一个新的OsBRI1弱等位突变体的鉴定及其调控种子大小的功能研究

水稻(Oryza sativa)籽粒大小是影响其产量的关键农艺性状, 克隆并研究水稻籽粒大小相关基因对于提高水稻产量具有重要意义。为深入探究水稻籽粒大小的调控机制, 通过EMS诱变品种宽叶粳(KYJ), 分离了一系列水稻籽粒大小改变的突变体, 其中smg12表现为籽粒变小, 株高变矮, 一级枝梗数和二级枝梗数减少。遗传分析表明, 该小粒突变体受隐性单基因控制。细胞学分析显示, 该突变体颖壳纵向细胞长度显著变短, 表明SMG12主要影响细胞扩展。利用Mutmap方法对候选基因进行克隆, 筛选出SMG12的候选基因OsBRI1, 该基因编码油菜素内酯受体激酶。OsBRI1外显子上的第2 074个碱基发生了由C到T的置换, 产生非同义突变, 使得该位置编码的脯氨酸变为丝氨酸, 从而影响OsBRI1的功能。综上, 该研究鉴定了OsBRI1基因的1个新等位变异, 揭示了油菜素内酯途径调控水稻籽粒大小的细胞和分子基础。     

图位克隆技术新型遗传学实验教学项目的设计与实践

染色体重组与连锁互换是遗传学教学的重点和难点。为使学生更好地理解此方面知识, 我们课题组前期利用图位克隆(map-based cloning)技术, 克隆了1个调控水稻(Oryza sativa)类病变表型的基因SPL5, 并基于此设计了一个新的综合型遗传学实验, 即利用DNA分子标记对基因进行定位。实验中学生利用水稻spl5突变体与野生型杂交获得的F₂代定位群体和多态性分子标记, 对spl5突变进行染色体连锁分析、初步定位和遗传作图。该教学实验不仅可有效促进学生对遗传学三大定律的理解, 而且对其开阔视野、提高解决问题和团队协作的能力也有促进作用。     

水稻雄性不育突变体ms102的鉴定和基因定位

水稻(Oryza sativa)隐性核雄性不育突变体是第三代杂交水稻技术的核心。为了挖掘优质雄性不育突变体, 该研究通过筛选优质籼稻黄华占(HHZ)的甲基磺酸乙酯(EMS)诱变突变体库, 获得1个雄性不育突变体ms102 (male sterility mutant 102)。该突变体营养生长正常, 但花药不开裂, 花粉败育。细胞学分析表明, 突变体花药绒毡层不能正常降解, 导致小孢子发育异常; 遗传分析表明, 该突变体的不育表型由1个已报道编码酰基转移酶的DPW2基因突变造成。研究获得了1个隐性核雄性不育突变体, 进一步证实了DPW2基因在水稻花药发育中的功能。     

一个新的OsBRI1弱等位突变体的鉴定及其调控种子大小的功能研究

水稻(Oryzasativa)籽粒大小是影响其产量的关键农艺性状,克隆并研究水稻籽粒大小相关基因对于提高水稻产量具有重要意义。为深入探究水稻籽粒大小的调控机制,通过EMS诱变品种宽叶粳(KYJ),分离了一系列水稻籽粒大小改变的突变体,其中smg12表现为籽粒变小,株高变矮,一级枝梗数和二级枝梗数减少。遗传分析表明,该小粒突变体受隐性单基因控制。细胞学分析显示,该突变体颖壳纵向细胞长度显著变短,表明SMG12主要影响细胞扩展。利用Mutmap方法对候选基因进行克隆,筛选出SMG12的候选基因OsBRI1,该基因编码油菜素内酯受体激酶。OsBRI1外显子上的第2 074个碱基发生了由C到T的置换,产生非同义突变,使得该位置编码的脯氨酸变为丝氨酸,从而影响OsBRI1的功能。综上,该研究鉴定了OsBRI1基因的1个新等位变异,揭示了油菜素内酯途径调控水稻籽粒大小的细胞和分子基础。     

水稻穗长基因PAL3的克隆及自然变异分析

穗型是决定水稻(Oryza sativa)产量的关键因素之一。我们从粳稻品种圣稻808 (SD808)的EMS诱变突变体库中发现4份短穗突变体, 这些突变体的穗长、一级枝梗数、二级枝梗数和穗粒数发生不同程度的降低。基因定位和图位克隆表明, 这些突变体的表型受同一基因控制, 将该基因命名为PAL3 (PANICLE LENGTH3)。PAL3编码一个含12个跨膜结构域的多肽转运蛋白。pal3-1和pal3-2的点突变造成保守区域的氨基酸发生非同义突变; pal3-3的点突变造成第1外显子和内含子拼接错误; pal3-4的点突变造成蛋白翻译提前终止, 导致第12个跨膜域缺失。对PAL3进行单倍型分析, 共鉴定出9个单倍型(Hap1-Hap9), 其中Hap1-Hap3为主要单倍型。Hap1以粳稻为主, Hap2同时包含籼稻和粳稻, Hap3则以籼稻为主。Hap1起源于普通野生稻(O. rufipogon), Hap2和Hap3可能起源于一年生普通野生稻(O. nivara)。统计分析结果表明, Hap3的穗长显著高于Hap1和Hap2, 其具有提高穗长的潜力。该研究揭示了多肽转运蛋白对水稻穗型的重要调控作用, 为水稻穗型改良奠定了理论基础。     

图位克隆技术新型遗传学实验教学项目的设计与实践

染色体重组与连锁互换是遗传学教学的重点和难点。为使学生更好地理解此方面知识, 我们课题组前期利用图位克隆(map-based cloning)技术, 克隆了1个调控水稻(Oryza sativa)类病变表型的基因SPL5, 并基于此设计了一个新的综合型遗传学实验, 即利用DNA分子标记对基因进行定位。实验中学生利用水稻spl5突变体与野生型杂交获得的F₂代定位群体和多态性分子标记, 对spl5突变进行染色体连锁分析、初步定位和遗传作图。该教学实验不仅可有效促进学生对遗传学三大定律的理解, 而且对其开阔视野、提高解决问题和团队协作的能力也有促进作用。     

水稻早衰突变体LS-es1的基因定位及候选基因分析

衰老是植物发育末期自主发生且不可逆的适应性反应, 叶片早衰相关分子机制研究对水稻(Oryza sativa)遗传改良以及抗衰老品种培育有重要意义。LS-es1是通过EMS诱变粳稻品种TP309获得的稳定遗传的早衰突变体。对LS-es1及其野生型的表型观察和生理生化分析表明, LS-es1叶片中积累了大量活性氧且细胞死亡更多, 同时LS-es1与产量相关的农艺性状均显著下降, 这也验证了LS-es1早衰的特征。对LS-es1及其野生型幼苗进行外源激素处理, 结果表明LS-es1对水杨酸(SA)、脱落酸(ABA)和茉莉酸甲酯(MeJA)更敏感。用图位克隆方法将LS-es1基因定位在水稻第7号染色体长臂46.2 kb区间内, 该区间共包括8个开放阅读框(ORF)。对该区间内的基因进行生物信息学分析, 结果发现Os07g0275300和Os07g0276000两个候选功能基因与早衰途径相关, 并且这2个基因的表达量在野生型和突变体中差异较大。研究结果为进一步克隆LS-es1基因并深入研究其生物学功能奠定了基础。     

水稻OsWRKY42是Xa21介导的抗白叶枯病途径新元件

Xa21对白叶枯病菌(Xanthomonas oryzae pv. oryzae, Xoo)具有广谱抗性, 是最早被克隆的水稻(Oryza sativa)抗白叶枯病基因。前期研究表明, OsWRKY42可能在Xa21介导的抗病反应中发挥作用。在Xa21基因遗传背景下制备了OsWRKY42的RNA干扰株系, 将经免疫印迹确认的转基因株系接种白叶枯病菌, 结果表明, 与抗病对照4021相比, 转基因株系的病斑长度增加, 说明OsWRKY42的丰度下调抑制了Xa21对白叶枯病的抗性反应。免疫印迹分析表明, 在OsWRKY42-RNAi转基因水稻中, OsPR6、OsPR15和OsPR16的蛋白质丰度降低, OsPR1A、OsPR1B、OsPR2和OsPR10A的蛋白质丰度升高, 表明这些病程相关蛋白质可能位于OsWRKY42基因的下游, 受OsWRKY42调控并参与Xa21介导的抗病性。研究结果表明, OsWRKY42是Xa21介导的抗白叶枯病途径新元件, 增进了对Xa21介导的水稻抗病机理的认识。     

玉米叶形相关性状的Meta-QTL及候选基因分析

叶长、叶宽、叶面积及叶夹角不仅影响玉米(Zea mays)光合效率, 也是株型的重要构成因素。通过对620个叶形QTL进行整合, 构建不同遗传背景下的叶形QTL整合图谱, 利用元分析发掘出22个叶长、22个叶宽、12个叶面积以及17个叶夹角mQTL; 进一步运用生物信息学手段, 确定44个与叶片发育密切相关的候选基因。分析发现, 仅有NAL7-like、YABBY6- like和GRF2等13个基因位于mQTL区间内, 而玉米中已克隆的KNOTTED1、AN3/GIF1、rgd1/lbl1、mwp1及SRL2-like、HYL1-like和CYCB2;4-like等水稻(Oryza sativa)和拟南芥(Arabidopsis thaliana)叶形同源基因位于未被整合的QTL内; 对44个候选基因在叶片长、宽、厚发育过程中基部-末端、中央-边缘、远轴-近轴的调控机理进行归纳分析, 发现玉米中仅有少数几个候选基因被报道, 揭示了叶形发育的部分分子机理。因此, 对玉米叶形相关mQTL/QTL及基因进行全面深入的分析, 不仅有助于增加对其遗传结构的了解, 发掘更多候选基因, 阐明叶形发育和形成的分子机制, 还可为耐密理想株型的分子标记辅助选择提供依据。     

水稻类病斑突变体研究进展

植物类病变突变体是一类在没有病原物侵染情况下就能自发产生坏死斑的突变体。这类突变往往导致植株的抗病增强和防御相关基因的组成性表达。水稻中已报道了将近200个来源不同的类病变突变体,截至2014年12月73个水稻类病变突变体已被鉴定和命名,其中11个控制类病变性状的基因被克隆,它们分别编码不同的蛋白,包括热激蛋白转录因子、E3泛素连接酶、质膜蛋白激酶、锌指蛋白、酰基转移酶。尽管这些蛋白不是直接与植物抗病途径相关,但是在已鉴定的水稻类病变突变体中,绝大多数提高了对白叶枯病或稻瘟病的抗性,表明这些类病变基因的突变激活了植株的防御系统,并且不同的类病变基因可能参与了不同的抗病信号传导途径。深入研究水稻类病变突变体对作物抗病的分子机理研究和栽培品种的遗传改良都具有重要的意义。     

Reduction to homozygosity is the predominant spontaneous mutational event in cultured human lymphoblastoid cells

Expression of recessive mutant phenotypes can occur by a number of different mechanisms. Inactivation of the wild-type allele by base-substitution mutations, frameshift mutations or small deletions occurs at both hemizygous and heterozygous cellular loci, while other events, such as chromosome level rearrangements, may not be detected at hemizygous loci because of inviabiltty of the resulting mutants. In order to assess the relative contribution of each type of mutational event, we isolated a human lymphoblastoid cell line that is heterozygous at the adenine phosphoribosyltransgerase (aprt) locus. The mutation rate for the expression of the mutant phenotype (aprt^+/−→aprt^−/−) was 1.3 × 10⁻⁵/cell/ generation. Molecular analysis of the DNA from 26 mutant clones revealed that 19% had undergone deletion of the entire wild-type allele. The aprt heterozygote carries a mutation in the coding sequence of the gene that results in the loss of a restriction site. Analysis of aprt^−/− mutants for this restriction fragment length difference reveales that 23% of the mutants contained point mutations or small ((< 100 bp) deletions. The remainder of the mutants (58%) resulted from reduction to homozygosity of the mutant allele. We suggest that, as in tumor cells in vivo, reduction to homozygosity is a major mechanism for the expression of recessive mutant phenotypes in cultured human cells.     

New mutations affecting induced mutagenesis in yeast

Previously isolated mutations in baker's yeast, Saccharomyces cerevisiae, that impair induced mutagenesis were all identified with the aid of tests that either exclusively or predominantly detect base-pair substitutions. To avoid this bias, we have screened 11 366 potentially mutant clones for UV-induced reversion of the frameshift allele, his4–38, and have identified 10 mutants that give much reduced yields of revertants. Complementation and recombination tests show that 6 of these carry mutations at the previously known REV1, REV1 and REV3 loci, while the remaining 4 define 3 new genes, REV4 (2 mutations), REV5 and REV6. The rev4 mutations are readily suppressed in many genetic backgrounds and, like the rev5 mutation, impart only a limited deficiency for induced mutagenesis: it is likely, therefore that the REV4⁺ and REV5⁺ gene functions are only remotely concerned with this process. The rev6 mutants have a more general deficiency, however, as well as marked sensitivity to UV and an increased spontaneous mutation rate, properties that suggest the REV6 gene is directly involved in mutation induction. The REV5 gene is located about 1 cM proximal to CYC1 on chromosome X.     

Influence of DNA repair on mutation spectra in Salmonella

This paper reviews the influence of DNA repair on spontaneous and mutagen-induced mutation spectra at the base-substitution (hisG46) and -1 frameshift (hisD3052) alleles present in strains of the Salmonella (Ames) mutagenicity assay. At the frameshift allele (mostly a CGCGCGCG target), ΔuvrB influences the frequency of spontaneous hotspot mutations (−CG), duplications, and deletions, and it also shifts the sites of deletions and duplications. Cells with pKM101+ΔuvrB spontaneously produce complex frameshifts (frameshifts with an adjacent base substitution). The spontaneous frequency of 1-base insertions or concerted (templated) mutations is unaffected by DNA repair, and neither mutation is inducible by mutagens. Glu-P-1, 1-nitropyrene (1NP), and 2-acetylaminofluorene (2AAF) induce only hotspot mutations and are unaffected by pKM101, whereas benzo(a)pyrene and 4-aminobiphenyl induce only hotspot in pKM101⁻, and hotspot plus complex in pKM101⁺. At the base-substitution allele (mostly a CC/GG target), the ΔuvrB allele increases spontaneous transitions in the absence of pKM101 and increases transversions in its presence. The frequency of suppressor mutations is decreased 4× by ΔuvrB, but increased 7.5× by pKM101. Both repair factors cause a shift in the proportion of mutations to the second position of the CC/GG target. With UV light and γ-rays, the ΔuvrB allele increases the proportion of transitions relative to transversions. pKM101 is required for mutagenesis by Glu-P-1 and 4-AB, and the types and positions of the substitutions are not altered by the addition of the ΔuvrB allele. Changes in DNA repair appear to cause more changes in spontaneous than in mutagen-induced mutation spectra at both alleles. There is a high correlation (r²=0.8) between a mutagen's ability to induce complex frameshifts and its relative base-substitution/frameshift mutagenic potency. A mutagen induces the same primary class of base substitution in TA100 (ΔuvrB, pKM101) as it does in Escherichia coli, mammalian cells, or rodents as well as in the p53 gene of human tumors associated with exposure to that mutagen. Thus, a mutagen induces the same primary class of base substitution in most organisms, reflecting the conserved nature of DNA replication and repair processes.     

Mutations by near-ultraviolet radiation in Escherichia coli strains lacking superoxide dismutase

Om wild-type Escherichia coli, near-ultraviolet radiation (NUV) was only weakly mutagenic. However, in an allelic mutant strain (sodA sodB) that lacks both Mn- and Fe-superoxide dismutase (SOD) and assumed to have excess superoxide anion (O₂⁻), NUV induced a 9-fold increase in mutation above the level that normally occurs in this double mutant. When a sodA sodB double mutant contained a plasmid carrying katG⁺ HP-I catalase), mutation by NUV was reduced to wild-type (sodA⁺ sodB⁺) levels. Also, in the sodA sodB xthA triple mutant, which lacks exonuclease III (exoIII) in addition to SOD, the mutations frequency by NUV was reduced to wild-type levels. This synergistic action of NUV and O₂⁻ suggested that pre-mutational lesions occur, with exoIII converting these lesions to stable mutants. Exposure to H₂O₂ induced a 2.8 fold increase in mutations in sodA sodB double mutants, but was reduced to control levels when a plasmid carrying katG⁺ was introduced. These results suggest that NUV, in addition to its other effects on cells, increases mutations indirectly by increasing the flux of OH^. radicals, possibly by generating excess H₂O₂.     

Mutagenesis of pea (Pisum sativum L.) and the isolation of mutants for nodulation and nitrogen fixation

Pea mutants for nodulation have been obtained by treating seeds with ethyl methane sulfonate (EMS) followed by 2 screening procedures. In one, mutants resistant to nodulation (nod⁻), or with ineffective nodules (nod⁺, fix⁻) were obtained, whilst in the other 4 hypernodulated mutants (nod⁺⁺) with 5–10 times more nodules than cv. Frisson and expressing a character of nitrate tolerant symbiosis (nts) were discovered. All mutations are under the control of single recessive genes. (nod⁻), (nod⁺, fix⁻) and (nod⁺⁺, nts) mutations result from mutation events at 6, 7 and 1 different loci respectively.

Grafting experiments showed the (nod⁻) and (nod⁺, fix⁻) phenotypes are associated with the root genotypes and that (nod⁺⁺, nts) phenotype is associated with the shoot genotype.