化学诱导激活型拟南芥突变体库的构建及分析

利用化学诱导激活XVE（LexA-VP16-ER）系统构建了一个包含40000余个独立转化株系的拟南芥突变体库,并对其中的18000余个株系进行了初步的遗传学和表型分析鉴定。卡那霉素抗性分离比表明,51．6％的株系为单位点插入株系,T-DNA插入的平均拷贝数为每株系1．38个。部分T1代和T2代植株表现出了可见的形态变异,包括下胚轴长度、根长度、植株大小和颜色、叶子颜色和形态、开花时间、种皮颜色及结实情况等对数个代表性突变株系表型及T—DNA插入位点侧翼序列进行了分析,结果表明突变体的表型是由于T—DNA的插入造成的,而且这些突变体中包括前人发现的AP2和AGAMOUS的等位基因。由于T-DNA标记或相邻的基因可被XVE系统诱导性的激活,或被T-DNA破坏导致功能缺失,该突变体库可以用于大规模筛选鉴定功能缺失性和功能获得性突变体。     

EMS诱变西瓜突变体库的构建及表型分析

采用1.0%诱变剂甲基磺酸乙酯(EMS)处理西瓜品系W1-17种子9h,然后对M1和M2代群体单株在叶、花、茎、育性、分支习性等方面进行表型变异观察,同时选取M2代典型变异株系,利用23对西瓜SSR引物进行分析鉴定,构建西瓜突变体库。结果表明:(1)EMS诱变使M1代幼苗形态呈现出叶畸形、叶褶皱、部分黄化、花畸形、雄花不散粉、卷须畸形、矮小、生长缓慢、不育等特异性状,获得由1 252个单株组成的西瓜突变体M1群体,群体总变异频率为18.33%。(2)M2代共筛选到205个突变植株,40种表型变异,表现在子叶性状(黄化、扭曲不对称、折叠等)、叶和茎性状(叶黄化、变小、裂刻变深,茎变细,节间变短,分支少等)、花性状(花变大,花色变浅,两性花,花瓣皱缩、部分退化、数目突变,柱头畸形,雄蕊不成熟等)和其他性状(生长缓慢、不育等)等方面,总的表型突变率达到了19.59%。(3)针对M2代10个典型变异植株,通过SSR引物分析发现有9份材料在DNA水平上有变异。本研究初步构建了含有120个M1代家系及1 051株M2代植株、40种表型变异的西瓜突变体库。     

一个黄绿色的水稻细胞核突变体

用甲基磺酸乙酯(EMS)处理粳稻品种8126离体培养早期的花药,从所产生的一个花粉植株的H,株系中分离到一个黄绿色突变体。突变体植株叶片的叶绿素含量仅为正常叶片的1/3。突变性状稳定,在连续自交4代中,没有出现任何绿色植株;由突变体通过花药培养产生的植株除部分白化苗外均呈黄绿色。突变体与正常植株杂交,无论其为父本或母本,杂种F_1均为绿色植株;杂种F_2分离的绿色与黄绿色植株数比值符合3:1;由杂种F_1植株通过花药培养产生的小苗中绿苗与黄绿苗数的比值符合1:1。证明突变性状是由单一的隐性细胞核基因所控制。     

银合欢和苏门答腊金合欢根瘤菌的分离和回接

从银合欢和苏门答腊金合欢的根瘤各分离出三个快生型的根瘤菌,对其形态作了初步的观察。这些根瘤菌可能是同一族的,它们对其寄主植物进行回接和互接均能引起银合欢和苏门答腊金合欢结根瘤,互接形成的根瘤形态与寄主植物的根瘤相同;箭舌豌豆和武宁苕子的根瘤菌不能引起银合欢和苏门答腊金合欢植株结根瘤;分离得到的根瘤菌对银合欢和苏门答腊金合欢均有反馈的增益作用,几个生物学指标均比对照的高;在无菌条件下,全氮培养抑制了根瘤菌对寄主植物结瘤,琼脂固体培养的植株根瘤固氮活性比沙培、水培的高。     

水稻Ds插入纯合体的筛选和鉴定

采用Basta抗性鉴定、潮霉素抗性鉴定和PCR检测相结合的方法筛选和鉴定了水稻Ds插入纯合体。在T1代236个转化株系中,有16个株系的全部植株表现出对Basta的敏感,其余220个株系的植株表现出对Basta的抗性。经过3代的纯合筛选,共鉴定出Ds插入纯合体203个．这些Ds插入纯合体可用于构建Ac／Ds系统和对Ds插入突变体进行筛选和鉴定,为水稻功能基因组学研究提供了材料。     

两个葡萄杂交后代根系抗葡萄根瘤蚜及抗寒性鉴定

【目的】为了筛选抗葡萄根瘤蚜Daktulosphaira vitifoliae Fitch且抗寒的葡萄砧木以适应我国葡萄生产需求。【方法】以山葡萄Vitis amurensis Rupr.左山1号×SO4杂种F1代的45个株系(A系列)和左山1号×101-1杂种F1代27个株系(B系列)为试材,采用离体根接种鉴定法进行抗葡萄根瘤蚜鉴定及抗性分级;采用差热分析系统(differential thermal analysis,DTA)进行各株系根系的低温放热(low temperature exotherms,LTE)分析,建立各株系根系韧皮部及木质部的温度-伤害度(LT-I)回归方程,评估各株系根系的抗寒性。【结果】葡萄根瘤蚜在杂交株系根系上的产卵量均显著低于敏感品种巨峰,筛选出被葡萄根瘤蚜侵染后不能形成根瘤,抗葡萄根瘤蚜级别为0级的A系列杂交株系18个和B系列株系11个。被葡萄根瘤蚜侵染后形成根瘤比例低于10%的抗葡萄根瘤蚜级别为1级的A系列杂交株系9个和B系列株系4个;筛选出A系列综合低温放热温度隶属度函数、韧皮部和木质部低温放热温度隶属度函数3个指标均低于贝达的株系27个,B系列各指标均低于贝达的株系3个。【结论】本研究筛选出抗寒性强且对葡萄根瘤蚜抗性强的A系列株系15个和B系列株系2个。其中,A14,A16,A18,A22,A23,A28,A34,A35,A38,A44,A50,B24和B26对葡萄根瘤蚜抗性级别为0;A11,A15,A17和A27对葡萄根瘤蚜抗性级别为1。     

抗拿捕净晋谷21突变体的筛选和分子鉴定

谷子(Setaria italica(L.)P.Beauv.)是我国重要的杂粮作物,具有抗旱耐贫瘠、水分利用率高、适应性广、营养丰富等特点,但谷子田间除草一直制约着谷子的集约化栽培和有机旱作产业规模化的推广与发展。为快速选育抗除草剂谷子品种,本试验以晋谷21突变体M2群体为试验材料,通过喷施0.33%拿捕净除草剂对晋谷21突变群体进行初次筛选和再次筛选,进而快速获得抗拿捕净除草剂突变体植株并对其进行分子鉴定。结果显示,在大田苗期喷施0.33%拿捕净除草剂的初次筛选中,筛选出抗拿捕净除草剂的晋谷21突变体共39个株系;对这39个突变体株系再次喷施0.33%拿捕净除草剂,筛选获得9个有抗拿捕净除草剂特性的株系。对筛选获得的抗拿捕净突变体株系的其中3个株系共15个植株进行分子鉴定,发现有4个突变体植株的ACCase基因编码的第1780个氨基酸-异亮氨酸突变为亮氨酸。该结果可为今后利用分子生物学手段改良名优谷子品种晋谷21、加速抗除草剂谷子品种的选育提供理论和材料基础。     

小麦黄化突变体叶绿体超微结构研究

利用透射电镜对小麦自然黄化突变体及其突变亲本(西农1718)叶片细胞叶绿体的数目、形态及超微结构进行比较分析。结果发现:(1)3种不同黄化程度突变体的叶绿体分布、数目、形状及大小与突变亲本无明显差异;(2)突变体叶绿素含量为野生型58%的黄绿植株与其突变亲本叶绿体超微结构无明显差异,基质类囊体与基粒类囊体高度分化,基粒数目以及基粒片层数目较多;(3)突变体金黄和绿黄植株的叶绿素含量分别为野生型的17%、24%,其叶绿体超微结构与突变亲本明显不同,突变体的叶绿体发育存在明显缺陷,其中突变体金黄植株的叶绿体内无基粒、基质片层清晰可见,有淀粉粒,嗜锇颗粒较多,而突变体绿黄植株的叶绿体内有基粒,但明显少于突变亲本,且基粒片层较少,基质类囊体较发达。结果表明该黄化突变体叶绿体超微结构的改变,是由于叶绿素含量降低造成,推测,该黄化突变是由于叶绿素合成受阻导致的。     

CRISPR/Cas9技术在水稻类受体激酶基因OsBAK1L突变体制备中的应用

植物类受体激酶在植物生长发育及响应外界环境信号刺激中具有重要作用,为了进一步研究水稻类受体激酶OsBAK1L的功能,我们利用CRISPR/Cas9技术对该基因进行定点编辑。OsBAK1L定位在细胞膜上且该基因预测编码蛋白包含三个功能结构域:胞外LRR结构域、跨膜结构域及胞内激酶域。为了进一步理解该蛋白不同结构域上的功能,我们分别在胞外LRR结构域(Target 1)和胞内激酶域(Target 2)上设计该基因定点编辑靶点。将合成的靶位点序列插入入门载体CH,然后与表达载体Cas9进行重组。重组载体通过农杆菌介导方法转入野生型水稻9522中,2个构建分别获得26棵和12棵潮霉素抗性植株。通过对转基因植株的测序分析,找到了3棵和2棵序列发生变化的杂合T_0代植株。T_0代杂合植株自交分离分别获得T_1代两靶位点处纯合子突变体,靶位点1处纯合子突变体株系缺失5个碱基,靶位点2处纯合子突变体株系插入1个碱基,均可造成该基因转录本翻译提前终止。该基因不同结构域上突变体的获得为进一步研究该基因功能提供了重要且稳定的遗传材料。     

拟南芥叶边缘锯齿状突变体的分离与鉴定

叶的极性建立直接决定叶的平展性发育,极性改变导致叶形态异常,影响植物体的各种正常生理活动。利用反向遗传学方法,从拟南芥基因激活标签突变体库中分离到一个叶片边缘锯齿状表型的突变体（命名为pCB1294）,该突变体同时表现出叶表皮腺毛形态发育异常。通过TailPCR方法成功定位突变基因为At5g41663,该基因编码miR319b基因。Real time PCR显示,pCB1294突变体植株中miR319b基因的表达量是野生型（col）植株的11倍多。所得结果为进一步研究miRNA调控叶极性的分子机制和进一步分析miR319b与叶形态发生的关系奠定了基础。     

Genetic analysis of leaf form mutants from the Arabidopsis Information Service collection

Although a vast inventory of morphological mutants of Arabidopsis thaliana is available, only some have been used for genetic studies of leaf development. Such is the case with the Arabidopsis Information Service (AIS) Form Mutants collection, assembled by A. R. Kranz and currently stored at the Nottingham Arabidopsis Stock Centre, which includes a large number of mutant lines, most of which have been little studied. With the aim of contributing to the genetic dissection of leaf ontogeny, we have subjected 57 mutant lines isolated by others to genetic analysis; 47 of which were from the AIS collection. These are characterized by vegetative leaves of abnormal shape or size, and were chosen as candidates for mutations in genes required for leaf morphogenesis. The mutant phenotypes studied were shown to be inherited as single recessive Mendelian traits and were classified into 10 phenotypic classes. These mutant strains were found to fall into 37 complementation groups, 7 of which corresponded to known genes. Results of the phenotypic analysis and data on the genetic interactions of these mutants are presented, and their possible developmental defects discussed.     

Genetic analysis of leaf form mutants from the Arabidopsis Information Service collection.

Although a vast inventory of morphological mutants of Arabidopsis thaliana is available, only some have been used for genetic studies of leaf development. Such is the case with the Arabidopsis Information Service (AIS) Form Mutants collection, assembled by A. R. Kranz and currently stored at the Nottingham Arabidopsis Stock Centre, which includes a large number of mutant lines, most of which have been little studied. With the aim of contributing to the genetic dissection of leaf ontogeny, we have subjected 57 mutant lines isolated by others to genetic analysis; 47 of which were from the AIS collection. These are characterized by vegetative leaves of abnormal shape or size, and were chosen as candidates for mutations in genes required for leaf morphogenesis. The mutant phenotypes studied were shown to be inherited as single recessive Mendelian traits and were classified into 10 phenotypic classes. These mutant strains were found to fall into 37 complementation groups, 7 of which corresponded to known genes. Results of the phenotypic analysis and data on the genetic interactions of these mutants are presented, and their possible developmental defects discussed. Received: 28 October 1998 / Accepted: 21 February 1999     

Developmental mutants showing abnormal organ differentiation in rice embryos

Summary Zygotes of rice (Oryza sativa L. cv Taichung 65) were treated with 1.0 mM solution of the chemical mutagen N-methyl-N-nitrosourea. Out of 1420 M₂ lines, 28 single-locus recessive mutants on embryogenesis were identified. Among them, we analyzed 11 mutants in the present study, which differentiated the shoot (plumule) and/or root (radicle) with abnormality. Of the 11 mutants, two showed no shoot differentiation with normal root. On the other hand, we could not detect any mutant which exhibited a normal shoot without a root. This suggests that shoot and root are genetically controlled by different loci and that the alleles associated with shoot formation mutate more frequently than do those of the root. Five mutants showed aberrant morphology of shoot when both the shoot and root developed. One of them, odm 5 (organ differententiation mutant 5) was germinable, but produced many fine and twisted leaves. This mutant was, however, lethal at the early post-germination stage under the usual cultural conditions. In another mutant (odm 4), shoot differentiation seemed to be initiated at an arbitrary position, resulting in a very abnormal morphology of the shoot when the position fronted the endosperm. The other two mutants showed abnormal morphology of both the shoot and root. One (odm 11) of the remaining two mutants showed a wide variation of abnormalities including no organ differentiation, either shoot or root differentiation and the development of both shoot and root with abnormalities. The last one (odm 16) was unique. It had an embryo with normal shoot and root but the embryo size was only one-third to one-half of normal embryos in length. Of course, the shoot and root are also small but viable. Therefore, odm 16 is considered to be a mutant in the size regulation of the embryo. Although an allelism test has not yet been done, most of these mutants are probably non-allelic, as the phenotypic abnormality differs largely with each one. In rice, the shoot and root highly differentiate in contrast to dicotyledonous embryo. Accordingly, these developmental mutants are very useful materials for investigating the regulatory mechanism of gene expression in organ differentiation.     

Mutations affecting the structure and function of immunoglobulin M.

Using a hybridoma cell line which secretes hapten-specific immunoglobulin M (IgM), we have isolated a variety of mutants which produce abnormal immunoglobulin. Immunoglobulin was tested for the size and composition of the component heavy and light chains and for variable and constant region related functional and serological activities. Some mutants secrete IgM which seems to be defective in hapten binding; others make IgM which appears not to activate complement. Many of the mutants secrete monomeric as opposed to pentameric IgM. In some cases, the defect apparently correlates with structural alterations in the mu heavy chain: partial deletion, polypeptide addition, and abnormal glycosylation have been observed. These mutant cell lines provide a means of identifying the structural basis of IgM function and of studying the biochemistry of IgM synthesis and processing.     

Expression of recessive Aprt- mutations in mouse CAK cells resulting from chromosome loss and duplication

Karyotypes of recessive mutants at the autosomal adenine phosphoribosyltransferase (Aprt) locus in a clone of the near-diploid mouse CAK cell line have been analyzed. The Aprt located on chromosome 8. One copy of chromosome 8 was morphologically abnormal in the parental clone (CAK-B3-Toyr13) from which Aprt- mutants were isolated. Among 22 mutants, there were ten in which one copy of chromosome 8 had been lost. Four of these were monosomic, and in the others duplication of the remaining homolog had occurred. These findings indicate that newly induced recessive mutations in cultured mammalian cells can be expressed as the result of loss of one chromosome carrying a wild-type allele with or without duplication of the homolog carrying the mutant allele. Loss and duplication would not be detected in cell lines lacking morphologically marked chromosomes.     

Integrated functions among multiple starch synthases determine both amylopectin chain length and branch linkage location in Arabidopsis leaf starch

This study assessed the impact on starch metabolism in Arabidopsis leaves of simultaneously eliminating multiple soluble starch synthases (SS) from among SS1, SS2, and SS3. Double mutant ss1- ss2- or ss1- ss3- lines were generated using confirmed null mutations. These were compared to the wild type, each single mutant, and ss1- ss2- ss3- triple mutant lines grown in standardized environments. Double mutant plants developed similarly to the wild type, although they accumulated less leaf starch in both short-day and long-day diurnal cycles. Despite the reduced levels in the double mutants, lines containing only SS2 and SS4, or SS3 and SS4, are able to produce substantial amounts of starch granules. In both double mutants the residual starch was structurally modified including higher ratios of amylose:amylopectin, altered glucan chain length distribution within amylopectin, abnormal granule morphology, and altered placement of α(1→6) branch linkages relative to the reducing end of each linear chain. The data demonstrate that SS activity affects not only chain elongation but also the net result of branch placement accomplished by the balanced activities of starch branching enzymes and starch debranching enzymes. SS3 was shown partially to overlap in function with SS1 for the generation of short glucan chains within amylopectin. Compensatory functions that, in some instances, allow continued residual starch production in the absence of specific SS classes were identified, probaby accomplished by the granule bound starch synthase GBSS1.     

Mutations in alpha- and beta-tubulin affect spindle formation in Chinese hamster ovary cells

Two Chinese hamster ovary cell lines with mutated beta-tubulins (Grs-2 and Cmd-4) and one that has a mutation in alpha-tubulin (Tax-1) are temperature sensitive for growth at 40.5 degrees C. To determine the functional defect in these mutant cells at the nonpermissive temperature, they were characterized with respect to cell cycle parameters and microtubule organization and function after relatively short periods at 40.5 degrees C. At the nonpermissive temperature all the mutants had normal appearing cytoplasmic microtubules. Premature chromosome condensation analysis failed to show any discrete step in the interphase cell cycle in which these mutants are arrested. These cells, however, show several defects at the nonpermissive temperature that appear related to the function of microtubules during mitosis. Time-lapse studies showed that mitosis was lengthened in the three mutant lines at 40.5 degrees C as compared with the wild-type cells at this temperature, resulting in a higher proportion of cells in mitosis after temperature shift. There was also a large increase in multinucleated cells in mutant populations after incubation at the nonpermissive temperature. Immunofluorescent studies using a monoclonal anti--alpha-tubulin antibody showed that the mutant cells had a high proportion of abnormal spindles at the nonpermissive temperature. The two altered beta-tubulins and the altered alpha-tubulin all were found to cause a similar phenotype at the high temperature that results in mitotic delay, defective cytokinesis, multinucleation, and ultimately, cell death. We conclude that spindle formation is the limiting microtubule function in these mutant cell lines at the nonpermissive temperature and that these cell lines will be of value for the study of the precise role of tubulin in mammalian spindle formation.     

大豆疫霉菌的EMS化学诱变

以甲基磺酸乙酯(ethylmethane sulfonate,EMS)为诱变剂,通过其对大豆疫霉菌Phytophthora sojae休止孢萌发的影响,确定化学诱变条件。通过收集单卵孢子,建立了包含640个单卵孢子系的突变体库,其中约有50%的诱变菌系在培养性状和菌落形态方面发生了明显变化,菌落形态多样,表现出较紧密或松散,近圆形或不规则;气生菌丝减少,生长速度较慢或快;在卵孢子产量方面,8.13%的菌系有增加,20.41%的菌系减少,27.82%的菌系极少或者没有卵孢子产生,43.64%的菌系卵孢子产量类似野生型。以质膜氢离子泵蛋白基因PsPMA1(plasma membrane H+-ATPase1)为对象,通过TILLING技术,从320个大豆疫霉菌突变体中获得9个突变体,进一步确认了EMS对大豆疫霉菌的诱变效果,并且估算EMS对大豆疫霉菌的诱变频率至多每115kb发生一个核苷酸变异。新构建的突变体库为开展大豆疫霉病菌的功能基因组研究奠定了遗传材料基础。     

Hormone-dependent insertional Arabidopsis thaliana mutants with decreased viability and fertility

We present data on the phenotype identification and genetic analysis of offspring in three lines of dominant morphological mutants of Arabidopsis thaliana having drastically reduced fertility (a sterile calluslike mutant, a flower mutant, and a dwarf mutant) and in five lines of recessive morphological mutants (four mutants with lethal seedlings and one pigmentation mutant). The mutants were selected from a collection of transgenic plants that had genomes carrying a T-DNA insertion of plasmid vectors pLD3 and pPCVRN4; the collection was created earlier via agrobacterial transformation of germinating seeds. The results presented here were obtained using compensation of hormonal imbalance in the insertional morphological mutants of A. thaliana by exogenous hormones.