marker based on this InDel was developed and designated as Ta COMT-3 BM. A total of 157 wheat cultivars and advanced lines grown in four environments were used to validate the associations between allelic patterns and SLC. The SLC of cultivars with TaCOMT-3 Ba was significantly(P<0.01) higher than that of those with TaCOMT-3 Bb, and the marker TaCOMT-3 BM could be effectively used in wheat breeding.

Geographical Distribution of Bacillus thuringiensis Strains and vip3 Genes in Different Climatic Zones in China

Vegetative Insecticidal Proteins(VIPs), a large family of insecticidal proteins, are produced from Bacillus thuringiensis(Bt) during the vegetative growth stage. VIPs represent the second generation of bio-insecticides that confer a wider insecticidal spectrum and have stronger activity. This work compared the geographical distribution of Bt strains and their vip3 genes in different climatic zones in China, the tropical(Hainan Province), subtropical(Guangxi Province) and temperate zones(Heilongjiang Province). A total of 156 Bt strains were isolated from 841 soil samples in Hainan Province tropical region, 356 Bt strains from 1 420 soil samples in Guangxi Province and 167 Bt strains from 1 010 soil samples in different geographical regions in Heilongjiang Province. Twenty-two out of 156 strains from tropical Hainan Province and two out of 356 from subtropical Guangxi Province were found to express vip3 genes, while vip3 genes were not expressed from temperate zone in Heilongjiang Province. Restriction Fragment Length Polymorphism(RFLP) was used to identify different types of vip3 genes that were within the same family and three fulllength vip3 genes were isolated. The genes cloned from Bacillus thuringiensis strain SL3 expressed in the transformed E. coli BL21 strain. Through SDS-PAGE, 88.6 ku insecticidal protein was expressed. The bioassays used two-instar larva of Lepidoptera insects(Spodoptera exigua and Agrotis ipsilon) were performed. The results of the bioassays showed that the protein strongly inhibited the body weight increasement on Spodoptera exigua and Agrotis ipsilon in a standard bioassay. Taken together, the results indicated that the distribution of Bt strains and vip3 genes had regional preference. Tropical and subtropical regions were the rich resources of Bt strains and vip3 genes compared with temperate region. These results would undoubtedly facilitate the studies of insecticidal proteins and expand the list of the pest-killing candidates to make fully use of the extremely rich microbial resources. The new vip3 genes isolated in the current study might also help resolve the emerging insecticidal resistance problems.