Molecular cloning, chromosomal mapping and expression analysis of disease resistance gene homologues in rice (Oryza sativa L.)

Resistance-like sequences have been amplified from first strand cDNA and genomic DNA of rice by PCR using oligonucleotide primers designed from sequence motifs conserved between resistance genes of tobacco andArabidopsis thaliana. 3 PCR clones, designatedOsr1, Osr2 andOsr3 which were 98% identical in nucleotide sequence level, have been found to be significantly homologous to known plant resistance genes and all contained the conserved motifs of NBS-LRR type resistance genes, such as P-loop, kinase2a, kinase3a and transmembrane domain.Southern hybridization revealed that rice resistance gene hornologueswere organized as a cluster in the genome. RFLP mapping using a DH population derived from anindica/japonka cross (Zhaiyeqing 8/Jingxi 17) and an RFLP linkage map assigned two copies ofOsrl and one copy ofOsr3 to the distal position of chromosome 12 where a blast resistance QTL has been mapped previously. Northern blot analysis showed thatOsrl gene was constitutively transcribed in rice leaves, shoots and roots. Further study concerning isolation of full-length cDNAs would be conducive to elucidating the functions of these genes.     

Cytogenetic Mapping of Disease Resistance Genes and Analysis of Their Distribution Features on Chromosomes in Maize

0　IntroductionMaizeisamongthemostintensivelystudiedspeciesingeneticsandoneofagronomicallythemostimportantplants.Therearemanydis easemicrobesandpeststoattackmaize,whichre sultsinlowproductionandbadquality .Withthedevelopmentofverydensegeneticmapconstruc tion ,avarietyoftheimportantdiseaseresistancegenesofmaizeincludingHelminthosporiumtur ciumPassresistancegenesHt1,Htn1andHt2 ,HelminthosporiummaydisNisikresistancegenesRhm1andRhm2 ,maizedwarfmosaicvirusresis tancegeneMdm1,wheatstreakmosaicvi…     

QTL mapping of resistance to sheath blight in maize（Zea mays L.）

Maize sheath blight (Rhizoctonia Solani) is a widely occurring fungus disease with great harm to corn-pro- ducing regions in the world. The first happening of sheath blight in China was reported in Jilin Province as early as in 1966[1]. Since the 1970s, the enlargement of corn- growing regions, the application of maize hybrids, the increasing use of fertilizers, especially the nitrogenous fertilizer, and a higher growth-density, all have caused a quick spread of sheath blight, the occurring …     

A novel tobacco gene coding for a product similar to bacterial two-component regulators

The two-component signaling system has been studied in bacteria. It takes part in signal transduction of adaptive behavior. Recent studies have shown that a similar two-component system is also present in eukaryotes. Examples of this areETRl andCKLl genes which may involve the signal transduction of plant hormone ethylene and cytokinin respectively. The cloning and characterization of a novel gene (NTHKl) fragment from tobacco are presented. Its partial sequence codes for a product which shows similarity to many two-component signaling proteins. Southern blot analysis indicated that there are 2 to 3 copies ofNTHKl gene in tobacco genome (allotetraploid). Homologous genes may also exist in other plants such as Arabidopsis, soybean and spinach. The expression ofNTHKl gene has also been analyzed in tobacco. Further studies on the isolation of full-length cDNA ofNTHKl gene will elucidate more clearly its function in signal perception and transduction.     

Fine mapping of the rice bacterial blight resistance gene Xa-4 and its co-segregation marker

An F₂ population developed from theXa-4 near isogenic lines, IR24 and IRBB4, was used for fine mapping of the rice bacterial blight resistance gene,Xa-4. Some restriction fragment length polymorphism (RFLP) markers on the high-density map constructed by Harushima et al. and the amplified DNA fragments homologous to the conserved domains of plant disease resistance (R) genes were used to construct the genetic linkage map around the geneXa-4 by scoring susceptible individuals in the population.Xa-4 was mapped between the RFLP marker G181 and the polymerase chain reaction (PCR) marker M55. The R gene homologous fragment marker RS13 was found co-segregating withXa-4 by analyzing all the plants in the population. This result opened an approach to map-based cloning of this gene, and marker RS13 can be applied to molecular marker-assisted selection ofXa-4 in rice breeding programs.     

黑子南瓜中STK类抗病基因同源序列的克隆及序列分析

 黑子南瓜是一种具有较强抗病性的葫芦科植物,依据所克隆的植物丝氨酸/苏氨酸蛋白激酶类抗病基因中的保守氨基酸序列合成相应的简并引物,从黑子南瓜基因组DNA中通过PCR克隆到了一些STK类的R-片段,经相关软件分析发现其中之一可以编码完整的氨基酸序列,可能来源于黑子南瓜中某个抗病或抗病相关基因.该基因片段与来源于其他已知基因的相应序列比较发现同源性都比较低,属于一个新的STK类R-片段家族成员.从黑子南瓜中克隆STK类片段,将为进一步从该植物材料中获得STK类抗病基因提供新线索.     

植物抗病基因克隆的研究进展

植物抗病基因的分子生物学研究已成为一个热点，抗病基因克隆研究突飞猛进的发展展示了诱人的应用前景。分别从功能克隆、定位克隆、序列克隆和表型克隆4个方面分析讨论了植物抗病基因克隆的研究进展，指出了存在的问题及今后可能解决的策略。     

Expression ofBcl-2 in cells with different telomerase activities

Both telomerase andBcl-2 are important genes in controlling apoptosis. The activation of telomerase and the abnormal regulation ofBcl-2 are also closely related to carcinogenesis. However, little is known about the linkage between telomerase andBcl-2. The effect of activated telomerase on the expression ofBcl-2 has been investigated. It is demonstrated that in tumor and transformed cells with higher telomerase activity,Bcl-2 expression is significantly lower than that in telomerase negative or less telomerose activity cells. Further study showed that in the telomerase gene-transformed 2BS-fibroblasts,Bcl-2 expression is inhibited significantly while the exogenous telomerase catalytic subunit gene is re-expressed in fibroblasts. Results indicated that there might be a certain linkage between the expression of telomerase andBcl-2, and overexpression of exogenous telomerase gene might down regulate the expression ofBcl-2. They contributed equally to this work.     

Genome-wide analysis of nucleotide-binding site disease resistance genes in Medicago truncatula

The class of nucleotide-binding site （NBS）- Leucine-rich repeat （LRR） disease resistance genes play an important role in defending plants from a variety of pathogens and insect pests. Consequently, many NBS-LRR genes have been identified in various plant species. In this study, we identified 617 NBS-encoding genes in the Medicago truncatula genome （Mt3.5v5） and divided them into two groups, regular （490） and non-regular （127） NBS- LRR genes. The regular NBS-LRR genes were character- ized on the bases of structural diversity, chromosomal location, gene duplication, conserved protein motifs, and EST expression profiling. According to N-terminal motifs and LRR motifs, the 490 regular NBS-LRR genes were then classified into 10 types： CC-NBS （4）, CC-NBS-LRR （212）, TIR-NBS （20）, TIR-NBS-LRR （160）, TIR-NBS-TIR （1）, TIR-NBS-TIR-LRR （2）, NBS-TIR （7）, NBS-TIR-LRR （1）, NBS （10）, and NBS-LRR （73）. Analysis of the phys- ical location and duplications of the regular NBS-LRR genes revealed that the M. truncatula genome is similar to rice. Interestingly, we found that TIR-type genes are more frequently expressed than non-TIR-type genes in M. trun- catula, whereas the number of non-TIR-type regular NBS- LRR genes was greater than TIR-type genes, suggesting the gene expression was not associated with the total number of NBS-LRR genes. Moreover, we found that the phylogenetic tree supported our division of the regular NBS-LRR genes into two distinct clades （TIR-type and non-TIR-type）, but some of the non-TIR-type lineages contain TIR-type genes. These analyses provide a robust database of NBS-LRR genes in M. truncatula that will facilitate the isolation of new resistance genes and breeding strategies to engineer disease resistance in leguminous crop     

Cloning and expression of putative ethylene receptor genes in soybean plant

Ethylene plays important roles in plant growth, development, and stress responses, and ethylene receptors have been identified and studied extensively in various plant species. Here we report the cloning of four ethylene receptor genes from soybean, i.e. GmETR1, GmERS1, GmETR2 and GmEIN4. Construction of the phylogenic tree showed that GmETR1 and GmERS1 belong to subfamily I whereas GmETR2 and GmEIN4 belong to subfamily II. The four ethylene receptor genes showed different tissue-specific expression patterns in roots, stems, leaves, cotyledons, flowers, pods and seeds of soybean. These genes were differentially regulated by various abiotic stresses and plant hormones. The possible roles of the four genes in soybean plant were also discussed.     

Cloning and expression of putative ethylene receptor genes in soybean plant

Ethylene plays important roles in plant growth, development, and stress responses, and ethylene receptors have been identified and studied extensively in various plant species. Here we report the cloning of four ethylene receptor genes from soybean, i.e. GmETR1, GmERS1, GmETR2 and GmEIN4. Construction of the phylogenic tree showed that GmETR1 and GmERS1 belong to subfamily I whereas GmETR2 and GmEIN4 belong to subfamily II. The four ethylene receptor genes showed different tissue-specific expression patterns in roots, stems, leaves, cotyledons, flowers, pods and seeds of soybean. These genes were differentially regulated by various abiotic stresses and plant hormones. The possible roles of the four genes in soybean plant were also discussed.     

Mutant acetolactate synthase （ALS） gene as a selectable marker for Agrobacterium-mediated transformation of soybean

Soybean is one of the crops most difficult to be manipulated in vitro. Although several soybean marker genes, all the selectable markers used were from bacteria origin. To find suitable selectable marker gene from plant origin for soybean transformation, a mutant acetolactate synthase （ALS） gene from Arabidopsis thaliana was tested for Agrobacterium-mediated soybean embryo axis transformation with the herbicide Arsenal as the selective agent. Transgenic soybean plants were obtained after the herbicide se- lection and the To transgenic lines showed resistance to the herbicide at a concentration of 100 g/ha. ALS enzyme assay of To transgenic line also showed higher activity compared to the wild type control plant. PCR analysis of the T1 transgenic lines confirmed the integration and segregation of the transgene. Taken together, our results showed that the mutant ALS gene is a suitable selectable marker for soybean transformation.     

Generation of a recombinant herpes simplex virus which can provide packaging function for recombinant adeno-associated virus

The generation of a recombinant HSV (rHSV) that can provide packaging function for rAAV production is described. A set of cosmids including cos48, cos28, cos6, cosl4 and cos56, which represents the HSV-1 genome was used for generation of this rHSV.Rep andcap genes of AAV-2 were inserted intoXba I site ofUL2 gene on cod, generating cos6rcΔUL2. After being digested withPac I, cos6-rcΔUL2 and the other 4 cosmids were cotransfected into BHK-21 cells. The recombinant virus HSV1-rc/ΔUL2 carryingrep andcap genes was generated due to the homologous recombination of the 5 cosmids. The results showed that the existence ofrep andcap genes on this rHSV was stable from passage to passage and the rHSV could support the packaging of rAAV either in cells transiently transfected with AAV vector or in stable cell line harboring AAV vector. Further modification of this rHSV and optimization of conditions involved in rAAV preparation may lead to a large-scale production of rAAV in the near future.     

Expression of two insect-resistant genescryIA (b&c)/GNA in transgenic tobacco plants results in added protection against both cotton bollworm and aphids

The synthesizedBacillus thuringiensis insecticidal protein gene cryIA(b&c) and the synthesized geneGNA, (the mannose specific lectin from snowdrop (Galanthus nivalis)), tumefaciens have been inserted into plant expression vector pGW4BAI. Leave stripes ofNicotiana tabacum var. K326 have been transformed withAgrobacterium tumefaciens strain LBA4404 harboring the plant expression vector. 28 kanamycin resistant tobacco plants have been obtained. PCR and Southern blot analyses show that the foreigncryIA andGNA genes have been inserted into the genome of transformed tobacco plants. Haemagglutination assays show thatGNA has a functional activity. Leaf disc bioassays against cotton bollworm (H. armigera) show that the transgenic tobacco plants have a high insecticidal activity. The inhibition of aphid population in leaf disc bioassays againstMyzus persicae shows that the fecundity of aphid on transgenic plants is lower than that on untransformed plants; the aphid population on the transgenic tobacco plants is 25%–70% that on untransformed tobacco plants. ELISA analysis of ClyIA protein in tobcco leaves provides similar data to bioassay results. Through the two bioassays againstH. armigera andM. persicae, several transgenic tobacco plants showing high insect-resistant activities to both pests have been obtained.     

Search for new antiphytovirucides

A convenient method to synthesize substituted 2-(2′-hydroxyphenyl) benzimidazoles is reported. Six title compounds have been synthesized by the reaction of salicylic acid and 4-substituted0-phenylenediamine in the presence pyridine-POCl₃. Three compounds were tested as plant virucide against tobacco mosaic virus and they exhibited some activities. Supported by the National Natural Science Foundation of China, and National Laboratory of Elemento Organic Chemistry of Naikai University Huang Xiaoling: born in 1938, Professer     

Variation induced by DNA rearrangement in a transgenicBt+CpTI cotton strain

In the development of transgenicBt + CpTI cotton cultivars, one male and female sterile mutant has been found in a homozygous T₄ strain in our laboratory. The mutant plant, as well as its leaves, buds and flowers, is only 1/2–1/3 as large as that of the wild transgenicBt + CpTI bivalant cotton plants. Cytological observation found that the chromosome number of the mutant is 2n = 52; however, there are 4–8 univalents observed in meiosis I of pollen mother cells. Laboratory bioassay indicated that the mutant was highly resistant to bollworm as the wild plants. PCR amplification revealed thatBt andCpTI genes in the mutant were still intactly inserted. However, small deletion of flanked area had been observed in the mutant by Southern blotting analysis. So it is proposed that the mutant phenotype might result from either the DNA deletion or T-DNA transferring in plant genome. No such report has been presented that the rearrangement of chromosome structure in a homozygous transgenic line occurred. Further analysis is ongoing.     

不结球白菜种质创新及资源信息管理研究进展

对不结球白菜(Brassica campestris ssp. chinensis Makino)抗性育种、细胞质雄性不育、基因克隆和转基因育种、遗传图谱和DNA分子标记辅助育种,以及种质资源信息管理进行了综述,以期为不结球白菜种质创新和管理提供参考和建议.