Cd、Cu污染胁迫对拟南芥幼苗错配修复相关基因表达的影响

土壤重金属污染问题已成为影响我国持续农业和生态环境质量的重要因素，引起了人们的广泛关注。由于传统污染诊断方法的缺点，急需建立土壤污染生态毒理学诊断方法，生物标记物技术则是其中的研究热点之一。本文采用营养液培养的方法，以模式植物拟南芥为试材，采用半定量反转录聚合酶链式反应（RT-PCR）技术，结合传统分析方法研究了Cd、Cu在不同胁迫水平下对拟南芥幼苗的形态、生理及分子水平的毒性效应，并在此基础上，比较和分析不同测试指标对Cd、Cu胁迫响应的敏感性，进而筛选对Cd、Cu胁迫响应敏感的生物标记物。主要结果如下： 1 不同浓度Cd和Cu污染胁迫下，拟南芥幼苗生长均受到不同程度的影响 幼苗初生根伸长均受到明显抑制，而地上部叶片数、地上部鲜重却没有显著的变化。重金属首先作用于植物的根系，根系的生长对胁迫响应的敏感性高于地上部。 2 幼苗地上部的可溶性蛋白含量受到不同程度干扰，而在不同浓度的Cd、 Cu处理下，叶绿素含量变化不明显，表明幼苗地上部可溶性蛋白质含量对胁迫的敏感性高于叶绿素含量的变化。 3 幼苗地上部错配修复（MMR）和增殖细胞核抗原（PCNA）基因都明显 地出现了表达诱导或表达抑制，表明MMR和PCNA基因表达的变化对Cd、Cu胁迫表现出较高的敏感性。 4 幼苗地上部的可溶性蛋白质含量及幼苗地上部MMR和PCNA基因表达 均对Cd和Cu污染胁迫具有较高的敏感性，两者均可用于指示Cd和Cu污染的敏感生物标记物。基因表达变化图谱虽然对污染胁迫响应比较敏感，是一种污染胁迫响应敏感的生物标记物，但其在生态毒理诊断中的应用还需进一步的实验对其予以证明。

镉胁迫对拟南芥幼苗错配修复相关基因表达及突变的影响

土壤重金属污染是影响农业可持续发展和生态环境的重要问题，而通过生物标记物方式对污染土壤进行早期诊断已成为环境科学领域的研究热点。本文以M&S营养液为培养介质，以拟南芥为供试材料，以错配修复基因MutS 2 homolog (atMSH2)，atMSH3，atMSH7，细胞增殖核抗原1 和2 (atPCNA1和atPCNA2)为检测目的基因，分别采用半定量反转录-聚合酶链式反应（RT-PCR）技术、克隆及测序技术研究了Cd在不同胁迫水平（0，0.125，0.25，1.0，3.0 mg•L-1 ）上对上述错配修复相关基因表达和atMSH2基因突变的影响，并与拟南芥幼苗形态、生理指标的毒性效应进行比较分析，筛选出对Cd污染胁迫敏感的生物标记物。主要结果如下： 1. 不同浓度（0，0.125，0.25，1.0，3.0 mg•L-1 ）Cd处理7天后，拟南芥幼苗叶片数、地上部鲜重变化与对照相比差异均不显著；而根长随Cd胁迫强度的增加明显降低； 2. 不同浓度（0，0.125，0.25，1.0，3.0 mg•L-1 ）Cd处理7天后，叶绿素含量变化与对照相比差异均不显著； 地上部可溶性蛋白含量随Cd浓度的增加变化明显，0.125 mg•L-1 Cd处理下，地上部可溶性蛋白含量显著增加，而在0.25，1.0和3.0 mg•L-1 Cd时降低，但仍高于对照； 3. 地上部atMSH2，atPCNA1，atPCNA2基因表达量的变化与Cd胁迫浓度呈明显的倒U字型关系，分别在0.125mg•L-1，0.25mg•L-1和0.125mg•L-1 Cd时达到最大值。地上部可溶性蛋白含量变化趋势与atMSH2，atPCNA1，atPCNA2基因表达量的变化相似，均可作为对Cd污染胁迫敏感的潜在生物标记物。 4. 对不同浓度（0，0.125，0.25，1.0，3.0 mg•L-1 ）Cd处理7天后，拟南芥atMSH2基因PCR后的扩增产物进行回收、纯化、克隆和测序。测序结果表明，0.25 mg•L-1 Cd处理拟南芥atMSH2基因在第8个和第9个外显子之间的内含子有一个碱基转换；在1.0 mg•L-1 Cd处理下，拟南芥在第10个外显子有一个碱基转换。

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.

Cytogenetic Effects of Low Doses of Energetic Carbon Ions on Rice After Exposures of Dry Seeds, Wet Seeds and Seedlings

In order to investigate the biological effects of heavy ion radiation at low closes and the different radiosensitivities of growing and non-growing plants. rice at different lift stages (dry seed, wet seed and seedling) were exposed to carbon ions at closes of 0 02, 0.2, 2 and 20 Gy. Radiobiological effects on survival, root growth and mitotic activity, as well as the induction of chromosome aberrations in root meristem. were observed The results show that radiation exposure induces a stimulatory response at lower close and an inhibitory response at higher dose on the mitotic activity of wet seeds and seedlings Cytogenetic damages are induced in both seeds and seedlings by carbon ion radiation at doses as low as 0.02 Gy Compared with seedlings. seeds are more resistant to the lethal damage and the growth rate damage by high doses of carbon ions, but are more sensitive to cytogenetic damage by low closes of irradiation Different types of radiation induced chromosome aberrations are observed between seeds and seedlings. Based on these results, the relationships between low close heavy ion-induced biological effects and the biological materials are discussed.

细菌二氯甲烷脱氯酶基因对拟南芥的转化

The dichloromethane dehalogenase gene (dcm) of Methylophilus was cloned into the plasmid pKYLX71-35S2 and fused with the 35S2 promotor,then transformed Arabidopsis by the infiltration mediated with Agrobacterium tumefaciens. Homozygous dcm seeds were obtained after several generations selection on the medium with 50 mg L -1 kanamycin.Northern blotting showed dcm mRNA was high in cytoplasm and the detection of DCM enzyme indicated that the lines containing high dcm mRNA expressed high DCM enzyme activities. Fig 4, Ref 9

一种拟南芥突变体对高浓度CO_2反应的研究

The study on the response of a mutant and a wild type of Arabidopsis to 660 μl·L -1 CO 2 and ambient CO 2 showed that under elevated CO 2,the stomatal numbers of the mutant increased,while those of the wild type decreased. The chlorophyll content and NR (nitrate reductase) activity of the mutant increased,but those of the wild type had no obvious response. The mutant was not reproductively mature after the continuous exposure to increased CO 2 for five months. The results provided evidence of plant response to the changes of atmospheric CO 2 concentration,and the clues to related studies on other plants.