水稻丙酮酸激酶基因OsPK1的过表达转基因株系的获得与鉴定

利用农杆菌介导的转化方法研究OsPK1过表达对水稻的影响。将CaMV 35S启动子驱动的OsPK1的全长CDS序列导入到日本晴基因组中。选择3个过表达转基因系作为代表进行鉴定和分析。结果显示,过表达植株在株高、分蘖数、穗长和千粒重四方面接近野生型水稻,结实率略下降,但每穗饱满种子数明显增加。通过定量RT-PCR分析,发现有4个代谢酶在过表达植株嫩叶中表达存在着上调或下调。用GC-MS方法检测糖含量,显示OsPK1过表达株系中葡萄糖、果糖、半乳糖和蔗糖的含量与野生型差异不大。总之,对OsPK1过表达株系的鉴定,有助于更好了解OsPK1的功能,并且每穗饱满种子数明显增加具潜在的应用价值。     

表达豌豆过氧化锰歧化酶转基因水稻的耐旱性研究

本试验研究了一种重要抗氧化酶——过氧化锰歧化酶在水稻耐旱性方面的作用。在氧化胁迫诱导SWPA2启动子的控制下，采用农杆菌介导转化法，我们将豌豆的过氧化锰歧化酶（Mn SOD）转到了水稻叶绿体中，培育出了耐旱水稻植株。在PEG6000诱导的干旱胁迫条件下，转基因水稻植株（T1）中豌豆MnSOD进行了功能表达。在经聚乙二醇（PEG）处理后，不管是否置于甲基紫精（MV）中，转基因叶片切片的电解质渗漏与原始类型（WT）相比都减少了，这说明转基因植株更抗MV或PEG诱导的氧化胁迫。当用PEG处理后，转基因植株还表现出所受损伤较少的特点（采用净光合率测定）。我们的研究结果表明SOD是植物叶绿体中ROS清扫机制的关键性成分，MnSOD的表达可改善水稻耐旱性。     

OsWRKY67负向调控水稻耐旱性的功能分析

WRKY转录因子在水稻生物胁迫及非生物胁迫中均发挥重要作用。本实验室前期发现OsWRKY67正向调控水稻应答病害胁迫,为研究OsWRKY67在水稻非生物胁迫中的作用,本研究对低温、干旱、高盐、ABA、高渗透压和活性氧等多种非生物胁迫处理下的OsWRKY67基因的表达量进行测定,结果表明该基因的表达受上述非生物胁迫的影响。此外,本研究对OsWRKY67基因的过表达株系(OX5-2和OX6-5)、基因沉默株系(R9-4, R11-2和R14-2)和野生型‘日本晴’进行模拟干旱胁迫处理,结果表明沉默OsWRKY67基因可显著增强水稻的耐旱性,过表达OsWRKY67基因会显著降低水稻的耐旱性。进一步研究发现OsWRKY67能够调控部分耐旱相关基因(DREB1A, DREB1B, DREB2A和DREB2B)、ABA合成相关基因(NCED3和NCED4)及ABA通路相关基因(RaB16A, LIP9和LEA3)的表达。综合以上研究表明,OsWRKY67参与调控水稻响应多种非生物胁迫,通过ABA信号通路介导负向调控水稻苗期耐旱性。本研究揭示了OsWRKY67在水稻耐旱性中的调控作用,为利用该基因进...     

水稻OsURGT1基因的生物信息学及表达谱分析

NSTs在细胞中负责将核苷酸单糖转运到高尔基体(Golgi)/内质网(ER)中,为细胞壁多糖分子的生物合成提供底物,水稻NSTs基因的研究将为深入了解植物NSTs蛋白的功能奠定良好基础。通过拟南芥AtURGT基因序列的同源比对,在水稻中发现了一个可能具有转运UDP-鼠李糖/UDP-半乳糖活性的基因,命名为OsURGT1(UDP-_L-Rha/UDP-_D-Gal transporters)。对其编码蛋白进行了氨基酸组成、进化关系、理化性质、跨膜结构域和高级结构的预测,对其启动子序列进行了顺式作用元件分析,同时还检测并分析了OsURGT1基因在水稻中的组织转录表达谱、激素及非生物胁迫处理诱导表达谱。结果显示:OsURGT1基因全长为3 421 bp,含有5个外显子;CDS全长为996 bp,编码331个氨基酸长度的蛋白,此蛋白被预测是一个疏水性蛋白,含有9个跨膜结构域;蛋白高级结构建模结果表明,其与一个NST模板蛋白高度相似。基因启动子顺式作用元件分析结果表明,OsURGT1基因有可能参与多种激素调控途径和逆境胁迫调控途径。转录表达谱分析结果表明,...     

水稻新生多肽结合复合体α链基因家族的表达分析

水稻新生多肽结合复合体α链(Osα-NAC)基因家族共有3个成员,分别位于第1,3,5号染色体。为研究Osα-NAC基因家族在逆境环境中的生物学功能,利用RT-q PCR和Western Blot技术,详细分析了Osα-NAC基因家族在不同组织中的表达情况,并进一步研究了Osα-NAC基因家族对非生物胁迫的应答模式。结果表明,在高盐和模拟干旱条件下,Os1g NAC和Os5g NAC基因表达上调,Os3g NAC基因表达下调,提示Osα-NAC基因家族各成员在水稻响应非生物胁迫的过程中发挥不同的作用。上述试验结果初步阐明该基因家族在非生物胁迫下的表达特性,为今后更深入研究该基因家族的功能提供了理论依据。     

异源过表达Atvip1基因增强转基因高粱对盐碱胁迫的抗性

为了研究异源过表达Atvip1基因的高粱对盐碱胁迫的耐受性及相应生长情况,采用NaHCO_3∶Na_2CO_(3 )为5∶1,75 mmol/L,pH值9.63的盐碱胁迫液在高粱三叶一心期处理,在胁迫0,4,12,24,72,120 h对根系的生长指标、叶绿素含量、抗氧化酶活性以及MDA含量进行测定。结果表明:异源过表达Atvip1基因能缓解盐碱胁迫对高粱幼苗生长的伤害,可使幼苗根表面积和根体积增大,根尖数和分叉数增多,高粱主根褐化出现较晚且症状轻,侧根发生早且数量多,在72 h后仍能保证新叶正常伸展,对地上部生长影响较轻。过表达Atvip1基因可提高转基因高粱根系抗O_2~(-·)活力,降低MDA的含量,抗氧化酶活性增强;胁迫早期(4～12 h),SOD、CAT和GR作用明显;胁迫中期(24～72 h),所有酶均发挥作用;胁迫后期(120 h),CAT和GSH-PX起重要作用。盐碱胁迫差异转录组分析中,差异表达基因COG分析表明,防御机制在不同时间段中占比较大,获得抗氧化酶相关差异表达基因42个。异源过表达Atvip1基因可通过缓解盐碱胁迫对光合作用和生长发育的影响,降低活性氧破坏及膜损伤来提高转基因高粱对盐碱胁迫的抗性。     

水稻(Oryza sativa)OsSPR1突变体及过表达株系对锰胁迫的响应

为了系统分析水稻OsSPR1基因突变体Osspr1及过表达株系对锰胁迫的响应情况。本研究以水稻短侧根突变体Osspr1及过表达株系为试验材料,研究不同锰胁迫条件下水稻生长,锰吸收和累积量及其抗氧化酶活性的差异。研究结果表明,水稻OsSPR1的过表达株系(OV1)的根系发育和植物生长已完全恢复至野生型水平。在锰胁迫条件下,spr1突变体的地上部和根系的锰含量、锰累积量均显著低于野生型。在高锰(4 mmol/L Mn2+)处理下过表达株系OV1的锰含量和锰累积量显著低于野生型,OV1锰转移系数显著低于野生型。锰胁迫条件下,突变体spr1和过表达材料OV1叶片的抗氧化酶过氧化物酶(peroxidase, POD)、过氧化氢酶(catalase, CAT)、超氧化物歧化酶(superoxide dismutase, SOD)的活性均高于或者显著高于野生型。综合以上研究表明Os SPR1基因突变和过表达均可以提高水稻对锰胁迫的耐受能力。本研究为水稻耐锰新品种的选育提供材料和基因资源。     

OsRPK1基因过表达和RNA干涉对水稻苗期耐盐性的影响

水稻OsRPK1基因属于富含亮氨酸重复序列的类受体蛋白激酶,在盐胁迫下其表达量暂时升高后出现明显下降。前期研究表明, OsRPK1基因过表达会造成拟南芥非生物胁迫耐受性明显降低。本研究对OsRPK1基因在水稻中实现过表达和RNA干涉,进而对OsRPK1的抗逆性功能加以探究。结果表明, OsRPK1基因表达量增加时,水稻幼苗表现为耐盐性下降;RNA干涉则使水稻幼苗表现为耐盐性增加。盐胁迫后OsRPK1过表达植株脯氨酸含量低于野生型、MDA含量和相对电导率显著高于野生型,而OsRPK1-RNAi水稻植株的脯氨酸含量显著高于野生型、MDA含量和相对电导率显著低于野生型。因此, OsRPK1基因的表达量对水稻耐盐性具有明显影响,脯氨酸含量及细胞质膜受破损程度的改变可能是造成转基因水稻耐盐性变化的内在原因。本研究为进一步阐明OsRPK1基因的抗逆作用机制奠定了基础,对于通过调整该基因表达改良水稻的耐盐性具有重要意义。     

水稻DUF760基因家族的全基因组鉴定与表达谱分析

为了探索DUF760基因家族在水稻生长发育中的潜在功能,对水稻DUF760基因家族进行了全基因组鉴定、分类、启动子序列分析和表达谱分析。通过生物信息学技术在水稻和拟南芥中分别鉴定了6,8个DUF760家族成员。系统进化树分析将这些家族成员分成2个亚家族,二者在蛋白保守基序和基因结构上也存在一些特征差别。水稻DUF760家族基因启动子区域存在多个响应逆境胁迫和植物激素的顺式作用元件,ABRE(脱落酸响应)元件存在于家族所有成员的启动子序列中,OsDUF760-1启动子区域拥有9个脱落酸(ABA)相关的响应元件。在ABA处理水稻后,OsDUF760-1的转录表达水平显著下调,而OsDUF760-3显著上调,二者在干旱胁迫处理水稻后的表达变化模式与ABA处理水稻后的表达变化模式一致,说明这2个基因可能通过ABA信号途径参与水稻干旱胁迫响应,并且扮演不同的角色。除对ABA和干旱胁迫处理具有较强响应外,水稻DUF760家族成员还对JA(茉莉素)、低温及稻瘟菌处理具有较强的响应表达变化。     

OsSP1基因编辑和过表达对水稻生长和干旱抗性的影响

OsSP1基因是水稻叶绿体定位基因,并且具有明显的干旱响应性。为确定OsSP1基因对水稻生长和干旱胁迫抗性的影响,分别采用CRISPR/Cas9基因编辑技术和基因过表达技术创建了水稻品种中花11 OsSP1基因编辑株系和过表达株系,并通过对靶序列进行片段扩增和测序确定基因编辑植株中OsSP1基因的突变方式,通过hpt序列扩增和潮霉素筛选获得单拷贝插入的纯合过表达植株。表型对比、叶绿素含量测定、过氧化氢测定和干旱抗性分析的结果表明,过表达OsSP1基因对水稻的生长和干旱胁迫抗性均无明显影响,但通过基因编辑引起OsSP1基因突变后,水稻的生长和结实受到了严重影响,在正常生长条件下,T_0和T_1双等位杂合突变和纯合突变的基因编辑植株均出现矮小、黄化、早衰、不分蘖等生长抑制表型。T_0植株结实数很低,T_1植株均未抽穗结实,叶片的叶绿素含量也显著或极显著低于野生型和过表达植株,上述结果显示了OsSP1基因在维持水稻正常生长和产量中的关键性作用,为进一步揭示OsSP1基因的作用机制提供了理论和材料基础。     

Overexpression of a small heat‐shock‐protein gene enhances tolerance to abiotic stresses in rice

Rice (Oryza sativa L.) is one of the most important crops in the world which survives from various abiotic stresses in natural environments with specific stress‐involved genes expressed. Plant sHSPs (small heat‐shock proteins) were reported to respond to abiotic stresses. To improve the understanding of sHSPs in rice, we characterized heat‐shock‐protein gene OsHSP18.6 here. OsHSP18.6 could be induced by diverse stresses, such as drought, salt and cold, especially under heat. The gene was found expressed in root, stem, leaf, internode and spikelet. Overexpression of OsHSP18.6 results in increased thermotolerance and exhibits universal tolerance to stresses tested, including heat, drought, salt and cold. Lower levels of malondialdehyde (MDA) and greater activities of catalase (CAT) and superoxide dismutase (SOD) were observed in OsHSP18.6‐overexpression rice under heat and drought. OsHSP18.6‐overexpression lines indicated decreased sterile rates under hot weather without remarkable changes in most of other agronomic traits compared with wild‐type plants.     

过表达ZmCIPKHT基因增强植物耐热性

高温胁迫对植物正常生长发育及产量的影响越来越显著。为了适应外界环境的变化,植物进化出了一系列应对高温胁迫的分子遗传机制。类钙调磷酸酶B蛋白(CBL)互作蛋白激酶(CIPK),在ABA信号转导途径上积极参与植物对高温胁迫的响应。在前期全基因组关联分析的基础上,本实验克隆了一个与玉米耐高温性状相关的候选基因ZmCIPKHT, qRT-PCR结果表明ZmCIPKHT基因受高温胁迫的显著诱导。室内表型鉴定的实验发现过表达ZmCIPKHT的转基因拟南芥植株在高温胁迫下,比野生型的存活率显著提高,生长状态更好。玉米原生质体瞬时转化实验证明ZmCIPKHT蛋白定位于细胞核中。酵母双杂交实验验证了ZmCIPKHT蛋白与玉米CBLs家族中的ZmCBL4蛋白存在互作关系。同时, ZmCIPKHT转基因拟南芥在高温胁迫条件下,脱落酸(ABA)通路相关基因的表达水平有其相应的变化,揭示了ZmCIPKHT可能依赖于ABA信号转导通路来增强植物的耐热性。这些结果为解析玉米CBL-CIPK信号通路依赖于ABA途径对植物非生物胁迫响应的分子机制提供了新的实验根据。     

不同耐旱性青稞叶片差异蛋白分析

为从蛋白质水平揭示不同青稞品种响应干旱胁迫的差异,分析抗旱蛋白质分子机制,本研究以干旱胁迫不敏感的旱地紫青稞(HDZ)和干旱胁迫敏感的大麻青稞(DM)为研究材料,以盆栽限量供水种植方法,对干旱处理不同梯度的青稞叶片进行叶绿素、可溶性蛋白、丙二醛含量及相对电导率4项生理指标的测定,同时利用iTRAQ技术对深度干旱胁迫青稞叶片全蛋白组进行差异蛋白分析。结果表明:随着干旱处理时间的延长,两种青稞的叶绿素和可溶性蛋白含量逐渐下降,电导率及丙二醛的含量逐渐升高,在相同处理条件下,大麻青稞的叶绿素和可溶性蛋白含量降低幅度、电导率及丙二醛含量的增高幅度明显高于旱地紫青稞;对两个青稞品种的干旱胁迫和正常培养的比较组进行iTRAQ分析,共定量出4163个蛋白(多肽),其中旱地紫青稞比较组中对比正常培养,筛选到表达上调的蛋白68个,下调的蛋白63个,在大麻青稞的比较组中筛选出表达上调蛋白21个,下调蛋白32个。KEGG通路分析表明,富集程度位于前3位的通路是代谢、氨基酸的生物合成以及次级代谢产物的生物合成,主要涉及到柠檬酸循环、碳循环等代谢通路,丙氨酸、精氨酸等氨基酸的合成降解以及花生四烯酸、亚麻酸等次级...     

ITRAQ-based quantitative proteomic analysis of japonica rice seedling during cold stress

Low temperature is one of the important environmental factors that affect rice growth and yield. To better understand the japonica rice responses to cold stress, isobaric tags for a relative and absolute quantification (iTRAQ) labeling-based quantitative proteomics approach was used to detected changes in protein levels. Two-week-old seedlings of the cold tolerant rice variety Kongyu131 were treated at 8°C for 24, 48 and 72 h, then the total proteins were extracted from tissues and used for quantitative proteomics analysis. A total of 5082 proteins were detected for quantitative analysis, of which 289 proteins were significantly regulated, consisting of 169 uniquely up-regulated proteins and 125 uniquely down-regulated proteins in cold stress groups relative to the control group. Functional analysis revealed that most of the regulated proteins are involved in photosynthesis, metabolic pathway, biosynthesis of secondary metabolites and carbon metabolism. Western blot analysis showed that protein regulation was consistent with the iTRAQ data. The corresponding genes of 25 regulated proteins were used for quantitative real time PCR analysis, and the results showed that the mRNA level was not always parallel to the corresponding protein level. The importance of our study is that it provides new insights into cold stress responses in rice with respect to proteomics and provides candidate genes for cold-tolerance rice breeding.     

Overexpression of cold-inducible wheat galactinol synthase confers tolerance to chilling stress in transgenic rice

Galactinol synthase (GolS) is considered to be a key regulator of the biosynthesis of Raffinose family oligosaccharides (RFOs). Accumulation of RFOs has been reported to play a role in protection against abiotic stresses. We identified two cDNAs encoding galactinol synthase from wheat (Triticum aestivum L.), which we designated as TaGolS1 and TaGolS2. Expression of the two TaGolS genes was induced by cold stress but not by drought, heat stress or ABA treatment in wheat. We generated transgenic lines of rice (Oryza sativa L.) constitutively overexpressing TaGolS1 or TaGolS2. These transgenic plants accumulated significantly higher levels of galactinol and raffinose than did wild-type plants and exhibited enhanced cold-stress tolerance. The results demonstrate the involvement of galactinol and raffinose in the development of chilling stress in rice and indicate that the genetic modification of the biosynthesis of RFOs by transformation with GolS genes could be an effective method for enhancing chilling-stress tolerance in rice.     

Ectopic expression of GmPAP3 enhances salt tolerance in rice by alleviating oxidative damage

GmPAP3, a purple acid phosphatase from soybean (Glycine max), was previously shown to alleviate salt stress in BY‐2 cells and Arabidopsis thaliana by reducing oxidative damage. To make use of GmPAP3 for crop improvement, we investigated whether the protective function of GmPAP3 is persistent in rice. Compared with the untransformed wild type, the transgenic rice plants exhibited enhanced germination rate, longer shoots and roots, and higher survival rate under salt stress, when compared to the untransformed control. In addition, the transgenic plants also showed increased activity of superoxide dismutase (SOD) and catalase (CAT), proline content, water content and chlorophyll content, but decreased electrolyte leakage and malondialdehyde content in leaves. These results indicate that the expression of GmPAP3 could be used to enhance salt tolerance in rice.     

Root pulling resistance in rice: Inheritance and association with drought tolerance

Summary Avoidance of drought stress is commonly associated with root system characteristics and root development. The inheritance of root pulling resistance in rice (Oryza sativa L.) was investigated and its relationship with visual field scores for drought tolerance was studied. Transgressive segregation for high root pulling resistance was observed in 3 crosses (high x high, low x high, and intermediate x intermediate). Both dominant and additive genes control the variation. F₁ superiority for high root pulling resistance was observed and could be exploited in an F₁ hybrid breeding program. F₂ distribution curves indicated that plants highly resistant to root pulling can be obtained not only from low x high and high x high crosses, but also from intermediate x intermediate crosses. Root pulling resistance in rice has a low heritability (39 to 47%). Thus, breeding for a high root pulling resistance may best be accomplished by selection based on line means rather than individual plant selection. Field screening showed significant differences in leaf water potential among random F₃ lines. F₃ lines with higher leaf water potential had better visual scores for drought tolerance. Visual drought tolerance scores were correlated with root pulling resistance. Plants with high root pulling resistance had the ability to maintain higher leaf water potentials under severe drought stress. The usefulness of the root pulling technique in selecting drought tolerant genotypes was confirmed.     

Evaluating the role of seed priming in improving drought tolerance of pigmented and non‐pigmented rice

This study was conducted to evaluate the influence of seed priming on drought tolerance of pigmented and non‐pigmented rice. Seeds of pigmented (cv. Heug Jinju Byeo) and non‐pigmented (cv. Anjoong) rice were soaked in water (hydropriming) or solution of CaCl₂ (osmopriming). Seeds were sown in soil‐filled pots retained at 70 (well‐watered) and 35% (drought) water‐holding capacity. Drought stress caused erratic and poor stand establishment and decreased the growth of both rice types. More decrease in plant height and leaf area under drought stress was noted in pigmented rice, whereas decrease in root length and seedling dry weight, under drought, was more obvious in non‐pigmented rice. Pigmented rice maintained more tissue water and photosynthesis and had more polyphenols, flavonoids and antioxidant activity than non‐pigmented rice. Seed priming was effective in improving stand establishment, growth, polyphenols, flavonoids and antioxidant activity; however, extent of improvement was more in pigmented rice under drought. In conclusion, drought caused erratic germination and suppressed plant growth in both rice types. However, pigmented rice had better drought tolerance owing to uniform emergence, and better physiological and morphological plasticity. Seed priming was quite helpful in improving the performance of both rice types under drought and well‐watered conditions.     

Comparison of selection indices to screen lowland rice for drought resistance

Summary Although many selection indices have been used to screen rices (Oryza sativa L.) for drought resistance, there has been little comparison of the relative merits of these indices. Research was conducted to compare drought resistance as estimated from grain yields, canopy-temperature-based stress indices, visual scoring, and uprooting force for 30 rice genotypes grown in the field with a puddled Maahas clay (Typic Tropaquept) and to evaluate traits related to drought resistance from nonstressed plants grown in the field and in aeroponic culture. Water deficit was imposed in the field by withholding irrigation from 45 to 75 days after transplanting compared to a continuously flooded control. Grain yields in the stress treatment were most strongly correlated with visual assessment of drought stress symptoms according to a standard evaluation system (r = 0.66). Canopy-temperature-based indices were also significantly correlated with grain yields of the stress treatment (r from –0.55 to –0.63). No trait of aeroponically grown plants was correlated with traits of stressed plants in the field. We conclude that visual scoring of stressed plants was the best method of screening for drought resistance, but if controlled water deficit cannot be imposed, then drought resistance may be estimated by measuring both uprooting force and grain yield.