不同抗旱性青稞LEA2/LEA3蛋白基因的克隆与表达

作物的抗旱性是一个多基因控制的、极为复杂的数量性状，植物对干旱在分子水平上的差异反应通过植物组织生理和细胞生物学水平，最终表现为植物抗旱性的不同。在我国，旱地农业超过耕地面积的50%，但水资源短缺，因此培育和选育抗旱高产作物是发展节水型农业最有效的途径。 青藏高原气候恶劣、年均降雨量少，也是世界大麦初生起源中心，因而蕴藏了十分丰富的与抗逆相关的种质资源材料，从这些特殊的资源材料克隆抗旱基因，不仅对培育抗旱、优质、高产大麦新品种具有重要理论意义和经济价值，而且对整个作物抗旱基础和育种应用研究都具重大促进作用。 为了筛选青稞（裸大麦，Hordeum vulgare ssp. vulgare）抗旱性材料，本研究选用来自青藏高原不同地区的84份青稞为材料，在叶片失水率(water loss rate, WLR)检测分析的基础上，选择失水率值差异显著的12个品种，通过相对含水量（relative water content, RWC）和反复干旱法评价其抗旱性，并通过植株对干旱胁迫下的丙二醛（MDA）含量和游离脯氨酸（free-proline）含量变化，了解不同抗旱性材料的生理反应特性。选择抗旱性强弱不同的品种各两份进行LEA2蛋白基因(Dhn6基因)、LEA3蛋白基因(HVA1基因)的克隆，比较LEA蛋白结构差异与作物抗旱性之间的关系。同时，对抗旱性不同的青稞品种受到干旱时间不同的失水变化率（dynamics water loss rate, DWLR）进行了检测；对抗旱性不同的青稞对照材料进行2 h、4 h、8 h和12 h的快速干旱处理，通过SYBR Green实时荧光定量RT-PCR技术对Dhn6基因、Dhn11基因、Dhn13基因和HVA1基因在不同抗旱性材料受到不同干旱时间处理后的相对表达水平进行了检测。本研究对LEA蛋白基因在抗旱性不同的青稞材料中的干旱胁迫分子水平上的差异反应进行了研究，也对植物的抗旱机理进行了初步探讨。主要研究结果如下： 1. 青稞苗期进行离体叶片失水率测定结果表明，来自青藏高原的84份青稞材料的WLR在0.086~0.205gh-1g-1DW之间。选择WLR低于0.1gh-1g-1DW和WLR高于0.18gh-1g-1DW的品种各6份，并对苗期分别进行未干旱及干旱12小时的处理。相对含水量检测结果表明，低失水率青稞材料干旱后的具有更高的相对含水量，盆栽缺水试验也显示叶片失水率低的材料耐旱能力强于失水率高的材料。通过水合茚三酮法测定离体叶片游离脯氨酸的含量，结果表明，所有品种未干旱处理时，游离脯氨酸含量差异不大（17.10~25.74 µgg-1FW）；干旱12小时后，低失水率的品种游离脯氨酸含量明显增高（32.99~53.45µgg-1FW），高失水率品种的游离脯氨酸含量与干旱前变化不明显(P<0.05)。硫代巴比妥酸法测定离体叶片丙二醛(MDA)含量，结果显示，12份所选对照品种中，丙二醛的含量在0.97~2.74nmolg-1FW，干旱12小时后丙二醛的含量显著上升（1.46~4.74nmolg-1FW），高失水率的6个品种的丙二醛含量在未干旱和干旱处理时都明显高于低WLR品种。本研究结果表明青稞的低失水率、低丙二醛含量、高相对含水量和高脯氨酸含量具相关性（P<0.05）。综上研究，我们认为作物失水率的测定可以作为快速检测作物抗旱性的指标之一，因此，强抗旱品种喜玛拉10号(TR1)、品比14号(TR2)和弱抗旱品种冬青8号(TS1)、QB24 (TS2)被选作抗旱基因克隆和表达分析的研究材料。 2. 高等植物胚胎发育晚期丰富蛋白(late embryogenesis abundant proteins, LEA proteins)与植物耐脱水性密切相关，为了探讨青稞LEA蛋白结构差异性与植物抗旱性的关系，本研究以强抗旱品种（喜玛拉10号、品比14号）和弱抗旱品种（冬青8号、QB24）为材料，利用同源克隆法，通过RT-PCR，分别克隆了与抗旱性密切相关的Dhn6基因和HVA1基因。Dhn6基因序列分析结果表明，强抗旱品种品比14号和弱抗旱品种冬青8号Dhn6基因所克隆到的序列为1026bp，它们之间只有5个碱基的差异；喜玛拉10号和QB24克隆到的序列长963bp。在强弱不同的抗旱品种中有22个核苷酸易突变位点，相应的脱水素氨基酸序列推导结果表明，22个核苷酸突变位点中，仅有8个位点导致相应的氨基酸残基的改变，其余的位点系同义突变，另外，21个富含甘氨酸序列的缺失并没有联系作物抗旱性特征。推测这些同义突变位点的氨基酸残基对维持青稞DHN6蛋白的正常结构和功能起着非常重要的作用，也可能DHN6蛋白对青稞长期适应逆境胁迫和遗传进化的结果。对HVA1基因的序列分析结果表明，冬青8号、QB24、品比14号和喜玛拉10号的目的基因核苷酸序列全长分别为661bp、697bp、694bp和691bp，它们都包含1个完整的开放阅读框。相应的LEA3蛋白氨基酸序列结果表明，11个高度保守的氨基酸残基组成基元重复序列的拷贝数与青稞抗旱性之间没有必然关系，在强抗旱品种（喜玛拉10号、品比14号）中三个共同的氨基酸突变位点Gln32、Arg33和Ala195可能对抗旱蛋白的结构和功能有影响；另外，强抗旱青稞品种LEA3蛋白质中11-氨基酸保守基元序列拷贝数和极性氨基酸占蛋白的比例更高，推测LEA3蛋白中基元序列拷贝数和极性氨基酸占蛋白的比例对该蛋白的结构和功能影响更大。 3. LEA蛋白基因的表达水平的上调与植物的耐脱水性密切相关，我们对强抗旱性材料（喜玛拉10号、品比14号）和弱抗旱材料（冬青8号、QB24）进行干旱处理2 h、4 h、6 h、8 h和10 h的失水变化率进行测定，结果表明弱抗旱品种在2~4小时之间失水率变化最明显，而四个对照品种的失水率在8小时后和24小时的失水率值变化不大。进一步提取青稞苗期进行2 h、4 h、8 h和12 h的干旱处理后的总RNA，通过SYBR Green实时荧光定量RT-PCR技术对青稞脱水素基因(Dhn6、Dhn11和Dhn13)和LEA3蛋白基因(HVA1)的相对表达水平受干旱时间和作物抗旱性的影响进行了检测。研究发现，抗旱性不同的青稞品种随干旱处理的时间延长，Dhn6、Dhn11、Dhn13和HVA1基因的相对表达水平不同。 Dhn6基因的相对表达水平在强抗旱青稞品种干旱8小时后快速上升，但在弱抗旱青稞品种干旱处理12小时后检测到更高表达量；Dhn11基因在对照青稞抗旱品种的表达累积水平随干旱时间的延长持续下降；整个干旱过程中，Dhn13基因的相对表达水平在弱抗旱品种持续上升，在强抗旱品种中干旱处理8小时快速上升并达到最高，干旱12小时后降低。与脱水素基因相比较，强抗旱青稞品种在干旱2小时后HVA1基因的相对表达水平显著升高，相对表达量随干旱处理的时间持续上升，在干旱12小时后达到最高；与之相比较，在整个干旱过程中，弱抗旱品种的相对表达水平显著低于强抗旱品种，在干旱8小时之前弱抗旱品种的相对表达水平变化不明显；在干旱8~12小时后却显著上升。上述结果表明，不同的LEA蛋白在植物耐脱水过程中的干旱表达累积水平不同；干旱不是诱导高等植物Dhn11基因表达的主要因素；植物的抗旱性不同，不同LEA蛋白基因对干旱的反应有差异。推测某些LEA蛋白基因的干旱胁迫早期表达累积程度与植物的抗旱性直接相关；其中，Dhn11基因和Dhn12基因不同的表达模式可能与干旱调控表达顺式作用成分(dehydration responsive element, DRE)的有无或结构上的差异有关。 本研究结果认为，(1)失水率和相对含水量可作为植物抗旱性检测的指标之一；(2) DHN6同义突变位点的氨基酸残基对维持该蛋白的正常结构和功能起着重要作用；(3) 11-氨基酸保守基元序列拷贝数和极性氨基酸的比例对LEA3蛋白结构和功能有重要影响；(4)LEA蛋白表达随着干旱胁迫程度而增加，但Dhn11基因并不受干旱诱导表达；(5)作物的抗旱性不同，LEA蛋白对干旱的累积反应并不相同，干旱早期LEA蛋白的累积程度可能会影响植物的抗旱性。 Drought resistance was a complex trait which involved multiple physiological and biochemical mechanisms and regulation of numerous genes. Because its complex traits, it is difficult to understand the mechanisms of drought resistance in plants. Plants respond to water stress through multiple physiological mechanisms at the cellular, tissue, and whole-plant levels. Tibetan hulless barley, a pure line, is a selfing annual plant that has predominantly penetrated into the Qinghai-Tibetan Plateau and remains stable populations there. The wide ecological range of Tibetan hulless barley differs in water availability, temperature, soil type and vegetation, which makes it possess a high potential of adaptive diversity to abiotic stresses. This adaptive genetic diversity indicates that the potential of Tibetan hulless barley serves as a good source for drought resistance alleles for breeding purposes. 12 contrasting drought-tolerant genotypes were selected to measure relative water content (RWC), maldondialdehyde (MDA) and proline content, based on values of water loss rate (WLR) and repeated drought methods from Tibetan populations of cultivated hulless barley. As a result of the screening, sensitive and tolerant genotypes were identified to clarify relationships between characteristics of LEA2/LEA3 genes sequences and expression and drought-tolerant genotypes, associated with resistance to water deficit. In addition, dynamics water loss rate (DWLR) was measured to observe the changes on diffrential drought-tolerant genotypes. Real-time quantitative RT-PCR was applied to detect relative expression levels of Dhn6, Dhn11, Dhn13 and HVA1 genes in sensitive and tolerant genotypes with 2 h, 4 h, 8h and 12 h of dehydration. In the present study, differential sequences and expression of LEA2/LEA3 genes were explored in Tibetan hulless barley, associated with phenotypically diverse drought-tolerant genotypes. 1. The assessments of WLR and RWC were considered as an alternative measure of plant water statues reflecting the metabolic activity in plants, and the parameters of MDA and proline contents were usually consistent with the resistance to water stress. The values of detached leaf WLR of the tested genotypes were highly variable among 84 genotypes, ranging from 0.086 to 0.205 g/h.g DW. The 12 most contrasting genotypes (6 genotypes with the lowest values of WLR and 6 genotypes with the highest values of WLR) were further validated by measuring RWC, MDA and free-proline contents, which were well watered and dehydrated for 12 h. Results of RWC indicated that the values of 12 contrasting genotypes RWC ranged from 89.94% to 93.38% under condition of well water, without significant differences, but 6 genotypes with lower WLR had higher RWC suffered from 12 h dehydration. The results indicated that lower MDA contents, lower scores of WLR and higher proline contents were associated with drought-tolerant genotypes in hulless barley. Remarkably, proline amounts were increased more notable in 6 tolerant genotypes than 6 sensitive genotypes after excised leaves were dehydrated for 12 h, with control to slight changes under condition of well water. Results of MDA contents showed that six 6 tolerant genotypes had lower MDA contents than the 6 sensitive genotypes under both stressed and non-stressed conditions. As a result of that screening, drought- resistant genotypes (Ximala 10 and Pinbi 14) and drought-sensitive genotypes (Dongqing 8 and QB 24) were chosen for comparing the differential characteristics of LEA2/LEA3 genes and their expression analysis. It was conclusion that measurements of WLR could be considered an alternative index as screening of drought-tolerant genotypes in crops. 2. Late embryogenesis abundant (LEA) proteins were thought to protect against water stress in plants. To explore the relationships between configuration of LEA proteins and phenotypically diverse drought-tolerant genotypes, sequences of LEA genes and their deduced proteins were compared in Tibetan hulless barley. Results of comparing Dhn6 gene in Ximala 10 and QB24 indicated that absence of 63bp was found, except that only 5 mutant nucleotides were found. While 22 mutant sites were taken place in Dhn6 gene between sensitive and tolerant lines, 14 synonymous mutation sites appeared in the contrasting genotypes. The additional/absent polypeptide of 21 polar amino acid residues was not consistent with phenotypically drought-tolerant genotypes in hulless barley. It was deduced that synonymous mutation sites would play important roles in holding out right configurations and functions on DHN6 protein. The sequencing analysis results indicated that each cloned HVA1 gene from four selected genotypes contained an entire open reading frame. The whole sequence of HVA1 gene from Dongqing 8, QB24, Pinbi 14 and Ximala 10 was respectively 661bp, 697bp, 694bp and 691bp. Results of DNA sequence analyses showed that the differences in nucleotides of HVA1 gene in sensitive genotypes were not consistent with that of tolerant genotypes, except for absence of 33 nucleotides from +154 to +186 (numbering from ATG) in QB24. Database searches using deduced amino acid sequences showed a high homology in LEA3 proteins in the selected genotypes. Multiple sequence alignments revealed that LEA3 protein from Dongqing 8 was composed of 8 repeats of an 11 amino acid motif, less the fourth motif than Pinbi 14, Ximala 10 and QB24. Consistent mutant amino acid residues appeared in contrasting genotypes by aligning and comparing the coding sequence region, including Gln32, Arg33 and Ala195 in tolerant genotypes as compared to Asp32, Glu33 and Thr195 (Thr184 in Dongqing 8) in sensitive lines. It was concluded that consistent appearance of Gln32, Arg33 and Ala195 would contributed to functions of LEA3 protein in crops, as well as higher proportion of 11-amino-repeating motifs and polar amino acid residues. 3. Most of the LEA genes are up-regulated by dehydration, salinity, or low temperature, are also induced by application of exogenous ABA, which increases in concentration in plants under various stress conditions and acts as a mobile stress signal. Higher levels of proteins of LEA group 3 accumulated was correlated well with high level of desiccation tolerance in severely dehydrated plant seedlings. Dehydrins (DHNs), members of LEA2 protein, are an immunologically distinct protein family, and Dhn genes expression is associated with plant response to dehydration. Dynamic water loss rate was measured between sensitive genotypes and tolerant genotypes after they were dehydrated for 2 h, 4 h, 6h and 8 h. Detailed measurements of WLR at the early stage of dehydration (2, 4, 6, and 8 h) showed that WLR was stabilizing after 8 h, and there were no significant changes between these values and WLR after 24 h. Drought stress was applied to 10-day-old seedlings by draining the solution from the container for defined dehydration periods. Leaf tissues of the selected genotypes were harvested from control plants (time 0); and after 2, 4, 8, and 12 h of dehydration. Differential expression trends of Dhn6, Dhn11, Dhn13 and HVA1 genes were detected in phenotypically diverse drought-tolerant hulless barleys, related to different time of dehydration. Results of quantitative real-time PCR indicated that relative level of HVA1 expression was always higher in tolerant genotypes, rapidly increasing at the earlier stages (after 2-4 h of dehydration). However, HVA1 expressions of sensitive genotypes had a fast increase from 8 h to 12 h of stress. Significant differences in expression trends of dehydrin genes between tolerant genotypes and sensitive lines were detected, mainly in Dhn6 and Dhn13 gene, depending on the duration of the dehydration stress. The relative expression levels of Dhn6 gene were significantly higher in tolerant genotypes after 8 h dehydration, by control with notable higher expression levels after 12 h water stress in sensitive ones. The relative expression levels of Dhn13 gene tended to ascend during exposure to dehydration in drought-sensitive genotypes. However, fluctuate trends of Dhn13 expression level were detected in drought-resistant lines, including in lower expression levels of 12 h dehydration as compared to 8 h water stress. It was conclusion that (1) diverse LEA proteins would play variable roles in resisting water stress in plants; (2) expression of Dhn11 gene was not induced by dehydrated signals because of the trends of expression descended in contrasting genotypes suffered from water deficit and (3) variable accumulations on LEA proteins would be appear in diverse drought-tolerant genotypes during dehydrations. It is deduced that higher accumulations of Dhn6 and Dhn13 expression in 8 h dehydration are related to diverse drought-tolerant lines in crops. The present results indicated that different dehydrin genes would play variable functional roles in resisting water stress when plants were suffered from water deficit. The authors suggest physiologically different reactions between resistant and sensitive genotypes may be the results of differential expression of drought-resistant genes and related signal genes in plants. In addition, contrarily induced expression of Dhn11 and Dhn12 was related to dehydration responsive element (DRE) in barleys. The present study indicated that (1) measurements of WLR and RWC could be considered as one index of drought-tolerant screenings; (2) synonymous mutation sites would play important roles in holding out right configurations and functions on DHN6 protein, (3) higher proportion of 11-amino-repeating motifs and polar amino acid residues would contribute to functions on LEA3 protein, (4) the longer drought, the more accumulation on LEA proteins, except for Dhn11 gene in crops and (5) differential responses on expression of LEA protein genes would result in physiological traits of drought tolerance in plants.

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.

Recent developments in intelligent biomedical polymers

Intelligent polymers or stimuli-responsive polymers may exhibit distinct transitions in physical-chemical properties, including conformation, polarity, phase structure and chemical composition in response to changes in environmental stimuli. Due to their unique 'intelligent' characteristics, stimuli-sensitive polymers have found a wide variety of applications in biomedical and nanotechnological fields. This review focuses on the recent developments in biomedical application of intelligent polymer systems, such as intelligent hydrogel systems, intelligent drug delivery systems and intelligent molecular recognition systems. Also, the possible future directions for the application of these intelligent polymer systems in the biomedical field are presented.

A Novel Biodegradable and Light-Breakable Diblock Copolymer Micelle for Drug Delivery

A facile approach to the preparation of light-responsive copolymer micelles is developed. This approach is based on the attachment of hydrophobic groups to one block of a diblock copolymer via a light-sensitive linkage. The micelles can be dissociated under light irradiation and release the encapsulated pyrene. The obtained polymeric micelles are expected to be of use as drug-delivery vehicles.

Synthesis and Characterization of Hyperbranched Poly(ether amide)s with Thermoresponsive Property and Unexpected Strong Blue Photoluminescence

A facile and efficient strategy for the syntheses of novel hyperbranched poly(ether amide)s (HPEA) from multihydroxyl primary amines and (meth)acryloyl chloride has been developed. The chemical structures of the HPEAs were confirmed by IR and NMR spectra. Analyses of SEC (size exclusion chromatography) and viscosity characterizations revealed the highly branched structures of the polymers obtained. The resultant hyperbranched polymers contain abundant hydroxyl groups. The thermoresponsive property was obtained from in situ surface modification of abundant OH end groups with N-isopropylacrylamide (NIPAAm). The study oil temperature-dependent characteristics has revealed that NIPAAm-g-HPEA exhibits an adjustable lower critical solution temperature (LCST) of about 34-42 degrees C depending on the grafting degree. More interestingly, the work provided an interesting phenomenon where the HPEA backbones exhibited strong blue photoluminescence.

Tethered diblock copolymer chains on platelets prepared by semicrystalline ABC triblock copolymers in toluene with trace amounts of water

Lamellar platelets of triblock copolymers grown in dilute toluene solution with trace amounts of water can be used as templates for tethered diblock copolymer chain preparation and analysis. Polystyrene-bpoly(2-vinylpyridine)-b-poly(ethylene oxide) (PS-b-P2VP-b-PEO) with two different block fractions were used as model templates to generate tethered P2VP-b-PS chains on the platelet basal surfaces. In toluene solution the aggregation states of PS-b-P2VP-b-PEO were sensitive to the water content in the solution. For toluene with trace amount of water, spherical micelles were formed in the early stage and large square platelets would gradually grow from these spherical micelles. The hydrogen bonding between water and EO units was responsible for the formation of micelles and subsequent square platelets in the solution. Tethered P2VP-b-PS chains on basal surface of PEO platelets could be regarded as diblock copolymer brushes and the density (or: 0.086-0.36) and height (d: 3.5-14.3 nm) of these tethered chains could be easily modulated by changing the crystallization condition and/ or the molecular weight of each block. The tethered P2VP-b-PS chains were responsive to different solvent vapor.

Direct formation of giant vesicles from synthetic polypeptides

This report describes direct formation of giant vesicles from a series of poly(L-lysine)-block-poly(L-phenylalanine) (PLL-b-PPA) block copolymers from their water solution. These polymers are prepared by successive ring-opening polymerization (ROP) of the two alpha-amino acid N-carboxyanhydrides and then removing the side chain protecting groups by acidolysis. The structures of the copolymers are confirmed by nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), and size exclusion chromatography ( SEC). The vesicles are studied by atomic force microscopy (AFM), field emission scanning electron microscopy (ESEM), and confocal laser scanning microscopy (CLSM). Rhodamine B is used as a fluorescent probe to confirm the existence of the vesicle with an aqueous interior. The vesicle size is in the range 0.55-6 mu m, depending on the absolute and relative lengths of the two blocks, on initial polymer concentration, and on solution pH. The vesicles are still stable in water for 2 months after preparation. Addition of the copolymer to DNA solution results in complex formation with it. The complex assumes the morphology of irregular particles of less than 2 mu m. It is expected to be used in drug and gene delivery.

Magnetic microsphere and its use in biochemical separation and analysis

Magnetic microsphere comprises a magnetically responsive metal or metal oxide core surrounded by a polymer shell with active groups. Nowadays, methods of directly coating polymer, monomer polymerazation, impregnation, extrusion and biological synthesis are generally used to prepare magnetic particles. This kind of superparamagnetic microspheres can be attached to chemical, biochemical and biological substances by their active groups, then applying a magnetic field to separate from the media. Preparation and utilization of magnetic microspheres in immunoassay, nucleic acid hybrization assay, gene sequencing, cell isolation, enzyme immoblization, receptor isolation and other Gelds are reviewed with 44 references in this paper. Also, the further development is outlooked.

Cloning and characterization of beta-carotene ketolase gene promoter in Haematococcus pluvialis

The unicellular green alga Haematococcus pluvialis accumulates a highly valuable ketocarotenoid, astaxanthin, under various environmental stresses. beta-carotene ketolase (BKT) plays a key role in astaxanthin biosynthesis in H. pluvialis. In this paper, an approximate 700 bp 5'-flanking region of the bkt gene containing a putative promoter was cloned through walking upstream. The results of the sequence analysis showed that this bkt 5'-flanking region might have cis-acting elements such as sterol regulatory element (SRE-1)-like motifs, the C-repeat/dehydration responsive element (DRE) and al-3 proximal element (APE)-like motifs, except for typical TATA and CCAAT boxes. The results of the P-galactosidase assay and the transient expression of lacZ driven by a series of sequential deletions revealed that a minimal promoter-like region might exist from -630 to -408 bp, and the highest promoter activity was observed to span the positions from -630 to -308 bp. The results of the site-directed mutagenesis of a C-repeat/DRE and two APE-like motifs in a promoter-like region (-630 to -308 bp) suggested that two APE-like motifs might be essential for transcriptional control of the bkt gene.

Cloning and expression of tumor necrosis factor (TNF alpha) cDNA from red seabream Pagrus major

A fragment of TNFalpha cDNA sequence from red seabream was cloned by homology cloning approach with two degenerated primers which were designed based on the conserved regions of other animals' TNF sequences. The sequence was elongated by 3' and 5' RACE to get the full length CDS sequence. This sequence contained 1264 nucleotides that included a 5' UTR of 85 bp, a 3' UTR of 514 bp and an open reading frame (ORF) of 666 bp which could encode 222 amino acids propeptide. In 3' UTR, there were several mRNA instability motifs and three endotoxin-responsive sequences, but the sequence lacked the polyadenylation signal. The deduced peptide had a clear transmembrane domain, a TNFalpha family signature and a TNF2 family profile. The cell attachment sequence and the glycosaminoglycan attachment sites were also found in the sequence. The red seabream TNF sequence shared relatively high similarity with both mammalian TNFalpha and TNFbeta by multiple sequence alignments. Phylogenetic analysis showed that the piscine TNFalpha were located independently in a different branch compared with mammalian TNFalpha and TNFbeta. Based on the primary and secondary structure analysis and gene expression study, we could concluded that the red seabream TNF should be a TNFalpha, not TNFbeta. RT-PCR was used to study TNFa transcript expression. 24 h after the red seabream was challenged by Vibrio anguillarum, the RS TNFalpha transcript expression were detected in blood, brain, gill, heart, head kidney, kidney, Ever, muscle and spleen. Results showed that TNFalpha mRNA was constitutively expressed in parts of the tissues both in stimulated and unstimulated fish and the expression could be enhanced after the pathogen infection.

Molecular cloning and characterization of proliferating cell nuclear antigen (PCNA) from Chinese shrimp Fenneropenaeus chinensis

The proliferating cell nuclear antigen gene was cloned from Fenneropenaeus chinensis (FcPCNA). The full-length cDNA sequence of FcPCNA encodes 260 amino acids showing high identity with PCNAs reported in other species. FcPCNA expressed especially high in proliferating tissues of shrimp such as haematopoietic tissue (HPT) and ovary. In order to understand the response of HPT to bacteria and virus challenge, mRNA level of FcPCNA in HPT was analyzed after shrimp were challenged by Vibrio anguillarum and white spot syndrome virus (WSSV). FcPCNA expression in HPT of shrimp was responsive to WSSV and Vibrio challenge, but different expression profiles were obtained after challenge by these two pathogens. The data provide additional information to understand the defense mechanisms of shrimp against virus and bacteria. (c) 2008 Elsevier Inc. All rights reserved.

Identification and molecular analysis of a stress-inducible Hsp70 from Sciaenops ocellatus

Hsp70 proteins are a family of molecular chaperones that are involved in many aspects of protein homeostasis. In this study, an Hsp70 homologue (SoHsp70) was identified from red drum Sciaenops ocellatus and analyzed at molecular level. The open reading frame of SoHsp70 is 1920 bp and intronless, with a 5'-untranslated region (UTR) of 399 bp and a 3'-UTR of 241 bp. The deduced amino acid sequence of SoHsp70 shares 84-92% overall identities with the Hsp70s of a number of fish species. In silico analysis identified in SoHsp70 three conserved Hsp70 domains involved in nucleotide and substrate binding. The coding sequence of SoHsp70 was subcloned into Escherichia coli, from which recombinant SoHsp70 was purified and, upon ATPase assay, found to exhibit apparent ATPase activity. Expressional analysis showed that constitutive expression of SoHsp70 was detectable in heart, liver, spleen, kidney, brain, blood, and gill. Experimental challenges with poly(I:C) and bacterial pathogens of Gram-positive and Gram-negative nature induced SoHsp70 expression in kidney to different levels. Stress-responsive analysis of SoHsp70 expression in primary cultures of red drum hepatocytes showed that acute heat shock treatment elicited a rapid induction of SoHsp70 expression which appeared after 10 min and 30 min of treatment. Exposure of hepatocytes separately to iron, copper, mercury, and hydrogen peroxide significantly unregulated SoHsp70 expression in time-dependent manners. Vaccination of red drum with a Streptococcus iniae bacterin was also found to induce SoHsp70 expression. Furthermore, recombinant SoHsp70 enhanced the immunoprotective effect of a subunit vaccine. Taken together, these results suggest that SoHsp70 is a stress-inducible protein that is likely to play a role in immunity and in coping with environmental and biological stresses. (C) 2010 Elsevier Ltd. All rights reserved.

Molecular cloning, characterization and evolutionary analysis of phytoene desaturase (PDS) gene from Haematococcus pluvialis

Phytoene desaturase is one of the most important enzymes necessary for the biosynthesis of carotenoids in some cyanobacteria, green algae and plants. In this study, genomic DNA and cDNA of pds were cloned from unicellular green alga Haematococcus pluvialis strain323 using PCR and RT-PCR methods. The cDNA was cloned into plasmid pET-28a and efficiently expressed in Escherichia coli BL21. The complete genomic PDS gene of H. pluvialis, 3.3 kb in size, included eight exons and seven introns. To locate transcriptional regulation elements, an approximate 1 kb of 5'-flanking region was isolated by genome-walking method. Results of bioinformatic analysis showed several putative cis-elements e.g. the ABRE motif (abscisic acid responsive element), the C-repeat/DRE (dehydration responsive element) motif and the GCN4 motif were located in 5'-flanking region of pds. Results of phylogenetic analyses reveal that different sources of PDS genes form a separate clade, respectively, with 100% bootstrap support. Moreover, a maximum likelihood approach was employed to detect evidence of positive selection in the evolution of PDS genes. Results of branch-site model analysis suggest that 7.9% of sites along the green algal branch are under positive selection, and the PDS gene in green algae exhibits a different evolutionary pattern from its counterparts in cyanobacteria and plants.

There are two 5 '-flanking regions of bkt encoding beta-carotene ketolase in Haematococcus pluvialis

The unicellular green alga Haematococcus pluvialis accumulates a commercially valuable astaxanthin, with levels reaching up to 4% dry weight under environmental stress. In recent years, much effort has been devoted to understanding the molecular mechanisms regulating astaxanthin biosynthetic pathways. Beta-carotene ketolase (bkt), with control being exhibited at the transcription level, plays an important role in astaxanthin biosynthesis by H. pluvialis. Here we demonstrate the presence of two separate 5'-flanking regions [1.5 kilobase (kb) and 2 kb] of bkt (bkt1 and bkt2) that possess regulatory elements similar to those of known stress-responsive genes in plants. Results of 5'-deletion constructs and transient beta-galactosidase expression assays demonstrate that there may be positive regulatory elements governing expression in the shorter promoter at -1060/-900 from the 1.5 kb 5' region, and in the longer promoter at -1838/-1219 and at -1046/ -734 from the 2 kb 5' region relative to each homologous ATG start codon. Furthermore, our present studies reveal that the first intron (+371/+497) downstream from the 1.5 kb 5' untranslated region of bkt1 may function as a negative regulatory element to regulate its own promoter.

Proteomic analysis of differentially expressed proteins in lymphoid organ of Fenneropenaeus chinensis response to Vibrio anguillarum stimulation

To gain an insight into the function of shrimp lymphoid organ at protein level, we analyzed the proteome of lymphoid organ in healthy Chinese shrimp Fenneropenaeus chinensis (F. chinensis) through two-dimensional gel electrophoresis (2-DE) based proteomic approach. A total of 95 spots representing 75 protein entries were identified by liquid chromatography tandem mass spectrometry (LC-MS/MS) with both online and in-house database. According to Gene Ontology (GO) annotation of biological process, the identified proteins were classified into 13 categories. Among them, approximately 36% of proteins related to cytoskeleton are noticeable. Then, a comparative proteomic approach was employed to investigate the differentially expressed proteins in lymphoid organ of Vibrio anguillarum-challenged F. chinensis. At 24 h post-injection (hpi), 17 differentially expressed protein spots were successfully identified, including 4 up-regulated protein spots (represent 4 proteins: cathepsin L protein similar to squid CG16901-PC, protein kinase C and protein similar to T-complex Chaperonin 5 CG8439-PA), and 13 down-regulated protein spots (represent 9 proteins: actin, beta-actin, cytoplasmic actin CyII, alpha tubulin, beta tubulin, protein similar to proteasome delta, vacuolar ATP synthase subunit B, elongation factor 2, carboxypeptidase B). These data may help us to understand the function of lymphoid organ and the molecular immune mechanism of shrimp responsive to pathogen infection. (C) 2010 Elsevier Ltd. All rights reserved.