Inheritance and expression of multiple disease and insect resistance genes in transgenic rice

2—3 anti-fungal disease genes are coinserted with hygromycin phosphotransferase in the same vector. Two insecticidal genes and PPT acetyl transferase genes are placed in another one. The vectors are co-delivered to rice embryonic cellus tissue at a molar ratio of 1︰1 using the particle gun method. 55 independent regenerated lines have been obtained through screening for hygromycin resistance. Of these, 70% transgenic plants harbor 6—7 foreign genes. The genes on the same vectors are always co-delivered to rice plant. Northern blot analysis has indicated that the multiple foreign genes give stable expression. In the 6 transgenic plants carrying 6—7 foreign genes, multiple foreign genes tend to integrate in 1 or 2 genetic loci. Progeny segregation is consistent with Mendel’s 3︰1 segregation law. 8 homozygous R₁ transgenic plants harboring 2—3 anti-fungal and 2 insecticidal genes are selected from large number of transgenic progeny screening for hygromycin and Basta resistance.     

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.     

JAK3 mediatesc-myc gene expression induced by interleukin-2

C myc gene expression can be rapidly induced by IL 2 through intracellular signal transduction which is triggered by the interaction between IL 2 and its receptor (IL 2R). JAK3 which associates to the intracellular domain of IL_2R γ may play a critical role in this process. To reveal the action of JAK3 in c myc induction, a chimeric receptor gene IL_2R α/γ/Δ NJAK3 is constructed which consists of extracellular domain derived from IL 2R α subunit (IL_2R α), the transmembrane sequence derived from IL_2R γ and the cytoplasmic domain derived from the catalytic domain of JAK3 (Δ NJAK3), and then transfected this chimeric gene into mouse fibroblast cell line L929 β which had been transfected with IL_2R β gene and stably expressed IL_2R β in high level. In the transfectants coexpressing IL_2R β and α/γ/Δ NJAK3, the stimulation of IL_2 could intensively induce c myc gene expression. Because the whole cytoplasmic domain of IL_2R γ which could recruit signaling molecules was replaced by JAK3 and the c myc could be still induced by IL_2 in this situation, the results here gave the direct evidence demonstrating that JAK3 plays an important role in c myc gene expression induced by IL_2.     

分子标记辅助选育含有抗稻瘟病基因和软米基因两系不育系水稻新品系

以"浦软粳S"为转育亲本,利用分子标记辅助常规育种技术成功培育出含有Pi9,Pita,Pib和Pigm稻瘟病抗性基因及软米基因(Wx~(mq)),同时表现柱头外露率高的两系不育系水稻新品系"2179S"."2179S"不育系茎秆粗壮,矮杆大穗,株高为63.8 cm,柱头外露率平均为60%.研究结果为今后培育具有稻瘟病抗性的优质两系杂交水稻新组合提供不育系亲本.     

Overexpression of rice OsLOL2 gene confers disease resistance in tobacco to Pseudomonas syringae pv. Tabaci

LSD1-related proteins of Arabidopsis with LSD1-like zinc finger domains regulate disease resistance and programmed cell death(PCD). We cloned a rice OsLOL2 gene, orthologous to LSDI of Arabidopsis and expressed it in a tobacco plant. Transgenic tobacco lines displayed enhanced disease resistance to a virulent bacterium Pseudomonas syringae pv. tabaci (Pst). RT-PCR analysis showed that overexpression of OsLOL2 in transgenic tobacco lines resulted in upregulation of two pathogenesis-related (PR) protein genes, PR2 and PR5. Our results suggest that overexpression of OsLOL2 in transgenic tobacco enhances the resistance through the induction of PR pro-teins and hypersensitive response-like reaction.     

稻瘟病抗性基因Pita2的克隆及其介导的防御相关基因表达分析

为了克隆位于水稻12号染色体着丝粒区域具有广谱抗性的稻瘟病抗性基因Pita2,研究构建并筛选了该基因供体品种PiNo.4的Fosmid文库,构建Pita2候选区域的物理图谱并互补验证了5个候选基因,克隆得到了稻瘟病抗性基因Pita2,确认了该基因就是Ptr基因.一组基因型接近、对Pita2致病性相反的稻瘟病菌菌株对水稻...     

Isolation of candidateR disease resistance genes from rice

Using a polymerase chain reaction (PCR) based method six distinct candidate disease resistant gene (R) homologs from rice have been isolated. The rice sequences are organized into two phylogenetic groups with contrasting genomic organization patterns. The first group, represented by a single sequence, Osh359-1, is more similar to non-riceR sequences than to rice ones and has a simple genomic organization. The second group, represented by Osh359-3, contains the remaining five rice sequences. Osh359-3 consists of a multi-gene family. The members of Osh359-3 family are further found to be clustered together in the genome.     

Mapping of a new gene for brown planthopper resistance in cultivated rice introgressed fromOryza eichingeri

Wild rice species is an important source of useful genes for cultivated rice improvement. Some accessions of Oryza eichingeri (2n = 24, CC) from Africa confer strong resistance to brown planthopper (BPH), whitebacked planthopper (WBPH) and bacterial blight (BB). In the present study, restriction fragments length polymorphism (RFLP) and simple sequence repeats (SSR) analysis were performed on disomic backcross plants between Oryza sativa (2n = 24, AA) and O. eichingeri in order to identify the presence of O. eichingeri segments and further to localize BPH-resistant gene. In the introgression lines, 1—6 O. eichingeri segments were detected on rice chromosomes 1, 2, 6, or/and 10. The dominant BPH resistant gene, tentatively named Bph13(t), was mapped to chromosome 2, being 6.1 and 5.5 cM away from two microsatellite markers RM240 and RM250, respectively. The transfer and localization of this gene from O. eichingeri will contribute to the improvement of BPH resistance in cultivated rice.     

Antisense expression of a rice cellular apoptosis susceptibility gene (OsCAS) alters the height of transgenic rice

Cellular apoptosis susceptibility (CAS) gene plays important roles in mitosis, development and export of importin α from the nucleus, but its function in plant is unknown. In this study, a rice CAS ortholog (OsCAS), which encodes a predicted protein of 983 amino acids with 62% similarity to human CAS, was identified. DNA gel blot analysis revealed a single copy of OsCAS in the rice genome. A 973 bp fragment at the 3′ end of OsCAS cDNA was cloned from rice cDNA library and transferred into rice in the antisense direction under the control of CaMV 35S promoter via Agrobacterium-mediated transformation method, 105 transgenic lines were obtained. Expression of OsCAS was suppressed in the antisense transgenic lines as revealed by semi-quantitative RT-PCR. The antisense transgenic lines showed dwarf phenotypes. The results indicated that OsCAS was involved in culm development of rice.     

Antisense expression of a rice cellular apoptosis susceptibility gene (OsCAS} alters the height of transgenic rice

Cellular apoptosis susceptibility (CAS) gene plays important roles in mitosis, development and export of importin a from the nucleus, but its function in plant is unknown. In this study, a rice CAS ortholog (OsCAS), which encodes a predicted protein of 983 amino acids with 62% similarity to human CAS, was identified. DNA gel blot analysis revealed a single copy of OsCAS in the rice genome. A 973 bp fragment at the 3' end of OsCAS cDNA was cloned from rice cDNA library and transferred into rice in the antisense direction under the control of CaMV 35S promoter via Agrobacterium-mediated transformation method, 105 transgenic lines were obtained. Expression of OsCAS was suppressed in the antisense transgenic lines as revealed by semi-quantitative RT-PCR. The antisense transgenic lines showed dwarf phenotypes. The results indicated that OsCAS was involved in culm development of rice.     

水稻抗条纹叶枯病分子标记在不同品种中的扩增比较

以感水稻条纹叶枯病品种＂日本晴＂、高抗品种＂kasalath＂以及14个目前在我国长三角地区水稻生产中被认为在抵抗条纹叶枯病方面具有较好效果的水稻品种为材料,分析比较了14对分子标记引物扩增16个水稻品种基因组获得的DNA产物.结果显示：在14对分子标记引物中,有8对在扩增感病品种＂日本晴＂和高抗品种＂Kasalath＂水稻基因组时可获得多态性条带.因此推断这8个标记可在以这两个品种为亲本的抗条纹叶枯病选育中使用;并且,本研究结果还将为利用本试验收集的14个水稻品种为亲本的分子标记辅助育种提供重要的背景信息.     

根际细菌诱导的系统抗性

在植物根际附近（包括根围和根内）存在大量的细菌,其中非致病性根际细菌能诱导植物产生系统抗性,称之为ISR,其表型与病菌诱导的系统获得性抗性（SAR）相似,根际细菌调控诱导的系统抗性对许多植物如拟南芥、豆类、黄瓜等上的真菌、细菌、病毒有抑制作用。诱导细菌和挑战病菌在空间上是相对隔离的,ISR的信号途径需要茉莉酸（JA）和乙烯的作用,ISR不产生PRs。在大田条件下ISR也是有效的,这就提供了一个植物病害生物防治的自然机制。     

水稻Os05g0442400基因启动子分析以及由Os05g0442400基因启动子引导GUS报告基因在转基因水稻中的表达

采用Plant CARE和PLACE软件分析预测水稻Os05g0442400基因启动子序列中可能存在的顺式作用元件.结果显示,在起始密码子ATG上游1500bp区域内,除了启动子基本的核心作用元件外,还存在一些与植物抵御非生物胁迫过程有关的作用元件、脱落酸应答元件、光诱导启动子作用元件、病原菌诱发因子作用元件,以及根特异性结合位点.采用根癌农杆菌介导法成功将Os05g0442400 promoter::gus构建导入"中花11"水稻.由不同组织部位GUS染液检测表明:在水稻苗期,内源Os05g0442400基因可能主要在根部表达;随着水稻生殖期的延续,水稻内源Os05g0442400基因在颖壳中的表达区域由上向下面积增大,并在抽穗后达到最大表达区域.这些结果可能与Os05g0442400基因启动子上分别存在根特异性结合位点和光诱导启动子作用元件具有一定的联系.     

Resistance to rice blast (Pyricularia oryzae) caused by the expression of trichosanthin gene in transgenic rice plants transferred through agrobacterium method

The gene of trichosanthin has been transferred into rice plants through agrobacterium method. The single copy insertion and the expression of foreign gene have been proved in regenerated plants. In antifungal assay the degrees of rice blast (Pyricularia oryzae) infection of the transgenic plants expressing trichosanthin and expressingGUS gene as control have been evaluated. The differences such as the time of disease symptom observed, the number of infected plants and damaged leaves, the growth of infected plants of the two transgenic plants after being inoculated by rice blast (Pyricularia oryzae) are significant. The transgenic plants with trichosanthin gene grew faster than the plants withGUS gene, even when humidity environment was removed. The results show that the transgenic plants that expressed trichosanthin are able to delay the infection of rice blast compared with the plants as control. In addition, no damage caused by the expression of trichosanthin gene in transgenic plants has been observed.     

Analysis of rice blast resistance genes by QTL mapping

Resistance to rice blast pathogen mostly shows a quantitative trait controlled by several minor genes. Its complexity and the mutable characteristic of rice blast isolates both hinder the development of the blast resistance research. The article here tried to explore the resistance gene distribution on rice chromosomes and the way of function. Totally 124 QTLs have been identified against 20 isolates using Cartographer software with a ZYQ8/JX17 DH population, which separately are at 100 loci of 72 marker intervals on 12 rice chromosomes. Of them, 16 QTLs were determined by the isolate HB-97-36-1. 82 QTLs (66.13%) are from the resistant parent alleles, ZYQ8, while 42 QTLs (33.87%) are from the susceptible parent alleles, JX17. In comparison of their positions on chromosome, most QTLs are clustered together and distributed nearby the major genes especially the regions on chromosomes 1, 2, 8, 10 and 12. Each QTL could account for the resistance variation between 3~2%-68.64%. And, a positional QTL might display the resistance to several different isolates with different contributions.     

Molecular tagging of a genic male-sterile gene in rice

The sterility of Pingxiang male-sterile rice (Pms), possibly derided from a spontaneous mutation in Pingxiang fertile rice (Pmf), was previously reported to be controlled by a single dominant nuclear gene. It can be restored to fertility either by a dominant epistatic gene or by higher temperature treatment at the early stage of inflorescence development. In order to tag the genic male-sterile gene, Pms, Pmf and Ce 64, a cytoplasmic male-sterile restoring line without the epistatic gene for Pms, were used to construct mapping populations. Two segregation populations, “(Pms/Ce 64) F1s (sterile plant)//Pmf ” F1 and “Pms//(Pmf/Ce 64) F1” F1, were simultaneously developed. Subsequently, the genic male- sterile gene was mapped between a simple sequence length polymorphism marker, RM228, and a restriction fragment length polymorphism marker, G2155, with distances of 14.9 and 2.6 cM, respectively. The tagged dominant genic male-sterile gene is temporarily designated Ms-p.