Improvement of Resistance to Rice Blast in Zhenshan 97 by Molecular Marker-aided Selection

Fungi blast is one of the most serious diseases of rice worldwide. Breeding resistant varieties have been proved to be the most effective and economical means to control the disease. This paper describes the molecular marker-assisted selection (MAS) procedure for a broad-spectrum blast resistant gene Pi1 integrated into an elite hybrid maintainer line, Zhenshan 97. A simple sequence repeat (SSR) based on molecular marker-aided selection system for Pi1 segment was established. Using a backcross population and a blast isolate F1829, Pi1 gene was mapped on the top of chromosome 11 between markers RZ536 and RM144, with a distance of 9.7 cM and 6.8 cM, respectively. Seventeen families derived from the recurrent parent Zhenshan 97 were obtained with homozygous Pi1 gene. The background of the 17 families was identified with inter simple sequence repeat (ISSR) amplification, the highest recovery of the Zhenshan 97 genetic background was 97.01% after the assay of 167 polymorphic bands.

Establishment and Application of a Multiplex PCR System for the Detection of Blast Resistance Genes Pi-ta and Pi-b in Rice

Rice blast is one of the important diseases in major rice producing areas of China. The main blast resistance genes Pi-ta and Pi-b showed broad-spectrum and durable resistance to rice blast in many rice growing areas of China, which have been widely utilized in rice breeding and commercial production. In this study, on the basis of detection and verification of the genotypes of 22 rice varieties har- boring known blast resistance genes （Pi-ta and Pi-b） and blast susceptibility genes （pi-ta and pi-b）, two multiple PCR systems for these genes were established by us- ing the functional markers of blast resistance genes Pi-ta and Pi-b as well as blast susceptibility genes pi-ta and pi-b, respectively. Specifically, multiple PCR system I could simultaneously detect blast resistance genes Pi-ta and Pi-b, while system II could detect simultaneously blast susceptibility genes pi-ta and pi-b. In addition, the genotypes of 336 high generation breeding materials were detected with these two multiple PCR systems. The results were highly consistent with those of conventional single mark detection, indicating that these two multiplex PCR systems were stable, reliable and time-saving. The established multiplex PCR systems may serve as a rapid and efficient method to identify and screen rice germplasm resources and can be applied in marker-assisted selection to polymerize multiple genes for blast resis- tance in rice breeding.

Real-Time PCR Technique and Its Application in Quantification of Plant Nucleic Acid Molecules

Real-time PCR is a closed DNA amplification system that skillfully integrates biochemical, photoelectric and computer techniques. Fluorescence data acquired once per cycle provides rapid absolute quantification of initial template copy numbers as PCR products are generated. This technique significantly simplifies and accelerates the process of producing reproducible quantification of nucleic acid molecules. It not only is a sensitive, accurate and rapid quantitative method, but it also provides an easier way to calculate the absolute starting copy number of nucleic acid molecules to be tested. Together with molecular bio-techniques, like microarray, real-time PCR will play a very important role in many aspects of molecular life science such as functional gene analysis and disease molecular diagnostics. This review introduces the detailed principles and application of the real-time PCR technique, describes a recently developed system for exact quantification of AUX/IAA genes In Arabidopsis, and discusses the problems with the real-time PCR process.

Screening and Taxonomic Status of a Highly Efficient Antifungal Strain against Cytospora chrysosperma

In order to look for Trichderma strains which have inhibiting effects on Cytospora chrysosperma, total 29 domestic and foreign Trichderma strains were screened by confrontation culture and growth rate method. The physiological ecology characteristics and taxonomic statuses of the screened highly efficient antifungal strains were studied. The results showed that the n-butanol extract from T-33 fer- mentation liquid showed the highest inhibition rate （94.2%） against mycelial growth, and its inhibition rate against spore germination was up to 100%. For the growth of T-33 strain, the optimum carbon source was glucose with optimum content of 3% （g/L）; the optimum nitrogen source was soy four with optimum content of 0.02% （g/L）; the optimum initial pH of culture medium was 6; the optimum culture temper- ature was 25 ℃. The 18S rDNA sequencing results showed that the full length of ITS sequence was 583 bp, and the T-33 strain was verified as Trichoderma viride.

Molecular Marker Assisted Selection for Fusarium Wilt Resistance Breeding in Watermelon(Citrullus lanatus)

Molecular identification on diploid and tetraploid watermelon breeding lines which were resistant to Fusarium wilt was carried out with the published dCAPS marker ＂4451_fon＂ which was closely linked with resistance gene of Fusarium wilt race 1. The results showed that all the diploid and tetraploid lines expressed as re- sistant genotype, which were defined as Fusarium wilt-resistant materials. The re- sults were consistent with that of artificial inoculation identification. Molecular identifi- cation results also indicated that the resistant lines were homozygote, and the Fusarium wilt-resistant gene would not separate or lose during the future self- crossed purification. Therefore, resistance selection would not be necessary in their progeny populations. The study results thought that dCAPS marker ＂4451_fon＂ could be applied on molecular marker assisted selection for Fusarium wilt resistance breeding in watermelon to increase breeding selection efficiency and accelerate breeding progress.

Genetic Inheritance and Molecular Marker of Clubroot Resistance Genes in Brassica campestris ssp. chinensis

Objective] This study was conducted to investigate the genetic inheritance of clubroot resistance in Chinese non-heading cabbage （Brassica campestris ssp. chinensis）. [Method] The clubroot resistance gene was introduced from a Brassica campestris ssp. pekinensis cultivar to non-heading Chinese cabbage, and the inheri-tance and molecular markers of clubroot resistance gene in parental lines, F1, F2 and BC1 of non-heading Chinese cabbage were studied through pathogen inoculation at seedling stage and ISSR-PCR. [Result] Clubroot resistance in non-heading Chi-nese cabbage was control ed by a single dominant gene. ISSR molecular markers with Bulk segregant analysis （BSA） found that primer-873 was linked to resistance gene, named CR-873, and the genetic distance between the marker and the resis-tance gene was 9.72 cM. [Conclusion] The results provide references for the molecular marker assisted breeding of non-heading Chinese cabbage.

Identification and Biological Characteristics of Blank Spot Pathogen from Lilium lancifolium

With the increasing planting area of Lilium lancifolium, the leaf fungal dis-ease of L. lancifolium is becoming more and more serious. In June and July of 2014, the excessive rainfal leads to the serious outbreak of leaf disease of L. lanci-folium. In mid-June, the wilting rate of L. lancifolium in seriously-infected field was even up to 50%-70%. In some fields, the shoots of L. lancifolium even al wilted. The pathogen was isolated from the infected leaves of L. lancifolium. Its pathogenici-ty, spore morphology, 18S rDNA sequence and biological characteristics were stud-ied. The results showed the isolated pathogen was Alternaria alternate. The lethal temperature of mycelial growth was 55 ℃. The optimum pH was 6-7. Among the tested carbon sources and nitrogen sources, the optimum carbon source was mal-tose, and the optimum nitrogen source was yeast extract.

Genetic Variation Analysis of 3D Gene and Molecular Detection of Porcine Kobuvirus in 2013-2014

[Objective] This study aimed to investigate the prevalence and variation of porcine kobuvirus （PKV） in suckling piglets in China. [Method] In 2013-2014, 224 feces samples from suckling piglets with diarrhea in 27 pig farms of five provinces in China were collected to detect 3D genes of PKV with RT-PCR method; the sequences and genetic variation of 29 PKV 3D genes were analyzed. [Result] Total positive rate of PKV in feces samples from suckling piglets with diarrhea was 65.18% （146/224）; total positive rate of PKV in pig farms was 85,2% （23/27）; nucleotide sequences and the deduced amino acid sequences of 29 PKV 3D genes shared 87.0%-100% and 92.7%-100% homologies with six PKV-related 3D sequences, respectively. [Conclusion] PKV infection is prevalent in suckling piglets in China; PKV 3D genes exhibit high diversity.

~1H and ^(13)C NMR Assignments for Amlodipine and Risperidone

Aim To investigate the NMR spectroscopy of amlodipine and risperidone.Methods 1D NMR and 2D NMR experimental techniques of gCOSY, gHSQC and gHMBC were wsed. Results Theassignments of the ~1H and ^(13) C NMR data for the two drugs were performed and confirmed by theevidence of J_(HF) and J_(CF). Conclusion The structures of amlodipine and risperidone wereconfirmed by careful analysis of regular 1D and 2D NMR spectroscopy.

Molecular Tagging of a New Resistance Gene to Maize Dwarf Mosaic Virus Using Microsatellite Markers

With joint analysis based on the parents, F 1, F 2 and backcrosses, the authors found that the resistance of the maize inbred line Huangzaosi to the maize dwarf mosaic virus strain B was conditioned by a major gene and polygene, and identified a new major gene. Bulked segregate and microsatellite analysis of a F 2 progeny from the combination of Huangzaosi×Mo17 were used to identify the resistance gene, mdm1(t), on the long arm of chromosome 6. This new resistance gene is tightly linked to and located between the microsatellite markers loci, phi077 and bnlg391. The linkage distances between phi077-mdm1(t) and mdm1(t)-bnlg391 are 4.74 centiMorgan (cM) and 6.72 cM respectively.

Sequence and Molecular Evolution Analysis of Ubiquitin Proteins Encoded by Baculoviruses

[Objective] The aim of this study was to analyze the sequence characteristics and molecular evolution of ubiquitins encoded by baculoviruses.[Methods]Clustal W software was used for multiple sequence alignment analysis,and neighbor-joining method（NJ）and maximum parsimony method（MP）were used for the construction of phylogenetic tree.[Results]The baculoviral ubiquitins showed 73%-86% sequence identity to eukaryotic ubiquitin.Two heterogeneous regions of baculoviral ubiquitins were observed：one was the residues from 15-32,the other was located from residues 53 to 60.The else parts were conserved,where many functional amino acids were also observed.Phylogenetic analysis indicated that baculoviral ubiquitins could be divided into three sub-families,including sub-family GV,sub-family I and sub-family II.The molecular evolution of baculoviral ubiquitins might be under negative selection to maintain their functional and structural stability.[Conclusion]The analysis had provided reference for the researches on functional characterization of baculoviral ubiquitins.

The Effect of As_2O_3 on the Epression of Drugresistance Molecule in Malignant Neoplasmas

Objective: To detect the action of arsenic trioxide (As_2O_3) on theexpression of Tumor drug-resistant molecule. Methods: APL cell line MR_2 resistant to all-transretin-oic acid (ATRA ) was put into research, while APL cell line NB_4 was used for control. Theim-munocytochemical assays were used to detect the expressions of P-glycoprotein (P_(gp)) andGluta-thione S-transferase ( GST) . Results: Not only the expression of P_(gp) in MR_2 cell line(30%-40%) was significantly higher than that in NB4 cell line (10%-20% ) (P < 0.001), but also theexpression of GST in MR_2 cell line (60. 4 +- 4.0 )-( 66.5 +- 4.4) was significantly higher thanthat in NB4 cell line (28.3 +- 5.6)-(31.2 +- 5. l)(P < 0.05). As_2O_3 at the concentration of0.5-2.0 μmol/l could significantly decrease the expression of P_(gp) and GSTπ, but could donothing about the expression of GSTα and GSTμ. Conclusion: The lower expression of P_(gp) andGSTπ might be the sensitive indications of frustrating drug-resistance, while GSTα and GSTμ mightnot be the case. ATRA might be the substrates of P_(gp) transmission and GSTπ catalysis .

Circulating adhesion molecules in patients with virus-related chronic diseases of the liver

AIM: In the inflammatory state, intercellular adhesion molecule-1 (ICAM-1) and vascular cellular adhesion molecule-1 (VCAM-1) play a key role in promoting migration of immunological cells from the circulation to target site.Aim of our study was to investigate soluble forms of these molecules in patients with virus-related chronic liver diseases, to assess their behavior in different pathologies and correlation with severity of liver damage.METHODS: Circulating ICAM-1 and VCAM-1 were assayed by EIA commercial kits (R&D System Co.,Abington, UK) in 23 patients with chronic active hepatitis (CH), 50 subjects affected by liver cirrhosis (LC) and 15 healthy controls comparable for sex and age. In patients, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were also detected by autoanalyzer.RESULTS: LC patients had significantly higher ICAM-1 values than CH patients (38.56±7.4 ng/mL vs 20.89±6.42 ng/mL; P＜0.001) and these ones had significantly higher values than controls (12.92±1.08 ng/mL; P＜0.001). In CH group, ICAM-1 levels were significantly related to inflammatory activity (P= 0.041) and ALT values (r= 0.77;P＜0.05). VCAM-1 values were significantly increased only in LC patients (P＜0.001) and related to severity of liver impairment.CONCLUSION: These findings suggest that the determination of serum ICAM-1 can be considered as an additional useful marker of hepatocellular necrosis and inflammatory activity in chronic hepatitis, while serum VCAM-1 is an indicator of liver fibrogenesis and severity of disease in cirrhosis.

Gallbladder cancer epidemiology, pathogenesis and molecular genetics: Recent update

Gallbladder cancer is a malignancy of biliary tract which is infrequent in developed countries but common in some specific geographical regions of developing countries. Late diagnosis and deprived prognosis are major problems for treatment of gallbladder carcinoma. The dramatic associations of this orphan cancer with various genetic and environmental factors are responsible for its poorly defined pathogenesis. An understanding to the relationship between epidemiology, molecular genetics and pathogenesis of gallbladder cancer can add new insights to its undetermined pathophysiology. Present review article provides a recent update regarding epidemiology, pathogenesis, and molecular genetics of gallbladder cancer. We systematically reviewed published literature on gallbladder cancer from online search engine Pub Med(http://www.ncbi.nlm.nih.gov/pubmed). Various keywords used for retrieval of articles were Gallbladder, cancer Epidemiology, molecular genetics and bullion operators like AND, OR, NOT. Cross references were manually searched from various online search engines(http://www.ncbi.nlm.nih.gov/pubmed,https://scholar.google.co.in/, http://www.medline.com/home.jsp). Most of the articles published from 1982 to 2015 in peer reviewed journals have been included in this review.

Serrated polyposis syndrome:Molecular,pathological and clinical aspects

Hyperplastic polyps have traditionally been considered not to have malignant potential.New pathological classification of serrated polyps and recent discoveries about the serrated pathway of carcinogenesis have revolutionized the concepts and revitalized the research in this area.Until recently,it has been thought that most colorectal cancers arise from conventional adenomas via the traditional tumor suppressor pathway initiated by a mutation of the APC gene,but it has been found thatthis pathway accounts for only approximately 70%-80% of colorectal cancer(CRC)cases.The majority of the remaining colorectal cancer cases follow an alternative pathway leading to CpG island methylator phenotype carcinoma with BRAF mutation and with or without microsatellite instability.The mechanism of carcinomas arising from this alternative pathway seems to begin with an activating mutation of the BRAF oncogene.Serrated polyposis syndrome is a relatively rare condition characterized by multiple and/or large serrated polyps of the colon.Clinical characteristics,etiology and relationship of serrated polyposis syndrome to CRC have not been clarified yet.Patients with this syndrome show a high risk of CRC and both sporadic and hereditary cases have been described.Clinical criteria have been used for diagnosis and frequent colonoscopy surveillance should be performed in order to prevent colorectal cancer.In this review,we try to gather new insights into the molecular pathogenesis of serrated polyps in order to understand their possible clinical implications and to make an approach to the management of this syndrome.

Hepatitis C virus proteins

Hepatitis C virus (HCV) encodes a single polyprotein, which is processed by cellular and viral proteases to generate 10 polypeptides. The HCV genome also contains an overlapping +1 reading frame that may lead to the synthesis of an additional protein. Until recently, studies of HCV have been hampered by the lack of a productive cell culture system. Since the identification of HCV genome approximately 17 years ago, structural, biochemical and biological information on HCV proteins has mainly been obtained with proteins produced by heterologous expression systems. In addition, some functional studies have also been confirmed with replicon systems or with retroviral particles pseudotyped with HCV envelope glycoproteins. The data that have accumulated on HCV proteins begin to provide a framework for understanding the molecular mechanisms involved in the major steps of HCV life cycle. Moreover, the knowledge accumulated on HCV proteins is also leading to the development of antiviral drugs among which some are showing promising results in early- phase clinical trials. This review summarizes the current knowledge on the functions and biochemical features of HCV proteins.

Inflammatory bowel diseases: From the mystical to the cellular and now the molecular

It is of interest in an era of increasing biomedical sophisticaton to recall that a relatively short time ago, early in the 20th century, ‘simple' ulcerative colitis was an obscure ‘medical curiosity' emerging slowly from an unknown past. Crohn's disease was yet unidentified as a separate entity although careful review of the IBD literature documented its early presence, masquerading as ‘intestinal tuberculosis'. Into the 1930s, the etiology and pathogenesis of ulcerative colitis and Crohn's disease were unknown, and investigative hypotheses were scarce. Therapeutic resources were limited and treatment was primitive. At a time of limited biomedical knowledge and minimal clinical awareness, unsubstantiated views prevailed, including ‘vague reactions to foods' (sugar,margarine, corn flakes), deficiency of a ‘protective factor'in pig intestine, and psychiatric disease.

Is diabetes a causal agent for colorectal cancer? Pathophysiological and molecular mechanisms

The possible relationship between diabetes mellitus (DM) and colorectal cancer (CRC), concerning pathophysiological and molecular mechanisms is highlighted in this review. The most recent and complete articles and developments in this particular field were thoroughly reviewed. Common risk factors, such as obesity, sedentary lifestyle, and Western diet between DM and CRC, led to the theory that DM might be a causal agent for CRC development. Various studies have connected type 2 DM and CRC, either proximal or distal, in both sexes. Additionally, chronic insulin treatment has been linked with increased colorectal tumor risk among type 2 diabetic patients. Interestingly, elevated hemoglobin A1c has been proven to be an independent predictor of aggressive clinical behavior in CRC patients. These mechanisms include the insulin-like growth factor-hyperinsulinemia theory and the participation of oncogenic intracellular signaling pathways. Furthermore, it has been proposed that Cox-2 inhibitors might have a role in decreasing the incidence of CRC. Finally, the use of statins to reduce the risk for colon cancer in patients with diabetes has remained controversial. Diabetic patients over 50 should receive counseling regarding their elevated risk for CRC, and screening colonoscopy should be recommended before initiating insulin therapy. However, there are no current guidelines, and this strategy is not yet applicable to some countries, as the corresponding risk would not allow screening colonoscopy to be adopted. There is strong evidence to indicate that DM is a causal agent for CRC development. This conclusion provides new impetus for re-evaluating CRC screening worldwide.

Carcinoma of the gastroesophageal junction in Chinese patients

Carcinoma of the gastroesophageal junction(GEJ) is defined as carcinoma that crosses the GEJ line,irrespective of where the tumor epicenter is located.This group of cancer is rare but controversial.Based on study results from the majority of epidemiologic and clinicopathologic investigations carried out in Western countries,this cancer is believed to arise from Barrett's esophagus(BE) and includes both distal esophageal and proximal gastric carcinomas because of similar characteristics in epidemiology,clinicopathology,and molecular pathobiology in relation to BE.As such,the most recent American Joint Committee on Cancer staging manual requires staging all GEJ carcinomas with the rule for esophageal adenocarcinoma(EA).This mandate has been challenged recently by the data from several studies carried out mainly in Chinese patients.The emerging evidence derivedfrom those studies suggests:(1) both BE and EA are uncommon in the Chinese population;(2) almost all GEJ cancers in Chinese arise in the proximal stomach and show the features of proximal gastric cancer,not those of EA;(3) application of the new cancer staging rule to GEJ cancer of Chinese patients cannot stratify patients' prognosis effectively;and(4) prognostic factors of GEJ cancer in Chinese are similar,but not identical,to those of EA.In conclusion,the recent evidence suggests that GEJ cancer in Chinese shows distinct clinicopathologic characteristics that are different from EA.Further investigations in molecular pathology may help illustrate the underlying pathogenesis mechanisms of this cancer in Chinese patients and better manage patients with this fatal disease.