小麦—偏凸山羊草6Mv/6B代换系的研究

小麦条锈病（Puccinia striiformis f. sp. tritici）是世界性小麦病害，可导致受害小麦减产30%以上，甚至绝收。小麦条锈病在我国西南、华北麦区危害严重，四川麦区是小麦条锈病发病最重的地区之一，每年因条锈病流行造成小麦产量损失巨大。利用抗条锈病品种是控制该病害最安全、经济的有效途径，因此挖掘利用抗病新基因，开展抗病遗传基础研究是当前育种工作中面临的重要任务。 偏凸山羊草（Aegilops ventricosa，DDMvMv，2n=28）是一年生草本植物，起源于地中海西部沿岸地区，具有对小麦白粉病、锈病等高抗或免疫、耐盐、抗寒、蛋白质含量高等优良性状，是小麦遗传育种很好的种质资源。本研究以高抗条锈病的小麦—偏凸山羊草6Mv/6B代换系（Moisson 6Mv/6B）为材料，对其含有的带条锈病抗性基因的偏凸山羊草6Mv染色体在四川小麦背景中的传递情况、与小麦—簇毛麦双端体附加系所具有的白粉病抗性的聚合以及对Moisson 6Mv/6B进行电离辐射诱变筛选抗条锈病的小麦—偏凸山羊草易位系三个方面进行了研究。取得的主要研究结果如下： 1. Moisson 6Mv/6B与高感条锈病的四川地区普通小麦品种绵阳26、绵阳93-124和SW3243的杂种F1与其普通小麦亲本分别作为父、母本回交，通过对其BC1和F2的结实率、根尖细胞有丝分裂中期染色体的观察以及对条锈病抗性的鉴定，发现含6Mv染色体的F1植株作母本时的回交结实率（83.10%）普遍高于含6Mv染色体的F1植株作父本（48.61%），结实率与普通小麦基因型密切相关（χ2=34.15>>χ20.05=5.99（df=2））；6Mv染色体在三种四川小麦中通过雌、雄配子传递的传递方式与其传递率间没有显著相关性，其传递率与普通小麦基因型呈显著相关性（χ2=6.42>χ20.05=5.99（df=2））。 2. Moisson 6Mv/6B与高抗白粉病的小麦—簇毛麦双端体附加系Pana（2n=42+2t）正反杂交，希望在聚合两者抗性的同时观察不同受体背景下的抗性反应。对Moisson 6Mv/6B和Pana正反杂交的结实率、杂交后代的农艺性状进行观察，并对杂交后代进行基因组荧光原位杂交（GISH）分析及条锈病和白粉病的抗性鉴定。结果表明Moisson 6Mv/6B作母本时杂交结实率（80.56%）高于Pana作母本时（58.33%），结实率与杂交方式间紧密相关（χ2=4.96>χ20.05=3.84（df=1））；Moisson 6Mv/6B和Pana杂交后代株高比最高亲本高约10cm，成熟期也较两亲本提前两个星期左右；正反杂交后代中具有偏凸山羊草6Mv染色体的植株具有条锈病抗性，具有簇毛麦端体的植株具有白粉病抗性，同时筛选到4株含有偏凸山羊草和簇毛麦遗传物质并对条锈病和白粉病兼抗的材料，证明来自偏凸山羊草6Mv染色体的条锈病抗性与来自簇毛麦端体的白粉病抗性已经聚合在一起，且没有产生相互抑制的作用，暗示通过这两个抗性基因的聚合是完全能获得兼抗条锈病和白粉病的小麦新种质。 3. 对Moisson 6Mv/6B在减数分裂时期的成株进行总剂量为6Gy、辐射频率为120rad/min的60Co-γ射线辐射，对辐射植株自交后代进行农艺性状及根尖细胞有丝分裂中期染色体形态观察和条锈病抗性鉴定。结果为辐射植株自交结实率为2.22%，根尖细胞有丝分裂中期的染色体存在明显碎片，辐射自交后代植株对条锈病具有成株期抗性。 小麦—偏凸山羊草6Mv/6B代换系对条锈病抗性稳定，是培育条锈病抗性品种的良好供体。本研究证明在四川小麦背景中要利用该品种抗性，在结实数满足需要时，可将其作父本，亦可作母本，但关键是要选择好一个优良的受体基因型；同时其条锈病抗性与来自簇毛麦的白粉病抗性没有相互抑制作用，可将两者抗性有效聚合用于小麦育种中。 Wheat stripe rust (Puccinia striiformis f. sp. Tritici) is a worldwide disease of wheat, and could lead to victims of 30 percent or even total destruction of wheat production. Wheat stripe rust harms badly in China's southwest and North China. Sichuan province is one of the regions damaged by wheat stripe rust heavily. The use of resistant varieties is the most secure and economical way to control the wheat stripe rust. Therefore, it is essential to identify new disease-resistant genes and genetically research of disease resistance. Aegilops ventricosa (DDMvMv, 2n = 28) is an annual herbaceous plant, originating in the coastal areas of the western Mediterranean, with good characters such as resistance of wheat powdery, rust, salt, cold and high protein content. It is a good germplasm resource. In this study, the wheat- Aegilops ventricosa 6Mv/6B substitution line Moisson 6Mv/6B (highly resistant to the wheat stripe rust) was used to study on the transmission of chromosome 6Mv of Aegilops ventricosa in different genetic background of Sichuan wheat varieties, hybridization with wheat- Haynaldia villosa ditelosomic addition line Pana (highly resistant to the powdery mildew) and screening of wheat- Aegilops ventricosa translocation line by exposuring Moisson 6Mv/6B under ionizing radiation. The main results are as following: 1. Moisson 6Mv/6B was crossed with Sichuan wheat varieties mianyang26, mianyang93-124 and SW3243 (highly susceptible to stripe rust), respectively. Their F1 hybrids were further backcrossed as male and female to corresponding wheat varieties. The seed-setting rate, chromosomes confirmation in the mitotic metaphase of root tip cells, and resistance to stripe rust of the subsequent BC1 and F2 plants were investigated. The average seed-setting rate of backcross via 6Mv as female donor (83.10%) was higher than that of backcross via 6Mv as male donor (48.61%), suggesting that the seed-setting rate was associated with the wheat genotypes（χ2=34.15>>χ20.05=5.99（df=2））. In all analyzed populations, transmission frequencies of chromosome 6Mv were not correlated with the ways of 6Mv through male or through female. However, transmission frequencies of chromosome 6Mv were significantly correlated with Sichuan wheat genotypes（χ2=6.42>χ20.05=5.99（df=2））. 2. To aggregating the resistances to stripe rust and powdery mildew, as well as research on the resistance reactions in different genetic background, Moisson 6Mv/6B was reciprocally hybrided with the wheat- Haynaldia villosa ditelosomic addition line Pana (highly resistant to the powdery mildew). The seed-setting rate, agronomic characters, genomic in situ hybridization (GISH) of hybrid progenies，and resistances to stripe rust and powdery mildew were investigated. The results showed that the seed-setting rate of hybridization via Moisson 6Mv/6B as female donor (80.56%) was significant higher than that via Pana as female donor (58.33%). The seed-setting rate was associated with the hybrid methods (χ2 = 4.96> χ20.05 = 3.84 (df = 1)). The plant height of hybrid progenies was about 10 cm higher than Pana, the parent with maximum height. And the maturity of hybrid progenies was about two weeks earlier than that of the parents. In the hybrid progenies, the plants with the 6Mv chromosome have the resistance to stripe rust and the plants with the telosome from Haynaldia villosa have the resistance to powdery mildew. It was found that four plants with both the 6Mv chromosome and the telosome from Haynaldia villosa were resistant to stripe rust and powdery mildew. It indicated that the resistance to stripe rust and powdery mildew aggregated, and no mutual inhibition was found. It implied that the aggregation of the two resistance genes was able to provide the new wheat germplasm with the resistances to stripe rust and powdery mildew. 3. Moisson 6Mv/6B was irradiated with 60Co-γ rays of 6Gy (120rad/min) during meiosis. The agronomic characters and chromosomes confirmation in the mitotic metaphase of root tip cells，as well as resistance to stripe rust were investigated. The seed-setting rate of irradiated plants was only 2.22%. The chromosomes in mitotic metaphase had clear fragments. The resistance to stripe rust of progeny of irradiated plants was the adult-plant resistance. The wheat- Aegilops ventricosa 6Mv/6B substitution line is a good stripe rust resistance donor for its stabile resistance. Our study demonstrated that the key for use the resistance is to choose a good receptor. There is no difference between Moisson 6Mv/6B be the female and be the male if the seed number meets the requirement. At the same time, the stripe rust resistance of Moisson 6Mv/6B did not have the mutual inhibition with the powdery mildew resistance from Haynaldia villosa. It is able to aggregate the two resistances for wheat breeding.

簇毛麦（ Dasypyrum villosum）矮秆突变体的研究

株高是农作物的重要农艺性状之一，适度矮化有利于农作物的耐肥、抗倒、高产等。20世纪50年代，以日本的赤小麦为矮源的半矮秆小麦的培育和推广，使得世界粮食产量显著增长，被誉为“绿色革命”。迄今为止，已报到的麦类矮秆、半矮秆基因已达70多个，但由于某些矮源极度矮化或者矮化的同时伴随不利的农艺性状，使得真正运用于育种实践的矮源较少。因此，发掘和鉴定新的控制麦类作物株高的基因，开展株高基因定位、克隆及作用机理等方面的研究，对实现麦类作物株高的定向改良，具有重要的理论意义和应用价值。簇毛麦(Dasypyrum villosum，2n＝14，VV)是禾本科簇毛麦属一年生二倍体异花授粉植物，为栽培小麦的近缘属。本课题组在不同来源的簇毛麦杂交后代中发现了一株自然突变产生的矮秆突变体。观察分析了该突变体的生物学特性，对矮秆性状进行了遗传分析，对茎节细胞长度、花粉的活力进行了细胞学观察，考察了该突变体内源赤霉素含量及不同浓度外施赤霉素对突变体的作用，分析了赤霉素生物合成途径中的内根贝壳杉烯氧化酶（KO）和赤霉素20氧化酶(GA20ox)的转录水平，对赤霉素20氧化酶和赤霉素3-β羟化酶(GA3ox)进行了克隆和序列分析，并对GA20ox进行了原核表达和表达的组织特异性研究。主要研究结果如下：1. 该突变体与对照植株在苗期无差异，在拔节后期才表现出植株矮小，相对对照植株，节间伸长明显受到抑制，叶鞘长度基本不变。在成熟期，对照植株的平均株高为110cm，而突变株的平均株高为32cm，仅为对照植株的1/3 左右。除了株高变矮以外，在成熟后期，突变株还表现一定程度的早衰和雄性不育。I2-KI染色法观察花粉活力结果表明，对照植株花粉90%以上都是有活力的，而突变植株的花粉仅20%左右有活力。2. 突变株与对照植株的杂交F1代均表现正常株高，表明该突变性状为隐性突变。F1代植株相互授粉得到的168株F2代植株中，株高出现分离，正常株高（株高高于80cm）与矮秆植株（株高矮于40cm）的株数比为130：38，经卡方检验，其分离比符合3：1的分离比，因此推测该突变体属于单基因的隐性突变。3. 用ELISA方法检测突变株和对照植株的幼嫩种子中内源性生物活性赤霉素（GA1＋3）含量，结果表明突变株的赤霉素含量为36 ng/ml，而对照植株的赤霉素含量为900 ng/ml。对突变株外施赤霉素，发现矮秆突变株的株高和花粉育性均可得到恢复。这些结果表明该突变株为赤霉素缺陷型突变。4. 用荧光定量PCR方法比较突变株与对照植株中内根贝壳杉烯氧化酶和赤霉素20氧化酶的转录水平，结果表明突变株的KO转录水平比对照植株分别提高了6倍（苗期）和16倍（成熟期），突变株的GA20ox转录水平与对照植株在苗期无明显差异，在成熟期突变株较对照植株则提高了10倍左右。这些结果表明该矮秆突变体与赤霉素的生物合成途径密切相关，而且极有可能在赤霉素的生物合成途径早期就发生了改变。5. 以簇毛麦总基因组为模板，同源克隆了GenBank登录号为EU142950，RT-PCR分离克隆了簇毛麦的GA3ox基因cDNA全长序列，分析结果表明该cDNA全长1206bp，含完整编码区1104bp，推测该序列编码蛋白含368个氨基酸残基，分子量为40.063KD，等电点为6.27。预测的氨基酸序列含有双加氧酶的活性结构，在酶活性中心2个Fe离子结合的氨基酸残基非常保守。该序列与小麦、大麦和水稻的GA3ox基因一致性分别为98%、96%、86%。基因组序列与cDNA序列在外显子部分一致，在478-715bp和879-1019bp处分别含238bp和140bp的内含子。6. 通过RT-PCR技术克隆了簇毛麦的GA20ox基因全长，命名为DvGA20ox，GenBank登录号为EU142949。该基因全长1080个碱基，编码359个氨基酸，具有典型的植物GA20ox基因结构。该基因编码的蛋白质与小麦、大麦、黑麦草等GA20ox蛋白的同源性分别为98%，97% 和91%。该序列重组到原核表达载体pET-32a(+)上，将获得的重组子pET-32a(+)-DvGA20ox转化大肠杆菌BL21pLysS后用IPTG进行诱导表达。SDS-PAGE分析表明，DvGA20ox基因在大肠杆菌中获得了高效表达，融合蛋白分子量为55kDa。定量PCR分析表明，该基因在簇毛麦不同器官中的表达差异明显：叶片中表达水平最高，根部表达水平次之，茎部和穗中表达较弱。在外施赤霉素后，该基因的表达水平在两小时以后急剧下降，表明该基因的表达受自身的反馈调节。本研究结果认为，(1)该簇毛麦矮秆突变体为单基因的隐性突变；(2)该矮秆突变体为赤霉素敏感突变，内源赤霉素含量检测表明突变体的内源性赤霉素含量仅为对照植株的1/30；(3)荧光定量PCR结果表明突变株的赤霉素生物合成途径的关键酶基因表达水平比对照植株高，而且突变植株的赤霉素生物合成改变很可能发生在赤霉素生物合成途径的早期；(4)GA20ox有表达的组织特异性，且受到自身产物的反馈调节。 Plant height is an impotrant agronomic trait of triticeae crops.Semi-dwarf cropcultivars, including those of wheat, maize and rice, have significantly increased grainproduction that has been known as “green revolution”. The new dwarf varieties couldraise the harvest Index at the expense of straw biomass, and, at the sametime, improvelodging resistance and responsiveness to nitrogen fertilizer. Moreover, dwarf traits ofplant are crucial for elucidating mechanisms for plant growth and development aswell. In many plant species, various dwarf mutants have been isolated and theirmodles of inheritance and physiology also have been widely investigated.The causesfor their dwarf phenotypes were found to be associated with plant hormones,especially, gibberellins GAs.Dasypyrum villosum Candargy (syn.Haynaldia villosa) is a cross-pollinating,diploid (2n = 2x = 14) annual species that belongs to the tribe Triticeae. It is native toSouthern Europe and West Asia, especially the Caucasuses, and grows underconditions unfavorable to most cultivated crops. The genome of D. villosum,designated V by Sears, is considered an important donor of genes to wheat for improving powdery mildew resistance, take-all, eyespot, and plant and seed storageprotein content. A spontaneous dwarf mutant was found in D. villosum populations.The biological character and modles of inheritance of this dwarf mutant are studied.The cell length of stem cell is observed. The influence of extraneous gibberellin tothe dwarf mutant is also examined; the transcript level of key enzyme of gibberellinbiosynthesis pathway in mutant and control plants is compared. GA3ox and GA20oxare cloned and its expression pattern is researched.1. The dwarf mutant showed no difference with control plants at seedlingstage.At mature stage, the average height of control plants were 110cm and the dwarfplants were 33cm. The height of the mutant plant was only one third of the normalplants due to the shortened internodes. Cytology observation showed that theelongation of stem epidermal and the parenchyma cells were reduced. The dwarfmutant also shows partly male sterile. Pollen viability test indicates that more than80% of the pollen of the mutant is not viable.2. The inheritance modle of this dwarf mutant is studied. All The F1 plantsshowed normal phenotype indicating that the dwarfism is controlled by recessivealleles. Among the 168 F2 plants, there are 130 normal plants and 30 dwarf plants, thesegregation proportion accord with Mendel’s 3:1 segregation. We therefore proposethat this dwarf phenotype is controlled by a single recessive gene.3. Quantitative analyses of endogenous GA1+3 in the young seeds indicated thatthe content of GA1+3 was 36ng/ml in mutant plants and 900ng/ml in normal plants.The endogenous bioactive GA1+3 in mutant plants are only about 1/30 of that innormal plants. In addition, exogenously supplied GA3 could considerably restore themutant plant to normal phenotype. These results showed that this mutant wasdefective in the GA biosynthesis.4. More than ten enzymes are involved in GA biosynthesis. KO catalyzes thefirst cytochrome P450-mediated step in the gibberellin biosynthetic pathway and themutant of KO lead to a gibberellin-responsive dwarf mutant. GA20ox catalyze therate-limited steps so that their transcript level will influence the endogenous GAbiosynthesis and modifies plant architecture. The relative expression levels of genesencoding KO and GA20ox were quantified by real time PCR to assess whether thechanges in GA content correlated with the expression of GA metabolism genes andwhere the mutant occurred during the GA biosynthesis pathway. In mutant plants,the transcript levels of KO increased about 6-fold and 16-fold at the seedling stage and elongating stage respectively comparing with the normal plants. For theseedlings, there was no notable difference in the expression of GA20ox betweenmutant and normal plants. At the elongating stage, GA20ox transcript increased 10times in mutant plants, suggesting that the GA biosynthesis pathway in mutant plantshad changed from the early steps rather than the late steps.5. A full length cDNA of D. villosum gibberellin 3β-hydroxylase homology(designated as DvGA3ox) was isolated and consisted of 1206bp containing an openreading frame of 1104bp encoding 368 predicted amino acid residues. Identityanalysis showed that the gibberellin 3β-hydroxylase nucleotide sequence shared 98%,96% and 86% homology with that of wheat, barley and rice. The predicted peptidecontained the active-site Fe of known gibberellin 3β-hydroxylase and the regionhomologous to wheat, barley and Arabidopsis. The genomic clone of gibberellin3β-hydroxylase has two introns.6. The full-length cDNA of D. villosum gibberellin 20 oxidase (designated asDvGA20ox) was isolated and consisted of 1080-bp and encoded 359 amino acidresidues with a calculated mol wt of 42.46 KD. Comparative and bio-informaticsanalyses revealed that DvGA20ox had close similarity with GA20ox from otherspecies and contained a conserved LPWKET and NYYPXCQKP regions. Tissueexpression pattern analysis revealed DvGA20ox expressed in all the tissues that wereexamined and the highest expression of DvGA20ox in expanding leaves followed byroots. Heterologous expression of this cDNA clone in Escherichia coli gave a fusionprotein that about 55KD. Transcript levels of DvGA20ox dramatically reduced twohours after application of biologically active GA3, suggesting that the biosynthesis ofthis enzymes might be under feedback control.

Characterization of airborne carbonate over a site near Asian dust source regions during spring 2002 and its climatic and environmental significance

IEECAS SKLLQG