转ZjAPX基因拟南芥对NaCl、干旱胁迫的耐性研究

旨在探讨枣树抗坏血酸过氧化物酶基因ZjAPX在植物渗透胁迫中的作用。将ZjAPX基因转入到模式植物拟南芥,以野生型(WT)、转ZjAPX拟南芥株系T2为试材,进行不同浓度NaCl胁迫和干旱胁迫。结果表明,转基因株系的种子萌发、植株生长均优于野生型株系;荧光定量PCR检测转基因拟南芥植株在干旱和盐胁迫处理10 d后目的基因ZjAPX的表达量显著高于野生拟南芥,表明ZjAPX的高表达明显提高了植株的抗旱和耐盐性。     

蒙古冰草MwMYB4基因功能鉴定

MwMYB4基因是从蒙古冰草中克隆得到的MYB类转录因子家族成员之一。该研究以转MwMYB4基因的拟南芥后代为材料,通过在干旱和低温胁迫下对转基因植株进行表型分析、理化指标测试和分子鉴定,分析并验证MwMYB4基因的功能。结果显示:(1)蒙古冰草MwMYB4基因已成功整合到转基因拟南芥T_1代的基因组中并实现转录水平的表达。(2)转基因拟南芥T_2代植株在干旱胁迫条件下,转基因植株叶片枯黄程度较轻,相对电导率较野生型变化幅度低,脯氨酸含量明显高于野生型对照,且MwMYB4基因的表达量随干旱胁迫时间延长而增加。(3)在低温胁迫条件下,转基因拟南芥叶片的枯白程度明显低于野生型,且MwMYB4基因的表达量随低温胁迫时间增加而增加。研究表明,过量表达蒙古冰草MwMYB4基因能够提高转基因拟南芥对干旱和低温的耐受性,该基因可能在干旱胁迫和低温胁迫调控机制中发挥调控作用,可作为改良农作物和其他牧草抗旱、抗寒性的重要候选基因。     

AtWRKY40参与拟南芥干旱胁迫响应过程

以拟南芥(Arabidopsis thaliana)野生型、AtWRKY40缺失突变体和过表达株系为材料,研究AtWRKY40在植物干旱胁迫响应过程中的作用及其生理和分子机制。结果显示,AtWRKY40受干旱胁迫诱导;AtWRKY40缺失导致干旱胁迫下种子萌发率降低,叶片失水加剧,而AtWRKY40过表达植株呈现出相反的表征;干旱胁迫下,At WRKY40缺失突变体植株叶片过氧化氢(H_2O_2)、超氧阴离子(O_2~-·)及丙二醛(MDA)含量显著高于野生型及其过表达株系,超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性、脯氨酸(Pro)和可溶性糖含量以及相关基因AtCu/ZnSOD、AtCAT1、AtP5C1S、AtG6PD5和AtBAM4表达量显著低于野生型,同时AtWRKY40过表达株系的渗透物质含量和保护酶活性及其基因表达量则高于野生型。由此说明,AtWRKY40通过调节植株抗氧化能力及渗透调节能力参与拟南芥干旱胁迫响应过程。     

转拟南芥P5CS1基因增强羽衣甘蓝的耐旱性

为提高羽衣甘蓝的耐旱性,本文将拟南芥Δ1-吡咯啉-5-羧酸合成酶（P5CS1）基因经农杆菌介导转入羽衣甘蓝植株中,检测转基因株系与野生型植株在干旱胁迫下P5CS1 mRNA表达量、幼苗脯氨酸含量、株系根系性状、整株干重、鲜重和整株存活率。结果表明,在15%PEG6000渗透胁迫下,转基因植株的P5CS1基因mRNA表达量明显增加,转基因植株脯氨酸含量是野生型的2.4倍;主根长、最长侧根长、侧根数目、整株干重和鲜重均高于野生型,干重/鲜重则低于野生型,转基因植株的平均存活率为78%,极显著高于野生型。数据显示,AtP5CS1基因在羽衣甘蓝中的表达明显改善了转基因植株的耐旱性。     

过量表达谷胱甘肽转移酶基因对转基因拟南芥抗旱能力的影响

盐地碱蓬谷胱甘肽转移酶基因（OST）在拟南芥中过量表达后,在干旱胁迫下,转基因拟南芥植株的干重比野生型植株高,其总谷胱甘肽含量和谷胱甘肽库的氧化水平都比野生型植株的高,而丙二醛含量则比野生型的低。这些显示转基因拟南芥的抗干旱胁迫能力有所增强。     

蔗糖合酶基因AtSUS3干涉后对拟南芥角果发育的影响

蔗糖合酶(SuSy)是植物蔗糖代谢关键酶之一,该研究利用反向遗传学手段,采用RNAi技术抑制拟南芥中AtSUS3基因的表达,测定纯系转基因植株的抽苔率,并对酶活性、糖含量等指标以及糖代谢相关基因的表达进行了检测,探讨SuSy在植物发育中的作用。结果显示:(1)转基因拟南芥的抽苔平均早于野生型植株2～3d,且优先3～4d完成抽苔。(2)开花后生长天数对角果蔗糖和葡萄糖含量有显著影响,而对果糖含量影响不显著;开花后5d时,野生型株系的葡萄糖含量显著高于转基因株系SUS3-2,至15d时,两种转基因株系葡萄糖含量均显著低于野生型株系。(3)开花后生长天数对SuSy、SPS、INV的活性均有显著影响,随开花时间延长,野生型株系SuSy活性显著低于转基因株系,而SPS和INV则相反。(4)AtSUS3基因沉默对其他糖代谢基因有不同程度的影响,开花后5d时,转基因植株的角果中AtCesA1、AtCesA7和AtCINV1的表达量较野生型都有所增加;开花后15d时,转基因植株的角果中AtCesA1、AtCesA7的表达量较野生型高,而AtCINV、AtCwINV的表达量比野生型低。研究表明,拟南芥AtSUS3基因沉默后,在正常生长条件下未造成植株发育异常,同时还可能通过同源家族中其他SuSy的表达水平增加,促进了该酶及糖代谢相关基因整体水平的增加,有助于角果成熟。     

转彩色马铃薯StAN1基因的烟草幼苗耐旱性分析

该研究以转彩色马铃薯StAN1基因烟草为材料、野生型烟草(WT)为对照,测定分析转StAN1基因烟草在种子萌发期、幼苗期和苗期对干旱(甘露醇)处理的耐受情况,并对苗期旱热共同胁迫的耐受情况进行测定分析,以探讨彩色马铃薯StAN1基因的功能,为耐旱彩色马铃薯育种提供新路径。结果显示:(1)转StAN1基因烟草鉴定显示,阳性率为82.6%,且转基因烟草的叶片明显变紫,花青素含量极显著高于野生型烟草。(2)在培养基甘露醇浓度为150 mmol/L时,点播在培养基上的转基因烟草种子第5天时的萌发率达到了7%,是野生型烟草萌发率的2.3倍。(3)在甘露醇浓度为0和100 mmol/L的培养基上竖直培养时,转基因烟草的根长分别是野生型烟草的1.46和1.30倍,根长比野生型烟草显著增长。(4)在干旱胁迫下,转基因烟草幼苗叶片中的脯氨酸含量以及超氧化物歧化酶活性均显著高于野生型烟草,丙二醛含量均显著低于野生型烟草。(5)转基因烟草LEA基因和ERF基因在干旱和旱热处理中的相对表达量均高于野生型烟草。研究表明,StAN1基因在提高植物花青素含量的同时也提高了植物的耐旱性。     

超表达AVP1基因提高甜菜的耐低磷和耐盐抗旱性

为探讨H⁺-焦磷酸酶编码基因对甜菜磷吸收和抗性的影响，实现优良基因在甜菜基因工程中的利用，研究在甜菜中超表达拟南芥液泡膜H⁺-焦磷酸酶编码基因AVP1,对转基因甜菜分析其耐低磷、耐盐性和抗旱性。结果显示，AVP1基因在甜菜植株的叶片和块根中表达，且在逆境胁迫下增强表达量响应胁迫；低磷处理条件下，转基因甜菜与野生型甜菜相比具有更高的含磷量，可提高甜菜对磷的吸收利用效率；干旱、盐胁迫处理条件下，AVP1基因在转基因甜菜中显著上升，在盐胁迫或干旱处理条件下，转基因植株的生长受抑程度相对较轻。随着盐和干旱胁迫的加剧，转基因植株体内MDA含量与野生型植株相比较低而脯氨酸含量显著增加，AVP1基因可通过减轻逆境对甜菜细胞膜的损伤及提高甜菜细胞的渗透调节能力，进而增强甜菜对高盐和干旱胁迫的抗性。     

转小拟南芥ApHRD基因烟草获得及其抗旱性鉴定

根据拟南芥(Arabidopsis thaliana)的HRD基因序列,采用PCR方法从新疆小拟南芥(A.pumila)中克隆了ApHRD基因,并构建了植物表达载体pBIN-ApHRD,通过农杆菌介导法转化烟草('NC89')获得转化植株,用PCR和RT-PCR法对转化烟草进行鉴定,并采用水分胁迫和PEG-6000模拟干旱进行抗旱性分析.结果显示:(1)克隆的新疆小拟南芥ApHRD基因与拟南芥AtHRD基因的核苷酸序列相似性为99.1%,对应氨基酸序列的同源性为98.37%,只有3个位点发生实义突变.(2)与野生型烟草植株相比,转ApHRD基因烟草植株主根粗壮,一级侧根较发达,移栽成活的植株生长快.(3)在水分胁迫和30%的PEG-6000模拟干旱胁迫条件下,转ApHRD基因烟草的叶片相对含水量和PSⅡ相对量子产率降低幅度、相对电导率和丙二醛含量的升高幅度均显著低于野生型烟草植株;与野生型植株相比,转ApHRD基因烟草植株叶片出现萎蔫症状的时间较迟、程度较轻,复水后恢复快且较完全,在干旱胁迫过程中受到的伤害较轻,表现出了较强的抗旱特性.研究表明,在干旱胁迫条件下,转小拟南芥ApHRD基因烟草植株表现出了优良的生理和生长优势,显示出较强的抗旱性特征,ApHRD基因在抗旱基因工程方面具有较好的应用前景.     

拟南芥黑芥子酶TGG1对抗旱性的作用

为阐明拟南芥中黑芥子酶TGG1对抗旱性的影响,构建了35S启动子驱动的TGG1过表达载体,并将其转入拟南芥获得了转基因植株。以野生型和过量表达TGG1的转基因植株为材料,进行干旱胁迫实验,结果显示,在甘露醇模拟的干旱胁迫下35S∶TGG1种子平均发芽率显著高于野生型,平均相对电导率则显著低于野生型;自然干旱胁迫下,35S∶TGG1的相对失水速率显著慢于野生型,而干旱复水后的平均存活率则显著高于野生型。对气孔的观察结果表明,过表达TGG1的转基因植株气孔对ABA处理具有更高的敏感性,气孔关闭程度显著高于野生型植株。以上研究结果表明,过量表达TGG1基因可显著提高拟南芥的抗旱能力,而且其抗性机制很可能与气孔在逆境下的关闭程度有关。     

Transgenic rice tolerant to high temperature with elevated contents of dienoic fatty acids

Transgenic rice plants in which the content of dienoic fatty acids was increased as a result of co-suppression of fatty acid desaturase were more tolerant to high temperatures than untransformed wild-type plants, as judged by growth rate and chlorophyll content. When untransformed wild-type and transgenic rice seedlings were incubated at 35 °C, seedlings of the transgenic rice lines showed approximately 1.6 and 2.1 times the growth of untransformed wild-type seedlings, as assayed by shoot and root mass, respectively. The chlorophyll content of the transgenic leaves after 9 d at 35 °C was also higher than that of wild-type rice. The maximum photochemical efficiency of photosystem 2 was also higher in transgenic plants than in wild-type plants upon high temperature stress.     

Tolerance of Mycorrhiza infected pistachio (Pistacia vera L.) seedling to drought stress under glasshouse conditions

The influence of Glomus etunicatum colonization on plant growth and drought tolerance of 3-month-old Pistacia vera seedlings in potted culture was studied in two different water treatments. The arbuscular mycorrhiza (AM) inoculation and plant growth (including plant shoot and root weight, leaf area, and total chlorophyll) were higher for well-watered than for water-stressed plants. The growth of AM-treated seedlings was higher than non-AM-treatment regardless of water status. P, K, Zn and Cu contents in AM-treated shoots were greater than those in non-AM shoots under well-watered conditions and drought stress. N and Ca content were higher under drought stress, while AM symbiosis did not affect the Mg content. The contents of soluble sugars, proteins, flavonoid and proline were higher in mycorrhizal than non-mycorrhizal-treated plants under the whole water regime. AM colonization increased the activities of peroxidase enzyme in treatments, but did not affect the catalase activity in shoots and roots under well-watered conditions and drought stress. We conclude that AM colonization improved the drought tolerance of P. vera seedlings by increasing the accumulation of osmotic adjustment compounds, nutritional and antioxidant enzyme activity. It appears that AM formation enhanced the drought tolerance of pistachio plants, which increased host biomass and plant growth.     

Engineering of enhanced glycine betaine synthesis improves drought tolerance in maize

Glycine betaine plays an important role in some plants, including maize, in conditions of abiotic stress, but different maize varieties vary in their capacity to accumulate glycine betaine. An elite maize inbred line DH4866 was transformed with the betA gene from Escherichia coli encoding choline dehydrogenase (EC 1.1.99.1), a key enzyme in the biosynthesis of glycine betaine from choline. The transgenic maize plants accumulated higher levels of glycine betaine and were more tolerant to drought stress than wild-type plants (non-transgenic) at germination and the young seedling stage. Most importantly, the grain yield of transgenic plants was significantly higher than that of wild-type plants after drought treatment. The enhanced glycine betaine accumulation in transgenic maize provides greater protection of the integrity of the cell membrane and greater activity of enzymes compared with wild-type plants in conditions of drought stress.     

The garlic NF-YC gene, <Emphasis Type="Italic">AsNF-YC8</Emphasis>, positively regulates non-ionic hyperosmotic stress tolerance in tobacco

To investigate the relationship between nuclear factor Y (NF-Y) and stress tolerance in garlic, we cloned a NF-Y family gene AsNF-YC8 from garlic, which was largely upregulated at dehydrate stage. Expression pattern analyses in garlic revealed that AsNF-YC8 is induced through abscisic acid (ABA) and abiotic stresses, such as NaCl and PEG. Compared with wild-type plants, the overexpressing-AsNF-YC8 transgenic tobacco plants showed higher seed germination rates, longer root length and better plant growth under salt and drought stresses. Under drought stress, the transgenic plants maintained higher relative water content (RWC), net photosynthesis, lower levels of malondialdehyde (MDA), and less ion leakage (IL) than wild-type control plants. These results indicate the high tolerance of the transgenic plants to drought stress compared to the WT. The transgenic tobacco lines accumulated less reactive oxygen species (ROS) and exhibited higher antioxidative enzyme activities compared with wild-type (WT) plants under drought stress, which suggested that the overexpression of AsNF-YC8 improves the antioxidant defense system by regulating the activities of these antioxidant enzymes, which in turn protect transgenic lines against drought stress. These results suggest that AsNF-YC8 plays an important role in tolerance to drought and salt stresses.     

过表达胡杨XTH基因能够提高烟草抗旱性

胡杨是典型的抗旱树种。挖掘和鉴定胡杨的耐旱基因对于提高植物抗旱性具有重要意义。木葡聚糖内转糖苷酶/水解酶（XTH）是植物细胞壁重构过程中的关键酶,在植物逆境胁迫响应中发挥重要作用。我们前期已从胡杨叶片中克隆了PeXTH基因。本文利用Real-time PCR检测PeXTH基因在干旱胁迫下的表达水平。在此基础上,构建植物表达载体pMDC85-PeXTH,通过农杆菌介导法将PeXTH基因转入烟草,分析过表达PeXTH基因烟草的抗旱性。研究发现,胡杨叶片中PeXTH基因的表达受干旱胁迫诱导。干旱处理后,转PeXTH基因烟草的萌发率明显高于野生型烟草;与野生型植株相比,转基因植株的叶片失水速率明显降低。干旱胁迫下,转基因烟草的气孔开度仅为野生型烟草的51.2%～53.6%。结果表明,过表达PeXTH基因能够提高烟草的抗旱性。本研究丰富了对胡杨PeXTH基因功能的认识,为植物抗旱分子育种提供了重要的基因资源。     

Enhanced proline accumulation and salt stress tolerance of transgenic <Emphasis Type="Italic">indica</Emphasis> rice by over-expressing <Emphasis Type="Italic">P5CSF129A</Emphasis> gene

Δ¹-pyrroline-5-carboxylate synthetase (P5CS) is a proline biosynthetic pathway enzyme and is known for conferring enhanced salt and drought stress in transgenics carrying this gene in a variety of plant species; however, the wild-type P5CS is subjected to feedback control. Therefore, in the present study, we used a mutagenized version of this osmoregulatory gene-P5CSF129A, which is not subjected to feedback control, for producing transgenic indica rice plants of cultivar Karjat-3 via Agrobacterium tumefaciens. We have used two types of explants for this purpose, namely mature embryo-derived callus and shoot apices. Various parameters for transformation were optimized including antibiotic concentration for selection, duration of cocultivation, addition of phenolic compound, and bacterial culture density. The resultant primary transgenic plants showed more enhanced proline accumulation than their non-transformed counterparts. This proline level was particularly enhanced in the transgenic plants of next generation (T₁) under 150 mM NaCl stress. The higher proline level shown by transgenic plants was associated with better biomass production and growth performance under salt stress and lower extent of lipid peroxidation, indicating that overproduction of proline may have a role in counteracting the negative effect of salt stress and higher maintenance of cellular integrity and basic physiological processes under stress.     

Expression of Arabidopsis DREB1C improves survival, growth, and yield of upland New Rice for Africa (NERICA) under drought

Dehydration-responsive-element-binding protein 1 genes have important roles in response to stress. To improve the drought tolerance of an upland rice cultivar NERICA1, we introduced Arabidopsis AtDREB1C or rice OsDREB1B driven by a stress-inducible rice lip9 promoter. Plants of some transgenic lines survived better than non-transgenic plants under severe drought. AtDREB1C transgenic plants had higher dry weights than non-transgenic plants when grown under moderate drought until the late vegetative growth stage. On the other hand, OsDREB1B transgenic plants had lower dry weights than non-transgenic plants under the same condition. Similar results were obtained under osmotic stress. The AtDREB1C transgenic plants headed earlier, had a larger sink capacity, and had more filled grains than non-transgenic plants. These results suggest that AtDREB1C expressed in NERICA1 improves not only survival under severe drought, but also growth and yield under moderate drought.     

RNAi-mediated disruption of squalene synthase improves drought tolerance and yield in rice

About one-third of the world's rice area is in rain-fed lowlands and most are prone to water shortage. The identification of genes imparting tolerance to drought in the model cereal plant, rice, is an attractive strategy to engineer improved drought tolerance not only rice but other cereals as well. It is demonstrated that RNAi-mediated disruption of a rice farnesyltransferase/squalene synthase (SQS) by maize squalene synthase improves drought tolerance at both the vegetative and reproductive stages. Twenty-day-old seedlings of wild type (Nipponbare) and seven independent events of transgenic RNAi lines showed no difference in morphology. When subjected to water stress for a period of 32 d under growth chamber conditions, transgenic positives showed delayed wilting, conserved more soil water, and improved recovery. When five independent events along with wild-type plants were subjected to drought at the reproductive stage under greenhouse conditions, the transgenic plants lost water more slowly compared with the wild type, through reduced stomatal conductance and the retention of high leaf relative water content (RWC). After 28 d of slow progressive soil drying, transgenic plants recovered better and flowered earlier than wild-type plants. The yield of water-stressed transgenic positive plants ranged from 14-39% higher than wild-type plants. When grown in plates with Yoshida's nutrient solution with 1.2% agar, transgenic positives from three independent events showed increased root length and an enhanced number of lateral roots. The RNAi-mediated inactivation produced reduced stomatal conductance and subsequent drought tolerance.     

Elongation and gravitropic responses of Arabidopsis roots are regulated by brassinolide and IAA

Exogenously applied brassinolide (BL) increased both gravitropic curvature and length of primary roots of Arabidopsis at low concentration (10(-10) M), whereas at higher concentration, BL further increased gravitropic curvature while it inhibited primary root growth. BRI1-GFP plants possessing a high steady-state expression level of a brassinosteroid (BR) receptor kinase rendered the plant's responses to gravity and root growth more sensitive, while BR-insensitive mutants, bri1-301 and bak1, delayed root growth and reduced their response to the gravitropic stimulus. The stimulatory effect of BL on the root gravitropic curvature was also enhanced in auxin transport mutants, aux1-7 and pin2, relative to wild-type plants, and increasing concentration of auxin attenuated BL-induced root sensitivity to gravity. Interestingly, IAA treatment to the roots of bri1-301 and bak1 plants or of plants pretreated with a BL biosynthetic inhibitor, brassinazole, increased their sensitivity to gravity, while these treatments for the BL-hypersensitive transgenic plants, BRI1-GFP and 35S-BAK1, were less effective. Expression of a CYP79B2 gene, encoding an IAA biosynthetic enzyme, was suppressed in BL-hypersensitive plant types and enhanced in BL-insensitive or -deficient plants. In conclusion, our results indicate that BL interacts negatively with IAA in the regulation of plant gravitropic response and root growth, and its regulation is achieved partly by modulating biosynthetic pathways of the counterpart hormone.     

干旱胁迫条件下AMF促进小马鞍羊蹄甲幼苗生长的机理研究

采用温室水分控制试验,在干旱胁迫条件下,定量化研究优势丛枝菌根真菌(AMF)影响优势乡土植物小马鞍羊蹄甲(Bauhinia faberi var.microphylla)幼苗生长的机理,主要通过研究干旱胁迫条件下摩西球囊霉菌(Funneliformis mosseae)与小马鞍羊蹄甲的共生关系,阐明AMF在植物生长初期的作用。结果表明,干旱胁迫条件下,摩西球囊霉菌能够很好地侵染幼苗,侵染率高达89%—97%,并且不受水分条件影响。接种的幼苗最大光合速率、水分利用效率随着干旱胁迫程度从重度到轻度(水分从低到高)逐渐增大,相反地,叶片脯氨酸含量逐渐减小。接种显著地促进幼苗株高、叶片数、叶面积、根长、根面积等生长指标,提高幼苗各部分生物量、地上地下磷(P)含量。当含水量为60%田间持水量时,AMF促进小马鞍羊蹄甲幼苗吸收P的效果最好。接种还显著影响幼苗的生物量分配,在重度干旱胁迫时影响P分配,水分条件也显著影响幼苗的生物量分配。此外,接种和水分的交互作用对叶生物量、总生物量、生长指标以及地上部氮(N)总量影响显著。结果表明干旱胁迫条件下菌根效应显著,并在干旱条件下显著促进了小马鞍羊蹄甲幼苗的生长,这为进一步干旱河谷植被恢复提供了理论依据。