一种简单高效的水稻体细胞诱变育种新方法

多样化种质资源的利用对于培育高产和理想农艺性状水稻品种显得非常重要.在本研究中,通过研究继代时间、2,4-D浓度和不同外植体对诱变和分化频率的影响,建立了幼穗为外植体、继代培养时间为3个周期(约75d)以及2,4-D的最佳诱变浓度为4.0mg/L的体细胞诱变育种方法.然后利用该方法诱变杂交水稻亲本株1S和中9B,分别成功选育了优良矮化突变株系SV14S和SV9B,为后续的杂交育种提供了优质种质资源.这些育种成果的取得进一步证实了该水稻体细胞诱变育种方法是一种简单高效的新型育种方法.     

水稻矮秆基因d-ss的遗传分析与克隆

通过γ射线诱变,从粳稻品种9522的M2代中筛选出一株矮秆水稻(Oryza sativa L.)突变体,定名d-ss.d-ss突变体表现为叶色深绿、短宽的叶片、以及小而圆的籽粒.以d-ss突变体与籼稻品种龙特普杂交的F2代群体为基因定位群体,利用InDel分子标记将d-ss突变位点定位在5号染色体上的InDel标记ZZ5-6和ZZ1343之间,物理距离为412kb.最终通过图位克隆的方法获得了此基因,测序结果表明此基因在编码区发生了两处缺失突变.     

籼型多分蘖矮秆突变体的遗传分析及其相关生理特性

籼型多分蘖矮秆突变体“佳禾丛矮”(Jiahecongai,JHCA)是本研究室在进行成熟花粉辐照诱变育种过程中发现的.遗传分析表明,JHCA同时携带互不等位的半矮秆基因sd-1和另一个由核基因隐性突变造成的多分蘖矮秆基因,暂命名为xmd(t).从JHCA与野生型高秆品种“广场13”(GC13)的杂交后代中分离出只带xmd(t)且遗传稳定的单基因型突变品系“新佳丛”( Xinjiacong,XJC).为揭示xmd(t)突变所造成的多分蘖与茎秆矮化协同出现的机理,对XJC的相关生理特性进行了分析.全生育期去除分蘖芽的试验表明,突变体植株的矮化是由于或部分由于过多分蘖的发生所造成的,该突变体的实质是多分蘖突变体.显微分析和田间分蘖动态观察表明,突变体多分蘖特性的形成是由于分蘖芽发生更早、分糵级数多、分蘖节位更高,且分蘖持续时间更长所造成的.本研究也表明,多分蘖矮秆突变体是研究水稻分蘖分子机理的理想材料.     

水稻白化突变体alb21生理特性和基因定位

高等植物叶绿体的正常发育需要叶绿体基因和核基因相互协调,这些基因的突变将导致叶绿体发育的缺陷．通过同位素诱变,获得了1例水稻的白化突变体alb21,其在幼苗时期就表现出白化性状,生长1个月左右逐渐死亡．遗传学分析表明,该突变属于单基因隐性突变;电镜观察表明,突变体细胞内完全丧失了叶绿体结构,只有一些空泡状的结构．突变体中既没有检测出叶绿素a或b,也没有检测出叶绿素合成的前体——原脱植基叶绿素a,说明此突变体的叶绿素合成途径受阻．因此,推测Alb21基因的突变,导致叶绿体发育受阻,叶绿素a或b以及叶绿素合成的前体——原脱植基叶绿素a不能合成．利用本实验室开发的水稻InDel分子标记,将该突变基因定位在第3条染色体上分子标记R3M51—2与R3M52—5之间约1520kb范围内．这些结果为该基因的克隆及叶绿体发育过程中的功能研究奠定了基础．     

60Co γ-Ray射线诱变水稻突变体的分离和遗传学初步分析

为了研究水稻发育的分子机制,我们利用γ射线对粳稻品种9522进行诱变,共诱变了3 000 g种子,M1代单株收种,M2代移栽5 963个株系于上海农科院.在M2代中筛选突变体,并在M3代中复选,最终得到了叶、株型育性等各类形态突变体666份.对其中162株突变体进行了遗传分析,有53.61%的突变体出现3∶1的分离,这些突变体是隐性的单位点突变.同时,这些突变体的分离也为水稻功能基因分离和功能鉴定等研究打下了良好的基础,并为水稻发育的研究提供了宝贵的材料.     

枯草杆菌JAS株α—淀粉酶的产生与其抗衣霉素的关系

为了研究Bacillus subtilis的产α-淀粉酶性状与其抗衣霉素性状之间的关系,进一步选择在诱变育种时能有效地筛选淀粉酶基因突变株的方法,我们对此作了初步探索.衣霉素敏感型的α-淀粉酶工业生产菌B.subtilis JAS经过紫外线诱变后,选得9株抗衣霉素突变株.通过对它们进行的多次发酵酶活力测定,发现其中3株是α-淀粉酶缺陷型、3株酶活力基本不变或降低,另外3株酶活力明显提高,其中1株酶活力是出发菌株的114%—123%.试验表明B.subtilis α-淀粉酶的产生与其抗衣霉素基因的表达有着密切关系.因此,在对该菌的诱变育种时,可以采用衣霉素为粗筛选择压力.     

拟南芥盐敏感突变体EMS85的快速基因定位与分析

土壤盐碱化是造成农作物减产的主要原因.作者通过对拟南芥(Arabidopsis thaliana)野生型种子Col-0进行甲基磺酸乙酯(EMS)诱变,筛选到1株盐敏感突变体EMS85.通过杂交和后代性状分离比确定该突变体的不耐盐性是受隐性单基因控制.采用图位克隆的方法,通过对拟南芥5条染色体上的SSLP标记的筛选,挑选了24对均匀分布于染色体上的SSLP标记作为基因初定位,同时设计了一系列精细定位分子标记,通过粗定位和精细定位发现,该基因位于拟南芥第5条染色体下臂的分子标记LUGSSLP847和5-13.58之间,进而测序结果表明该突变体是SOS2基因的等位突变体,突变位点为该基因1 521~1 522 bp处两碱基的缺失,造成移码突变,使SOS2基因功能丧失.本实验室建立的图位克隆体系加快了克隆基因的进程,对EMS85突变体的定位只在短短半年时间就已完成,为其他突变体的快速确定候选基因,加速基因分离进程奠定了基础.     

水稻黄绿叶基因YGLOSH的定位克隆

本文利用~(60)Co γ射线诱变光温敏感型核不育系广占63S(GZ63S),在后代中获得一个稳定遗传的黄绿化叶色突变体黄广占63S,突变体从苗期至成熟期均显示叶片黄化特征.遗传分析表明该性状受一对隐性核基因控制,命名为yglosh.利用BSA方法分析突变体黄广占63S与正常绿叶对照蜀恢881构建的F2群体,将该黄化基因定位于水稻2号染色体分子标记RM279和Pm6之间,物理距离约68kb.定位区间的cDNA测序分析发现,突变体中LOC_Os02g05890基因发生单碱基突变,形成终止子提前终止该基因的翻译.转基因互补实验确定LOC_Os02g05890为目标基因,可能参与叶绿体发育或者叶绿素生物合成途径.     

一个新矮秆水稻突变体的光合特性研究

从T-DNA插入的水稻突变体库发现了一个新的水稻矮秆突变体，平均株高约是野生型的37％，茎节间长度明显低于野生型，其叶片直立生长并呈暗绿色，边缘卷曲的性状。光合特性分析表明，单位重量叶片ChlaxChlb和B-Car色素含量显著高于野生型，Chla／b与野生型没有差异。与野生型相比，突变体最大光化学效率（Fv／Fm）、实际光化学效率（φesu）和光化学淬灭系数（qP）与野生型并没有显著差异，但其非光化学淬灭系数显著低于野生型，说明突变体光合效率优于野生型。电镜分析发现了突变体细胞内部出现衰老症状但叶绿体结构正常。     

一个新的水稻矮秆突变体sd-sl的遗传与基因定位研究

新的矮秆基因的发掘、研究和利用对水稻育种和植物生长发育机制研究有重要的作用.用60Coγ射线辐照粳稻9522,获得一个能稳定遗传的突变体.该突变体表型为株高较野生型矮,叶片短而微卷.将该突变体与籼稻广陆矮杂交,F2代呈3∶1分离,说明该突变体受隐性单基因控制.通过InDel分子标记对F2代分离群体进行遗传定位,将该基因定位于第6染色体InDel标记OS604附近.随后又发展了多对有多态性的InDel分子标记,将该基因座位精细定位在InDel标记XL6-6和XL6-1之间,AP003490和AP005619上,两个引物之间的物理距离为118 kb.本研究为该克隆基因及其作用机理的探究奠定了基础.     

Function analysis of a semi-dwarf sdg in rice with near isogenic lines

The physiological phenotype of the rice semi-dwarf mutant,sdg,was characterized in details using a pair of near isogenic .Neither gibberellin*deficient nor gibberellin-insensitive is characteristic of the sdgphenotype.Byanalyzing the secretion of aamylas and the promotion of stem growth caused by eogenous gibberellin(GA),the sdg plant was found to have decreased sensitivity to the GA at the seedling stage. The dwarfism stature of the sdg mutant can be attributed to the shortened internodes. Increase of the cell sults indicate the protein encded by the wild type gene of sdg may be a regulator for cell elogation.     

Molecular analysis of spontaneous somatic mutants.

Eukaryotic structural gene mutations occurring spontaneously in a mouse myeloma cell line offer the opportunity to study somatic mutation in animal cells at the molecular level. Studies on the myeloma protein and on mRNA have enabled us to characterise four such mutants representing four different mutation mechanisms. The results may have some bearing on the origin of antibody diversity.     

Isolation and mutation rate of the spontaneous PUR box and purR mutants

In our previous research in purine metabolism in Salmonella typhimurium, it was observed that the mutation frequency of the PUR box was one order of magnitude higher than that of purR under mutagenesis. In order to investigate further into this phenomenon, large amounts of independent PUR box and purR spontaneous mutants were isolated on lactose minimal medium by using a super-repressing mutant of purR (purRs). The mutational regions of 5 PUR box mutants and 4 purR mutants were cloned. Nucleotide sequence analysis showed that all the mutants had mutations at the expected sites. The comparison of the two types of mutations indicated that, although the purR gene was two orders of magnitude larger than the box). Thus, we concluded that high mutation frequency of the PUR box did not result from mutagenesis. Under spontaneous conditions, the mutation frequency of PUR box was also high. Some tentative explanations of this interesting phenomenon are given in this report.     

Complete mutagenesis of the HIV-1 protease

Retroviruses encode a protease which needs to be active for the production of infectious virions. A disabling mutation in the protease results in the production of non-infectious virus particles and examination of proteins from these mutant virions reveals unprocessed Gag and Gag-Pol precursor proteins, the substrates of the viral protease. Each amino acid of the HIV-1 protease was individually mutated using a simple mutagenesis procedure which is capable of introducing and identifying missense mutations in each residue of a protein. Phenotypic screening of these mutants in a heterologous assay system reveals three regions within the protease where multiple consecutive amino-acid residues are sensitive to mutation. These results show that random mutagenesis can be used to identify functionally important regions within a protein. Mutants with conditional phenotypes have also been identified within this collection.     

Drastic change in idiotypic but not antigen-binding specificity of an antibody by a single amino-acid substitution

In proliferating B lymphocytes, somatic mutation of rearranged antibody variable (V)-region genes occurs at high frequency and may have a key role in the selection of these cells. It is of interest in this context to learn in which way single mutations can affect antigen binding and/or idiotypic specificity of an antibody. Previous investigations have analysed spontaneous mutants of myeloma and hybridoma cells in which the mutation affected the antigen-binding specificity of the antibody. Here we describe an antibody mutant that has fully retained antigen-binding specificity but has lost or drastically changed all V-region antigenic determinants (idiotopes) of the wild type as defined by monoclonal anti-idiotope antibodies. The mutant phenotype is generated by a glycine to arginine exchange in the middle of the diversity (D) element, at position 103 of the heavy chain.     

水稻突变体B1-24的细胞学观察及其激素响应

以半矮化水稻突变体B1-24及其野生型惠阳珍珠早(Hyz)幼苗为材料,探究其细胞学特征及其对激素的响应.在光下生长的B1-24幼苗比Hyz矮14.62%,第一真叶长度短11.18%,黄化苗植株的中胚轴长度增长41.89%.B1-24黄化苗的胚芽鞘细胞长度仅为Hyz的45.95%,而中胚轴细胞长度是其118.28%.B1-24分别经激素GA3(600μg/mL)和IAA(7μg/mL)处理,其细胞长度显著增长,50μg/mL PAC处理使其严重缩短.推测B1-24的半矮化突变可能与激素合成或响应有关.     

Development of gene-tagged molecular markers for starch synthesis-related genes in rice

Improving grain quality is an important goal in breeding new elite rice varieties, requiring effective tools for the identification of target genotypes. Molecular marker-aided selection (MAS), combined with conventional breeding approaches, enables us to precisely identify the individual genotypes that are associated with different grain quality features, which can dramatically improve the breeding efficiency. However, to date, the number of molecular markers used in MAS for grain quality improvement is still somewhat limited. In this study, based on our previous study that rice grain quality is strongly associated with starch synthesis in the endosperm, we developed 51 gene-tagged molecular markers according to sequence variations in 18 starch synthesis-related genes from 16 typical rice cultivars. These markers can discriminate the different alleles among rice germplasms. These novel markers will provide effective tools in improving grain quality via the breeding new elite rice varieties.     

Green revolution: a mutant gibberellin-synthesis gene in rice

The chronic food shortage that was feared after the rapid expansion of the world population in the 1960s was averted largely by the development of a high-yielding semi-dwarf variety of rice known as IR8, the so-called rice 'green revolution'. The short stature of IR8 is due to a mutation in the plant's sd1 gene, and here we identify this gene as encoding an oxidase enzyme involved in the biosynthesis of gibberellin, a plant growth hormone. Gibberellin is also implicated in green-revolution varieties of wheat, but the reduced height of those crops is conferred by defects in the hormone's signalling pathway.