Development of Hybrid Rice Variety FY7206 with Blast Resistance Gene Pid3 and Cold Tolerance Gene Ctb1

Hybrid rice Fanyou 7206（FY7206）, derived from the cross between a sterile line Fanyuan A and a restorer line Fuhui 7206, was bred by the Rice Research Institute, Fujian Academy of Agricultural Sciences, China. FY7206 was characterized by moderate blast resistance, cold tolerance, as well as wide adaptability, and high yields. The blast resistance results indicated that the frequencies of blast races in race B, race C and the total resistance frequency for FY7206 were 95.5%, 100.0% and 97.2%, respectively. The disease resistance results showed that the leaf blast grade for FY7206 was level 1 and panicle blast was level 5. The indoor spray results indicated that FY7206 was resistant to 11 isolates of Magnorpathe oryzae. The blast resistance of FY7206 might be derived from the high expression of blast resistance gene Pid3. The results for simulated cold resistance in an artificial climate chamber indicated that the cold tolerance for FY7206 was moderate at the booting and flowering stages. The cold tolerance results also indicated that FY7206 could be tolerant to temperatures as low as 10 °C at the seedling stage. The q RT-PCR results showed that the expression of cold tolerance gene Ctb1 in FY7206 was relatively high. These results suggested that FY7206 is a hybrid indica rice variety with good comprehensive characteristics, including blast resistance and cold tolerance.

A new method to identify and improve the purity of hybrid rice with herbicide resistant gene

There is a close relationship between the hy—brid rice production and seed purity.Two-linehybrid rice with higher heterosis is producedthrough the hybridization between a photo-thermo sensitive genetic male sterile(GMS) rice line and a paternal variety.But the fertili-

Breeding lines with neutral genes to improve fertility of intersubspecific hybrid rice

Since the sterility-neutral allele S~n has been incorporatedinto indica or japonica varieties, many intersubspecifichybrids have been released commercially. These hybridsshowed high heterosis, but some of them exhibited unstableseed setting rate under low temperature. When the hybridsflowered at low temperature, the fertility of female gamete

Construction and analysis of tree models for chromosomal classification of diffuse large B-cell lymphomas

AIM: To construct tree models for classification of diffuse large B-cell lymphomas (DLBCL) by chromosome copy numbers, to compare them with cDNA microarray classification, and to explore models of multi-gene, multi-step and multi-pathway processes of DLBCL tumorigenesis. METHODS: Maximum-weight branching and distancebased models were constructed based on the comparative genomic hybridization (CGH) data of 123 DLBCL samples using the established methods and software of Desper et al . A maximum likelihood tree model was also used to analyze the data. By comparing with the results reported in literature, values of tree models in the classification of DLBCL were elucidated. RESULTS: Both the branching and the distance-based trees classified DLBCL into three groups. We combined the classification methods of the two models and classified DLBCL into three categories according to their characteristics. The first group was marked by +Xq, +Xp, -17p and +13q; the second group by +3q, +18q and +18p; and the third group was marked by -6q and +6p. This chromosomal classification was consistent with cDNA classification. It indicated that -6q and +3q were two main events in the tumorigenesis of lymphoma. CONCLUSION: Tree models of lymphoma established from CGH data can be used in the classification of DLBCL. These models can suggest multi-gene, multistep and multi-pathway processes of tumorigenesis. Two pathways, -6q preceding +6q and +3q preceding+18q, may be important in understanding tumorigenesis of DLBCL. The pathway, -6q preceding +6q, may have a close relationship with the tumorigenesis of non-GCB DLBCL.

LOCALIZATION OF TYPE I AND TYPE Ⅲ PROCOLLAGEN mRNAs IN BREAST SCIRRHOUS CARCINOMA BYIN SITU HYBRIDIZATION

Scirrhous carcinoma is charactertzed by reinarkable amount of collagen fibrils, mainly type I and type III collagens. The origin of collagens is still under debate.cDNA fragments of type I and type III procollagens were subcloned into Gemini pGEM vectors to synthesize the 35Slabeled cRNA probes. By in situ hybridization, we have found the fibroblasts surrounding the tumor cells and cords contained abundent type I and type III procollagen mRNAs which decreased with the distance of fibroblasts from the tumor cells. In all freshly prepared tissues, the tumor cells also contained significant pro α1 (I) and pro α1 (III) mRNAs, but no or little pro α2 (I) mRNA. The results indicated that type I and type III collagens in human scirrhous carcinoma of breast are mainly produced by fibroblasts. Tumor cells also perticipate in the disposition of collagen fibrils, probably type I trimer and type III collagens in accordance with what was observed in biochemical

Screening and identification of binding proteins to interferon-α from a cDNA library by yeast-two hybrid system

The aim of this study is to screen proteins interacting with interferon-α （IFN-α）. The IFN-α gene was amplified by polymerase chain reaction （PCR） and cloned into pGBKT-/vector, then the resulted pGBKT-/-IFN-α vector was transformed into yeast strain AH109. The transformed yeast AH109 was mated with yeast Y187 containing liver cDNA library plasmid in 2 × YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium （SD/-Trp-Leu-His-Ade and SD/-Trp-Leu-His） con- taining X-α-gal for selection. After plasmid extraction and enzyme cutting analysis, the blue colonies were subjected to sequence analysis and the results were analyzed by bioinformatics. The results showed that IFN-α was successful cloned into the pGBKT7 vector. IFN-α was expressed and there was no selfactivation and toxicity in AH109. Thirty-four positive colonies were obtained after yeast-two hybrid technique screening. After sequence analysis, eight clones were found to have a binding effect with IFN-α protein. IFN-α was successfully cloned into the pGBKT7 vector. IFN-α protein was expressed and there was no self-activation and toxicity in AH109. Eight proteins that interacted with IFN-α, including vitronectin, fibrinogen A alpha polypeptide, HIV-1 Tat interactive protein 2, arginase, NADH dehydrogenase 1 beta subcomplex, transferrin receptor 2 alpha （TFR2）, HCC-1, alcohol dehydrogenase IB （ADHIB） have been identified as IFN-α-binding proteins.

Suppression subtractive hybridization for identifying differentially expressed genes in renal cell carcinoma

Objective To construct a renal cell carcinoma (RCC) cDNA subtractive library using suppression subtractive hybridization.Methods Polyadenylated RNA ［Poly (A)+ RNA］ was isolated from tissues of RCC and normal kidney, and single-strand cDNAs and double-strand cDNAs were synthesized in turn. RCC cDNAs were divided into two groups and ligated to the specific adaptors l and 2, and then hybridized with normal kidney cDNA twice with two rounds of suppression PCR. Second round PCR products were cloned to T/A plasmid vectors to set up the subtractive library. One hundred clones were randomly picked to perform enzyme digest analysis, and some underwent sequence analysis and Northern blot to identify RCC specifically expressed genes. SMART RACE procedure was operated to clone full length novel RCC specifically expressed genes.Results A human RCC subtractive library with high subtractive efficiency was successfully set up. The amplified library contains 350 positive clones. Random analysis of 100 clones with enzyme restriction showed that 85 plasmids in the clones contained 50-400?bp inserts. Sequence analysis was performed for 10 clones. All the 10 sequences were unknown before and derived from 6 unique, novel genes among which the cDNA insert RCC18 had five copies. Northern blot analysis showed that RCC18 cDNA was highly expressed in RCC, but no signal could be detected in normal kidney. Using SMART RACE technique, we obtained the full length of the novel gene RCC18.Conclusions The constructed cDNA subtractive library of human RCC is a highly efficient one and lays a solid foundation for large scale screening and cloning new and specific oncogenes or tumor suppressor genes of RCC. The novel specifically expressed genes provided an important clue for studying the mechanisms of occurrence and development of RCC.

Elevated interleukin-13 in patients with active lupus nephritis

Objective To investigate the significance of interleukin-13 (IL-13) in patients with active lupus nephritis (LN).Methods Ten healthy volunteers and 16 patients with active LN were included in this study. The protein level of IL-13 in plasma was examined by enzyme linked immunosorbent assay (ELISA), and gene expression of IL-13 in peripheral blood mononuclear cells (PBMCs) by reverse transcription polymerase chain reaction (RT-PCR). Expression of IL-13 mRNA in renal tissue was studied by in situ hybridization (ISH) techniques.Results The level of IL-13 in plasma and the expression of IL-13 mRNA in PBMCs were significantly higher in LN patients than those in the controls ( P ＜ 0.001 ). Increased expression of IL-13 mRNA was detected in renal tissue of active LN patients compared to those in the controls ( P ＜ 0.001 ). Analysis of the linear correlation indicated that the level of IL-13 mRNA in the tubulointerstitial area in patients with active LN correlated with the concentration of serum creatinine (Scr), the glomerular activity index (GAl),the activity index of tubulointerstitium, and the level of serum C3 ( P ＜ 0.05 for each).Conclusion The elevation of IL-13 may play an important role inthe molecular pathogenesis of active LN.

Construction of the subtractive cDNA library of injured adult and fetal rabbit skins

Objective: Early gestational mammalian fetuses possess the amazing ability to heal cutaneous wounds in a scarless fashion. Over the past years, scientists have been working to decipher the mechanisms underlying this regenerative repair. The remarkable phenotypic differences between fetal and adult healings behoves us to learn their characteristics in genetics, which represents potentially important mechanisms involved in wound repair observed in fetal versus adult tissues. In this sense, it is reasonable to construct subtractive cDNA library for future research. Methods: Middle laparotomy and hysterotomy were performed on pregnant rabbits at 20 day gestation to expose the fetal back, and a longitudinal incision through the skin was made on the back of the fetus. The traumatized fetal skin was harvested 12 hours post operation, the fetus control and traumatized adult skin specimens were taken at the same time. dscDNA was synthesized from total RNA of skin samples with SMART technology. Taking one of the three samples as Tester respectively and the other two as Drivers, we obtained 1 forward and 2 reverse hybridization products. After being amplified with selective polymerase chain reaction, the products were inserted into a vector, and then transferred into E.coli HB101. The colonies were screened afterwards. Results: The wounded fetuses were alive for a long time even after birth. Every determinant step, such as RNA isolation, cDNA synthesis, Rsa I digestion, adaptor ligation and hybridization, was well operated. Subtractive efficiency identification demonstrated that the suppression subtractive hybridization (SSH) was successful. Insertion into vector and transferring to E.coli were satisfactory. Conclusions: Instead of classic SSH, an improved SSH with 2 Drivers was applied for the experiment. Results confirmed that the improved program was reasonable and correct in both theory and practice. The subtractive cDNA library we have obtained is going to be used for future researches to reveal scarless healing related gene(s) and its (their) expression.

Hybrid retroviral vector with MCK enhancers inserted in LTR for stable and specific expression of human factor IX in skeletal muscle

Background Retroviral vectors have been widely used to introduce foreign into various target cells in vitro,thus showing relatively high systemic delivery efficiency of various transgene products. The authors investigated the stability and efficiency of skeletal muscle-specific hybrid retroviral vectors in expression of human factor IX (FIX) in vitro and iv vivo. Methods FIX cDNA in LIXSN vector was replaced with a FIX minigene containing splicing donor and splicing acceptor sequence of first intron of human FIX gene. Two copies of muscle creatine kinase enhancer (MCK,Me2) were inserted in forward or reverse orientation at NheI site of 3’ long terminal repeat (LTR),resulting in two hybrid vectors,which were designated as LMe2IXm_2SN(F) and LMe2IXm_2SN(R),respectively. The vectors were tested in vitro and in vivo for stability and muscle-specificity of factor IX expression with SCID mice. Results Muscle cells carrying vector with Me2 expressed significantly higher levels of FIX (up to 1800 ng/106.24h) than those without Me2, thus suggesting that Me2 could specifically increase expression level of FIX in muscle cells. Myoblasts transduced with LMe2IXm_2SN(R) produced much less FIX in vivo in SCID mice than LMe2IXm_2SN(F). One or two copies of Me2 sequence were deleted in myoblasts transduced with LMe2IXm2SN(R) without changing the orientation of Me2. Conclusions LTR inserted with MCK enhancers can specifically increase human FIX expression in skeletal muscle cells in vitro and in vivo,and MCK enhancer should be positioned in the same orientation as that of LTR promoter.

Identification of chromosomal imbalances in pancreatic carcinoma using comparative genomic hybridization

Objective To identify genetic abnormalities in primary pancreatic carcinoma in humans.Methods Comparative genomic hybridization (CGH) was used to investigate genomic imbalances in 27 cases of pancreatic carcinomas. Multiple deletions and gains were observed in all tumor specimens.Results Losses affecting chromosomes 9p,17p,4q and 6p and gains involving 8q,7q,3q and 1q were commonly observed.Conclusions There are multiple regions of chromosomes with changes copy number in pancreatic carcinoma. The altered chromosomal regions may contain several candidate genes which are involved in the development and progress of pancreatic carcinogenesis.