Generating of rice OsCENH3-GFP transgenic plants and their genetic applications

In order to investigate rice functional centromeres, OsCENH3-GFP chimeric gene was constructed and transformed into the indica rice variety, Zhongxian 3037, mediated by Agrobacturium. The integration of the exogenous genes in the transgenic plants was confirmed by PCR and Southern blotting. The transgenic plants grow normally during their whole life time, just like Zhongxian 3037. No significant defects were detected in either mitosis or meiosis of the transgenic plants. The overlapping of GFP signals and anti-CENH3 foci in both mitotic and meiotic cells from T0 and T1 generation plants indicated that GFP had been successfully fused with CENH3, so the GFP signals can well represent the CENH3 locations on each chromosome. To evaluate the applicability of the transgenic plants to other genetic studies, fluorescence in situ hybridization （FISH） using rice centromeric tandem repetitive sequence CentO as the probe was conducted on the zygotene chromosomes of pollen mother cells （PMCs）. It has been revealed that the GFP signals are overlapping with CentO FISH signals, showing that CentO is one of the key elements constituting rice functional centromeres. Immunofluorescent staining using anti-o-tublin antibody and anti-PAIR2 antibody on the chromosomes during mitosis and meiosis stages of the transgenic plants further reveals that OsCENH3-GFP transgenic plants can be widely used for studying rice molecular biology, especially for tagging functional centromeres in both living cells and tissues.     

Physical location ofHelminthosporium carbonum susceptibility gene hm1 by FISH of a RFLP marker umc119 in Maize

A fluorescencein situ hybridization (FISH) procedure was adopted to physically map a RFLP marker, umc119 near the centromere of the long arm of linkage group1 in maize. The hm1 gene (Helminthosporium carbonum susceptibility gene) was linked closely with the marker umc119. RFLP markers are very good landmarks for mapping genes. Therefore, we also determined the position of the gene hm1 on the chromosome based on the physical location of umc119. The disease induced by infection ofHelminthosporium carbonum is one of the serious maize diseases and it distributes in many countries including China. Hybridization sites were showed on 1 L (long arm of chromosome1) and 5 L. The percentage distance from centromere to the hybridization site was 22.86 on 1 L and 58.23 on 5 L the detection rate was about 12% for mitotic cells. In interphase nuclei five hybridized sites were detected. It demonstrated that umc119 was multiplicated sequences. FISH has more advantages overin situ hybridization (ISH) detected by DAB for increasing the detection ratio and contrast between chromosomes and hybridization signals. The ability to detect the hybridization signal of a small low copy DNA sequence is a very important key towards wide application of FISH for plant genome mapping. Supported by the National Natural Science Foundation and Doctorate Vesting Point Foundation of the Education Committee of China Li Lijia: born in 1967. Ph. D.     

利用粳稻基因组DNA和C_ot-1 DNA探针对普通野生稻和亚洲栽培稻的比较分析

以粳稻日本晴基因组DNA和Cot-1 DNA为探针,分别对日本晴、籼稻广陆矮4号和普通野生稻的染色体组进行了基因组原位杂交(GISH)和Cot-1 DNA荧光原位杂交(FISH)分析,并对3种染色体组进行了同源聚类和比较研究.结果表明:粳稻基因组DNA和Cot-1 DNA探针信号在3种水稻染色体组中的分布状况和覆盖率相似,Cot-1 DNA的覆盖率分别为(47.13±0.18)%、(45.89±0.22)%、(44.24±0.21)%,3种水稻基因组同源性高,亲缘关系接近.Cot-1 DNA在3种水稻染色体上的杂交信号分布各有特点,中高度重复序列的变异在普通野生稻向栽培稻进化和亚洲栽培稻籼、粳分化过程中具有重要意义,中高度重复序列含量较低的2、5、8号染色体是水稻染色体组进化过程中相对活跃的成分.     

Comparative physical mapping of rice BAC clones linked to resistance genes Glh,Bph-3 and xa-5 in Oryza sativa L. and O. granulata Nees et Arn. ex Watt.

Oryza granulata Nees et Arn. ex Watt. is one of the three wild relatives of rice,which are the most valuable for study and utilization in China.In this study,the homology and physical locations of three rice resistance genes,Glh,Bph-3 and xa-5 are comparatively analyzed between O.sativa and O. granulata by Southern blotting and fluorescence in situ hybridization (FISH).The results of Southern blotting indicate that there exist homologous sequences of the tested RFLP markers in O. granulata.By using three bacterial artificial chromosome (BAC) clones scanned by the tested RFLP as probes, FISH signals are detected on both mitotic and pachytene chromosomes in O.sativa and O.granulata.Dual-color FISH demonstrates that two of the three BAC clones (14E16 and 38J9) are located on the short arm of the same chromosome pair in O. granulata. Additionally, colinearity is shown for the two clones between O.sativa and O.granulata. Another BAC clone 44B4 is located on the end of the short arm of other chromosome pair in these two species.Although the phylogenetic relationship between O.sativa and O.granulata is the most distinct in Oryza and these two species have evidently different biological features and ecological habits, the relative lengths and arm ratios of the detected chromosomes and the relative positions of the tested clone signals on chromosomes in O. granulata are quite similar to those in O. sativa.     

Physical location of the ricePi-5(t), Glh andRTSV genes by ISH of BAC clones

Mapping of low or single-copy sequences on plant chromosomes has proven difficult because of very low frequency of signal detection. Rice BAC library is being used widely in rice genome research due to its distinctive advantages over other library systems. In this study, two biotin-labeled rice BAC clones closely linked to a rice blast resistance, green leafhopper resistance and tungro spherical virus resistance gene,Pi-5(t), Glh, RTSV, werein situ hybridized to rice chromosomes. They were located on the long arm and short arm of chromosome 4 with FL value of 40% and 100% respectively. The frequency of signal detection reached 46.8% and 59.2%. The signal location were consistent with the selective marker on rice saturated molecular map. The results demonstrated the advantages to locate BAC clones to chromosomes byin situ hybridization and will facilitate the rice low or single-copy gene location by using the BAC library. Supported by the National Natural Science Foundation of China and the Doctorate Vesting Point Foundation of the Education Department of the People's Republic of China Yan Huimin: born in 1964, Lecturer     

Physical location of riceGm-6, Pi-5(t) genes inO. officinalis with BAC-FISH

A fluorescence in situ hybridization (FISH) procedure was adopted to physically map two rice BAC clones 24E21 and 4F22 linked to Gm-6 and Pi-5(t) in O. offi-cinalis. FISH results showed that the two BAC clones were located at 4L. The percentage distance from the centromere to the hybridization sites was 72 + 2.62 for 24E21 and 54+ 5.43 for 4F22, the detection rates were 52.70% and 61.2%. The results obtained from the BAC and plasmid clones, RG214 and RZ565 of cultivated rice and O. officinalis were the same. This suggested that the markers, RG214 and RZ565 of cultivated rice and O. officinalis were in the same BAC clones. The homologous sequences of Gm-6 and Pi-5(t) in O. officinalis were positions that signals existed on the 4L. Many signals were observed when no Cot-1 DNA blocked. This also showed that repetitive sequences were some ho-molgous between cultivated rice and O. officinalis. The identification of chromosome 4 of O. officinalis is based on Jena et al. (1994). In our study, we discussed the possibility of physical map in O. officinalis with rice BAC clones.     

Comparative analysis of A, B, C and D genomes in the genus Oryza with Cot-1 DNA of C genome

Fluorescence in situ hybridization （FISH） was applied to somatic chromosomes preparations of Oryza officinalis Wall. （CC）, O. sativa L. （AA）xO. officinalis F1 hybrid （AC）, backcross progenies BC1 （AAC and ACC）, O. latifolia Desv. （CCDD）, O. alta Swallen （CCDD） and O. punctata Kotschy （BBCC） with a labelled probe of Cot-1 DNA from O. officinalis. In O. officinalis, the homologous chromosomes showed similar signal bands probed by Cot-1 DNA and karyotype analysis was conducted based on the band patterns. Using no blocking DNA, the probe identified the chromosomes of C genome clearly, but detected few signals on chromosomes of A genome in the F1 hybrid and two backcross progenies of BC1. It is obvious that the highly and moderately repetitive DNA sequences were considerably different between C and A genomes. The chromosomes of C genome were also discriminated from the chromosomes of Dand B-genome in the tetraploid species O. latifolia, O. alta and O. punctata by Cot-1 DNA-FISH. Comparison of the fluorescence intensity on the chromosomes of B, C and D genomes in O. latifolia, O. alta, and O. punctata indicated that the differentiations between C and D genomes are less than that between C and B genomes. The relationship between C and D genomes in O. alta is closer than that of C and D genomes in O. latifolia. This would be one of the causes for the fact that both the genomes are of the same karyotype （CCDD） but belong to different species. The above results showed that the Cot-1 DNA had a high specificity of genome and species. In this paper, the origin of allotetraploid in genus Oryza is also discussed.     

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.     

抗除草剂旱稻转基因植株的获得

以旱稻丹粳旱５－５５、６－２４、６－３７的悬浮细胞及未成熟胚为受体材料，采用基因枪法将含ＢＡＲ基因的ｐＤＭ３０２质粒ＤＮＡ导入旱稻细胞中，经ＰＰＴ筛选获得抗性愈伤组织，筛选出的愈伤组织在分化培养基上再生出完整的旱稻转基因植株。Ｓｏｕｔｈｅｒｎ分子杂交分析表明，外源ＢＡＲ基因已整合到水稻基因组内，抗除草剂试验结果表明，转化植株对０．０１％Ｂａｓｔａ（有效成分为ＰＰＴ）有一定程度的抗性，说明外源ＢＡＲ     

着丝粒串联重复序列RCS2对亚洲栽培稻和非洲栽培稻的比较分析

为了揭示中高度重复序列在同为AA基因组的亚洲栽培稻和非洲栽培稻基因组中的差异以及重复序列在.栽培稻种的分化过程中可能起到的作用,利用水稻着丝粒串联重复序列RCS2作为探针分别对籼稻广陆矮4号、粳稻日本晴和非洲栽培稻的体细胞染色体进行荧光原位杂交(FISH)实验,并对其核型进行同源性聚类和比较分析,杂交结果显示:RCS2序列位于在3种栽培稻染色体组中,RCS2序列位于每条染色体的着丝粒位置,但有不同的分布特点,表明该3种栽培稻基因组的RCS2序列有不同的进化方向.探讨了RCS2序列结合Cot-1 DNA FISH方法对水稻染色体组进行核型分析的可行性和优势.     

Tagging major quantitative trait loci for sheath blight resistance in a rice variety, Jasmine 85

This study was conducted with a clonal F₂ population of rice from a cross between Jasmine 85, a resistant variety, and Lemont, a susceptible cultivar. The rice plants belonging to each F₂ clone were divided into two plots, which were put in two replicates, respectively. Clonal parents were tested as controls. The plants were inoculated by short toothpicks incubated with RH-9, a virulent isolate of the pathogenic fungus,Rhizoctonia solani, which causes rice sheath blight. The extreme resistant and susceptible clonal lines were selected for construction of resistant and susceptible DNA pools, respectively. A total of 94 polymorphic markers evenly distributed on 12 rice chromosomes were used for bulked segregant analysis, three positive ones were found polymorphic between the two DNA pools, and three major QTLs for sheath blight resistance, Rh-2, Rh-3 and Rh-7, were identified. The three major QTLs were located on chromosomes 2, 3 and 7, and could explain 14.4%, 26.1% and 22.2% of the phenotypic variation.     

Expression of hepatitis B surface antigen gene (HBsAg) in Laminaria japonica (Laminariales, Phaeophyta)

A transformation model for Laminaria japonica was established from 1993 to 1998, on the basis of which the transgenic kelp with heterologous gene encoding hepatitis B surface antigen (HBsAg) was obtained by using the microparticle bombardment transformation method. Results of quantitative ELISA showed that HBsAg in transgenic kelp was 0.529 μg/mg soluble proteins on average and the highest value was 2.497 μg/mg, implying that recombinant HBsAg had natural epitope. Further support for the integration of HBsAg gene into kelp genome was obtained by PCR-Southern and total DNA hybridization. Prospect of kelp bioreactor producing high value materials such as edible HBV vaccine was discussed as well.     

Repetitive DNA sequences from the X chromosome of the spiny eel (Mastacembelus aculeatus)

To investigate the characters of repetitive DNA sequence in the sex chromosomes of the spiny eel (Mastacembelus aculeatus), the X chromosomal library was screened and a family of repetitive sequence, consisting of Ma 1-Ma 6, was isolated. The fluorescence in situ hybridization (FISH) result confirmed that Ma 1 - Ma 5 dispersed over sex chromosomes and all autosomes, whereas, Ma 6 is sex chromosome-specific and distributed only on the C-band positive regions of X chromosome, and Ma 6 maybe the main components of the heterochromatic regions of X chromosome. This study provides additional information about the evolution of sex chromosomes in lower vertebrates such as fish.      

Studies of Improving the Frequency of Indica Rice Transformation by Biolistic Bombardment

In order to improve the frequency of indica rice transformation by biolistic bombardment,suitable culture conditions for embryonic calli,an optimal selection scheme for resistant calli and seedling,and optimum bombardment parameters a investigated by using 14 commercially important indica rice cultivars.The main results show that the CC medium with 36g/L mannitol is a scheme subculture medium in which the browning of indica rice calli can be mitigated significantly;The concentration of 30-40mg/L Hyg or 150-200mg/L G418 or 10-20mg/L Basta is suitable for selection of resistant calli;The transformation parameters of 100μg gold powder absorbing 0.2μg DNA per shot and 900 psi helium pressure and 6 cm bombardment distance and bombarded twice for each plate give the best result;Keeping the target calli on osmotic medium containing 60g/L mannitol from 12-24h before bombardment to 24-48h after it can increase the efficiencies of transformation.Furthermore,some transgenic indica rice plants are obtained using this optimized transformation system.     

Salt tolerance of transgenic rice (Oryza sativa L.) withmtlD gene andgutD gene

Southern blot analysis indicated thatmtlD gene (encoding mannitol-1-phosphate dehydrogenase) andgutD gene (encoding glucitol-6-phosphate dehydrogenase) had been integrated into the rice genome mediated byAgrobacterium tumefaciens LBA4404(pBIGM). The expression of the above two genes in transgenic rice plants was demonstrated by Northern blot analysis and enzymatic activity assay. Analysis of sugar alcohol showed that transgenic rice plants could produce and accumulate mannitol and sorbitol. The salt tolerance of transgenic plants was much higher than that of their controls.     

转基因猪外源hDAF基因的定位

应用非同位素原位杂交技术研究转基因猪外源ｈＤＡＦ基因在受体基因组染色体上的整合及其定位,结果表明,外源基因在转基因狸中呈随机多位点整合,转基因分别定位在２Ｐ、６Ｐ＾１４、７Ｐ＾１１－Ｐ＾１３、７ｑ＾２６、１１ｑ＾１２、１３ｑ＾４４－ｑ＾４８、１４ｑ＾２６。     

Centromere inactivation in a dicentric rice chromosome during sexual reproduction

T0135 is a variant selected from the progeny of a rice line telotrisomic for the short arm of chromosome 11 （2n＋IIS＇）. Fluores- cent in situ hybridization （FISH） results indicated that T0135 contained two telocentric chromosomes, which have two centro- mere-specific molecular markers （5S rDNA） for chromosome 11; thus T0135 is a newly-described rice chromosome variant with two dicentric chromosomes, named 22＋11L-＋11L＇＋I IS.11S-＋I 1S-11S. （22 represents the 22 chromosomes excluding chromo- some 11 in the rice genome, ＂-＂ represents the centromere）. To investigate the genetic stability of the rice dicentric chromosomes during sexual reproduction, we observed the chromosome types in the progeny. Ninety-four percent of the progeny had the same chromosome type as the parental line. This result indicates that the dicentric chromosomes are mostly stable during mitosis and meiosis. Immunofluorescence analysis for centromere specific histone H3 （CENH3） revealed that only one centromere is active and the other centromere is inactivated in the rice dicentric chromosomes.     

Distribution of highly repeated DNA sequences in Haynaldia villosa and its application in the identification of alien chromatin

Haynaldia villosa (L.) is a wild relative species of common wheat that possesses many beneficial genes that can be used for wheat improvement. The accurate detection of H. villosa chromosomes in the genetic background of wheat is critical for transferring its beneficial genes to common wheat by chromosome engineering. The aim of the present study was to investigate the distribution patterns of two repeated DNA sequences, pSc119.2 and pAs1, as well as two rDNA multigene family sequences, 45S rDNA and 5S rDNA, in the individual chromosomes of H. villosa for the future precise identification of alien chromatin in germplasm development and breeding programs. A set of common wheat-H. villosa disomic addition 1V-7V lines was used to determine these specific signals on individual chromosomes of H. villosa. The results showed that two rDNA probes, pTa71 (45S rDNA) and pTa794 (5S rDNA), were located on 1VS and 5VS, respectively, and the signal could be discriminated exclusively in the common wheat background as effective markers of 1VS and 5VS. Furthermore, all seven chromosomes of H. villosa could be distinguished clearly by fluorescence in situ hybridization using pSc119.2 and pAs1 as probes in combination. The utilization of these cytogenetic markers of repetitive sequences, combined with other molecular markers sometimes, will make it possible for a precise identification of alien chromosomes with high efficiency.     

Tracing the location of powdery mildew resistance-related gene Stpk-V by FISH with a TAC clone in Triticum aestivum-Haynaldia villosa alien chromosome lines

Bacterial artificial chromosomes(BACs)or yeast artificial chromosomes(YACs)containing large inserts as probes for fluorescence in situ hybridization(FISH)have been used in the physical mapping of specific DNA sequences,especially for single-or low-copy sequences.Our earlier study identified Stpk-V,a powdery mildew resistance-related gene located on the 6VS chromosome arm of the wild grass Haynaldia villosa(tribe Triticeae),and obtained several Triticum aestivum–H.villosa alien chromosome lines carrying the Stpk-V gene.However,the precise physical location of the Stpk-V gene on chromosome 6VS is not known.In this study,we used TAC-FISH with TAC15 as the probe coupled with sequential genomic in situ hybridization(GISH)to determine the physical location of the Stpk-V gene in different T.aestivum–H.villosa 6V alien chromosome lines,including addition,substitution and translocation lines.The result indicated that the fraction length of the Stpk-V locus is 0.575±0.035 on the 6V chromosome short arm and this was confirmed by FISH using TAC15 as the probe for tracing the Stpk-V gene in other genetic stocks.The cytological mapping strategies used in this study will be of benefit for tracing the alien gene location in the course of introducing desirable traits from wild species.