水分亏缺下玉米根系ZmPIP1亚族基因的表达

在PEG-6000胁迫条件下,以微管蛋白基因为内参基因、水通道蛋白基因ZmPIP1-1和ZmPIP1-2为检测基因,采用半定量逆转录聚合酶链式反应（RT-PCR）体系检测它们在玉米根系中的表达情况。实验结果是：胁迫条件下,ZmPIP1-1的表达量在杂交F,代‘户单4号’（抗旱）和母本‘天四’（抗旱）根系中增多,它的表达量与品种的抗旱性呈正相关,并且胁迫不同时间段它的表达量有差异;而ZmPIP1-2在3个玉米品种的不同水分处理条件下,表达量均没有明显变化。这提示,水分胁迫条件下根系中某些种类的水通道蛋白基因的表达量增多,并且与品种的抗旱性有关;而另一些水通道蛋白基因的表达不受水分亏缺的影响。     

丛枝菌根真菌通过上调根系及自身水孔蛋白基因表达提高玉米抗旱性

 在模拟干旱条件下, 研究了接种丛枝菌根(AM)真菌Glomus intraradices对玉米(Zea mays)根部13种质膜水孔蛋白基因表达的影响, 同时观测了AM真菌自身水孔蛋白基因的表达情况。结果表明, 干旱条件下, 除Zm PIP1;3、Zm PIP1;4、Zm PIP1;5和Zm PIP2;2之外的接种处理能显著提高根部其他8种质膜水孔蛋白基因的表达(Zm PIP2;7表达量未检测出), 并且AM真菌菌丝中水孔蛋白基因GintAQP1表达也显著增强。与此同时, 接种处理明显改善了植物水分状况, 提高了叶片水势。AM真菌增强宿主植物根部及自身的水孔蛋白基因的表达对于提高植物抗旱性具有潜在的重要贡献。     

定点整合人Bcl-2基因的CHO/dhfr-细胞株的建立

构建重组载体质粒pMCEfrt—Bcl-2,利用FIp—In^TM定点重组系统,在CHO—dhfr^-细胞内定点整合人Bcl-2基因,通过Western印迹检测重组细胞Bcl-2蛋白的表达。通过流式细胞仪和DNA Ladder检测在高NH4C1条件下细胞的凋亡情况;用台盼蓝染色检测在无血清IMDM培养基中细胞的活细胞数目和活细胞比例。结果获得了稳定表达Bcl-2基因的细胞株CHO—Bcl-2,该细胞株能高水平表达Bcl-2蛋白。在无血清培养过程中,CHO—Bcl-2细胞比对照细胞保持高约15％的活细胞比例,细胞总数高25％。CHO-Bcl-2在高NH4^＋（50mmol／L）培养条件下具有较低的凋亡水平。建立了能够高表达Bcl-2基因并具有一定的抗凋亡能力的重组CHO／dhfr^-细胞株。     

红树林植物木榄水通道基因的克隆和表达

根据植物水通道基因保守区设计简并引物,采用RT-PCR方法,从木榄树叶中分离出水通道基因的cDNA片段;3′RACE获得3′端cDNA序列;再经5′RACE获得5′端部分cDNA序列,命名为PIP2,GenBank登录号为EF126757。该基因全长843个碱基,编码281个氨基酸,具有典型的植物水通道基因结构。该基因编码的蛋白质与含羞草(PIP2;5)、欧洲葡萄(PIP)、拟南芥(PIP3)等水通道蛋白的同源性分别为90%、91%、88%。Northern杂交分析表明,该基因在木榄树不同器官中的表达差异明显:根部有较高的表达水平,茎部较弱,而在叶中只能检测到微弱的信号。     

PSF不同功能片段融合蛋白的构建及其在细胞内的分布

目的将人类PSF基因的不同功能片段定向连入pEGFP—C2质粒,使PSF蛋白的各功能片段与绿色荧光蛋白在HeLa细胞内融合表达,观察其在HeLa细胞中的表达及定位。方法以重组质粒pEGFP—C2-PSF为模板,PCR法扩增出目的基因,将扩增片段双酶切后连接到质粒pEGFP—C2上,构建重组质粒pEGFP—C2-PSF（I—V）。将构建成功的pEGFP—C2-PSF（I—V）质粒脂质体法转染HeLa细胞,Western印迹检测融合蛋白的表达,并在荧光显微镜下观察融合蛋白的定位与分布。结果成功构建质粒pEGFP—C2-PSF（I～V）,并在HeLa细胞中实现表达;Western印迹检测到融合蛋白GFP—PSF（I～V）;在激光共聚焦显微镜下观察到绿色的融合蛋白表达和定位。结论人类PSF基因的不同功能片段的重组质粒pEG—FP—C2-PSF（I～V）构建成功,可用于标记PSF蛋白的不同功能片段,为进一步研究PSF在信号转导中的作用机制以及其生物学功能奠定基础。     

陆地棉PIP亚家族基因的克隆及表达特征分析

该研究以陆地棉苯基香豆满苄基醚还原酶(phenylcoumaran benzylic ether reductase,PCBER)氨基酸序列为探针,利用Blastp从陆地棉基因组数据库中发现了6个同源性较高的基因。根据6个基因序列设计引物,利用RT-PCR技术从陆地棉纤维细胞中克隆出了这6个基因的全长cDNA序列,分别命名为GhPCBER1、GhPCBER2、GhPCBER3、GhIFR、GhPLR1和GhPLR2。多重序列比对和进化树分析发现,6个蛋白均含有PIP类型蛋白的所有保守性基序和活性残基,属于PIP亚家族。实时荧光定量PCR结果显示,除GhPLR1之外其他5个PIP亚家族基因均在纤维细胞中优势或特异表达;在纤维发育过程中,GhPCBER1、GhPCBER2、GhPCBER3和GhIFR的表达均表现为先上升后下降,GhPCBER1和GhPCBER2在花后21d表达量达到最高,GhPCBER3和GhIFR在花后18d达到最高,GhPLR1和GhPLR2在纤维中的表达量呈持续上升趋势。根据基因的表达特征,推测PIP亚家族可能在棉纤维的发育过程中发挥着重要作用。     

麻疯树水通道蛋白新基因JcPIP干旱胁迫下的功能分析

采用RT-PCR和RACE技术,从大戟科(Euphorbiaceae)耐旱植物麻疯树(Jarropha curcas)cDNA中克隆得到了一个麻疯树质膜内膜蛋白(PIP)新基因,命名为JcPIP.聚类分析表明,麻疯树PIP蛋白与蓖麻、葡萄和菠菜的PIP蛋白在进化上有最近的亲缘关系.将JcPIP基因在非洲爪蟾卵母细胞(xenopus oocytes)中异源表达发现细胞膨胀率扩大了10倍,表明JePIP基因编码的是一个水通道蛋白.合成亲水性、抗原性好的JcPIP保守序列多肽,制备并纯化JcPIP多克隆抗体.Western-bbt检测显示JcPIP蛋白富集在29 kDa区段,且在麻疯树各组织中均有大量表达.PEG-6000干旱胁迫下麻疯树叶片JcPIP蛋白丰度增加,复水后丰度开始下降,表明JcPlP与麻疯树的耐旱性相关.     

盐穗木水通道蛋白基因的克隆与功能鉴定

非生物胁迫(如盐渍和干旱)会引起植物水分代谢的紊乱,导致植物细胞水分丧失,直接抑制植物的生长发育。研究水通道蛋白(aquaporins,AQPs)在非生物胁迫下对植物生理功能的影响十分重要。本研究利用RACE技术克隆获得一个盐穗木(Halostachys caspica)水通道蛋白家族亚类质膜内嵌蛋白(plasma intrinsic protein,PIPs)基因,命名为Hc PIP1。Hc PIP1基因全长序列为1 244 bp,包含858 bp的开放阅读框(open reading frame,ORF)序列,编码285个氨基酸,分子量大小约为30.6 k D。q RT-PCR检测表明Hc PIP1在盐穗木根部的表达量显著高于同化枝中,盐胁迫诱导其上调表达。在酿酒酵母INVSc1中Hc PIP1的异源过表达显著提高了重组菌的耐盐能力;和野生型植株相比过表达Hc PIP1的拟南芥能够显著缓解渗透胁迫和离子胁迫对生长的抑制,并且根长明显比野生型的长。结果表明Hc PIP1在胁迫时能够通过增加根的生长以抵御胁迫的影响。     

番茄水通道蛋白基因家族的生物信息学分析

植物水通道蛋白不仅仅在根系输送水分,而是具有底物和细胞定位的多样性的基因家族,因而对植物生理和发育过程,包括种子发芽、侧根发生、碳固定和营养吸收等都有重要的贡献。番茄基因组中共有44个水通道蛋白基因。这些基因可分为TIP、PIP、NIP和SIP 4个亚家族。这4个亚家族具有不同的功能和亚细胞定位。在番茄十号染色体水通道蛋白聚集位点上的5个基因与一号染色体上的一个基因是直系同源水通道蛋白,相似度都很高,同属于PIP亚家族。番茄水通道蛋白大多在根部表达量较高,在茎、叶、花等部位表达较低。不过Solyc03g096290.2.1和Solyc01g094690.2.1基因在花中表达量最高。此外,有个别基因表达量稳定在较高的水平,包括Solyc06g074820.2.1和Solyc08g081190.2.1。这些基因的功能可能不仅是吸收和转运水分,是否有其他的转运底物,在植物抗逆和发育过程中是否有调控作用,是下一步研究的重点。番茄全基因组中的水通道蛋白进行检索和分析,对番茄分子生物学研究和种质改良都有重要的意义。     

牙龈卟啉单胞菌HA2基因和白细胞介素IL-15基因重组质粒的构建

构建抗牙龈卟啉单胞菌的牙周炎基因疫苗p VAX1-HA2、pVAX1-HA2/IL-15,体外检测其在293T细胞的表达。以HA2基因(牙龈卟啉单胞菌牙龈素—血凝素基因编码区的核心功能区)为目的基因与IL-15基因为免疫佐剂构建真核表达质粒,用Lip2000介导瞬时转染293T细胞,RT-PCR检测目的基因mRNA水平及酶联免疫吸附试验检测IL-15蛋白表达水平。重组质粒p VAX1-HA2、pVAX1-HA2/IL-15经酶切及DNA测序鉴定构建正确,转染的293T细胞能够检测到目的基因的表达,也可以检测到IL-15蛋白的表达。说明我们成功构建了真核共表达质粒pVAX1-HA2和p VAX1-HA2/IL-15,为下一步研制抗牙龈卟啉单胞菌DNA疫苗奠定了基础。     

Overexpression of PIP2;5 aquaporin alleviates effects of low root temperature on cell hydraulic conductivity and growth in Arabidopsis

The effects of low root temperature on growth and root cell water transport were compared between wild-type Arabidopsis (Arabidopsis thaliana) and plants overexpressing plasma membrane intrinsic protein 1;4 (PIP1;4) and PIP2;5. Descending root temperature from 25°C to 10°C quickly reduced cell hydraulic conductivity (L(p)) in wild-type plants but did not affect L(p) in plants overexpressing PIP1;4 and PIP2;5. Similarly, when the roots of wild-type plants were exposed to 10°C for 1 d, L(p) was lower compared with 25°C. However, there was no effect of low root temperature on L(p) in PIP1;4- and PIP2;5-overexpressing plants after 1 d of treatment. When the roots were exposed to 10°C for 5 d, L(p) was reduced in wild-type plants and in plants overexpressing PIP1;4, whereas there was still no effect in PIP2;5-overexpressing plants. These results suggest that the gating mechanism in PIP1;4 may be more sensitive to prolonged low temperature compared with PIP2;5. The reduction of L(p) at 10°C in roots of wild-type plants was partly restored to the preexposure level by 5 mm Ca(NO(3))(2) and protein phosphatase inhibitors (75 nm okadaic acid or 1 μm Na(3)VO(4)), suggesting that aquaporin phosphorylation/dephosphorylation processes were involved in this response. The temperature sensitivity of cell water transport in roots was reflected by a reduction in shoot and root growth rates in the wild-type and PIP1;4-overexpressing plants exposed to 10°C root temperature for 5 d. However, low root temperature had no effect on growth in plants overexpressing PIP2;5. These results provide strong evidence for a link between growth at low root temperature and aquaporin-mediated root water transport in Arabidopsis.     

Aquaporin JcPIP2 is involved in drought responses in Jatropha curcas

Water channel proteins, aquaporins, play fundamental roles in transmembrane water movements in plants. A new full-length cDNA encoding aquaporin was isolated from the seedlings of Jatropha curcas. The gene of the plasma membrane intrinsic protein （PIP） from J. curcas （JcPIP2） contained an 843 bp open reading frame encoding a protein of 280 amino acids. The amino acid sequence showed 94% identity with Ricinus communis PIP. Injection of JcPIP2 complementary RNA into Xenopus oocytes increased 10-fold the osmotic water permeability of the oocytes. Immunodetection of JcPIP2 with anti-JcPIP2 antibody indicated that this protein is ubiquitously located in all tested tissues of the plant. To investigate the relationship between aquaporins and drought resistance in J. curcas, the abundance of JcPIP2 was examined in seedlings of two J. curcas populations, GaoYou CSC63 and YanBian S 1, under water deficit with PEG6000. Under field conditions, those two populations, GaoYou CSC63 was resistant to water deficit, but YanBian S 1 was sensitive to water deprivation. With the increasing degree of drought stress, JcPIP2 level increased in seedlings of GaoYou CSC63, whereas there was no significant change in seedlings of YanBian S 1. Compared with YanBian S 1, GaoYou CSC63 also showed higher root hydraulic conductivity and lower decreasing trend in the seed- lings under water deficit. These results indicated that JcPIP2 probably played a role in drought resistance in J. curcas.     

Plasma membrane aquaporins play a significant role during recovery from water deficit

The role of plasma membrane aquaporins (PIPs) in water relations of Arabidopsis was studied by examining plants with reduced expression of PIP1 and PIP2 aquaporins, produced by crossing two different antisense lines. Compared with controls, the double antisense (dAS) plants had reduced amounts of PIP1 and PIP2 aquaporins, and the osmotic hydraulic conductivity of isolated root and leaf protoplasts was reduced 5- to 30-fold. The dAS plants had a 3-fold decrease in the root hydraulic conductivity expressed on a root dry mass basis, but a compensating 2.5-fold increase in the root to leaf dry mass ratio. The leaf hydraulic conductance expressed on a leaf area basis was similar for the dAS compared with the control plants. As a result, the hydraulic conductance of the whole plant was unchanged. Under sufficient and under water-deficient conditions, stomatal conductance, transpiration rate, plant hydraulic conductance, leaf water potential, osmotic pressure, and turgor pressure were similar for the dAS compared with the control plants. However, after 4 d of rewatering following 8 d of drying, the control plants recovered their hydraulic conductance and their transpiration rates faster than the dAS plants. Moreover, after rewatering, the leaf water potential was significantly higher for the control than for the dAS plants. From these results, we conclude that the PIPs play an important role in the recovery of Arabidopsis from the water-deficient condition.     

Over-expression of PIP2;5 aquaporin alleviates gas exchange and growth inhibition in poplars exposed to mild osmotic stress with polyethylene glycol

The effects of mild osmotic stress conditions on aquaporin-mediated water transport are not well understood. In the present study, mild osmotic stress treatments with 20 and 50 g L^?1 polyethylene glycol 6000 (PEG) in Hoagland’s mineral solution were applied for 3 weeks under controlled environmental conditions to transgenic Populus tremula × Populus alba plants constitutively over-expressing a Populus PIP2;5 aquaporin and compared with the wild-type plants. The PEG treatments resulted in growth reductions and triggered changes in net photosynthesis, transpiration, stomatal conductance and root hydraulic conductivity in the wild-type plants. However, height growth, leaf area, gas exchange, and root hydraulic conductivity were less affected by the PEG treatments in PIP2;5-over-expressing poplar lines. These results suggest that water transport across the PIP2;5 aquaporin is an important process contributing to tolerance of mild osmotic stress in poplar. Greater membrane abundance of PIP2;5 was most likely the factor that was responsible for higher root hydraulic conductivity leading to improved plant water flux and, consequently, greater gas exchange and growth rates under mild osmotic stress conditions. The results also provide evidence for the functional significance of PIP2;5 aquaporin in water transport and its strong link to growth processes in poplar.     

Root growth and water uptake during water deficit and recovering in wheat

Root growth and soil water content were measured in a field experiment with wheat subjected to two periods of water deficit. The first period was induced early in the season between the early vegetative stage (22 DAS) and late terminal spikelet (50 DAS), the second period at mid-season between terminal spikelet (42 DAS) and anthesis (74 DAS). Total root growth was reduced under water deficit by a reduction in the top 30 cm, while the root system continued to grow in the deeper soil profile between 30 and 60 cm. Shortly after rewatering, the growth pattern reverted to fastest root growth rates in the shallow soil layers. In relative terms, the total root system increased in relation to the above ground dry matter under water shortage. The early-, the mid-season water deficit treatments, and the control treatment had total root length of 27.4, 19.4 and 30.6 km m^-2, respectively, about 2 wk before maturity. Evapotranspiration declined under water deficit, but water uptake in deeper layers increased. Water uptake per unit root length was reduced with water deficit and was still low shortly after rewatering. Remarkable was the increase in water uptake at 2–3 weeks after rewatering, both deficit treatments exceeded the control by almost 100%. This increase in water uptake followed the burst of new root growth in the upper regions of the soil. However, water uptake rates subsequently declined towards maturity, being between 0.15 L km^-1 d^-1 and 0.17 L km^-1 d^-1 for the early and mid-season water deficit treatments, slightly higher than the control, 0.12 L km^-1 d^-1. The results showed that the crop subjected to early water deficit could compensate for some of the reductions in root growth during subsequent rewatering, but the impact of the mid-season water deficit treatment was more severe and permanent.     

Upland rice and lowland rice exhibited different PIP expression under water deficit and ABA treatment

Aquaporins play a significant role in plant water relations. To further understand the aquaporin function in plants under water stress, the expression of a subgroup of aquaporins, plasma membrane intrinsic proteins （PIPs）, was studied at both the protein and mRNA level in upland rice （Oryza sativa L. cv. Zhonghan 3） and lowland rice （Oryza sativa L. cv. Xiushui 63） when they were water stressed by treatment with 20% polyethylene glycol （PEG）. Plants responded differently to 20% PEG treatment. Leaf water content of upland rice leaves was reduced rapidly. PIP protein level increased markedly in roots of both types, but only in leaves of upland rice after 10 h of PEG treatment. At the mRNA level, OsPIP1,2, OsPIP1,3, OsPIP2;1 and OsPIP2;5 in roots as well as OsPIP1,2 and OsPIP1;3 in leaves were significantly up-regulated in upland rice, whereas the corresponding genes remained unchanged or down-regulated in lowland rice. Meanwhile, we observed a significant increase in the endogenous abscisic acid （ABA） level in upland rice but not in lowland rice under water deficit. Treatment with 60 μM ABA enhanced the expression of OsPIP1;2, OsPIP2;5 and OsPIP2;6 in roots and OsPIP1;2, OsPIP2;4 and OsPIP2;6 in leaves of upland rice. The responsiveness of PIP genes to water stress and ABA were different, implying that the regulation of PIP genes involves both ABA-dependent and ABA-independent signaling oathways during water deficit.     

Water deficit during root development: effects on the growth of roots and osmotic water permeability of isolated root protoplasts

The effect of low water potentials on root growth of flax (Linum usitatissimum L. cv. Ariane), rape (Brassica napus L. de Candolle, cv. Bristol), hard wheat (Triticum turgidum L. cv. Cham1) and soft wheat (Triticum aestivum L. cv. Ritmo) was studied by measuring the osmotic water permeability (Pos) of root protoplasts and the protein abundance of PIP1 and PIP2 aquaporins. These different species require more or less water, the most sensitive to water deficit being flax and rape. Ritmo, is a cultivar of wheat adapted to temperate zones, while the other cultivar Cham1 is adapted to low-rainfall areas. The seedlings were germinated and grown in water, salt or sugar solutions at different water potentials. The values of Pos for flax, rape and Chaml wheat were normally distributed and could be characterized by mean +/- SD. Root protoplasts from water-grown seedlings had Pos values of 485+/-159 microm s(-1) (flax), 582+/-100 microm s(-1) (rape), and 6.3+/-3.5 microm s(-1) (Cham1). At the same age, the protoplasts from Ritmo exhibited a much wider range of values than the protoplasts of Cham1. When seedlings were grown under conditions of osmotic or salt stress, the mass of the roots was reduced for all species. With 0.25 mol kg(-1) sorbitol or 0.125 M NaCl, the Pos for flax, rape and Cham1 remained constant or slightly increased, while for Ritmo the reduction in the mass of the roots was paralleled by a reduction in Pos. Only Cham1 and Ritmo were able to germinate at a lower potential (0.5 mol kg(-1) sorbitol). For Ritmo the reduction in the mass of the roots was paralleled by a reduction in Pos when grown in this stress condition and both wheats exhibited low Pos values. The expression of the PIP1 and PIP2 aquaporins families was also studied by immunoblotting. We did not observe any difference in protein expression for any of the species, whatever the growing conditions. We suggest that the high Pos values for flax and rape could play a role in the sensitivity of these plants at low water potential. The low native Pos for Cham1 in spite of the expression of both families of aquaporins may reflect its adaptation to low-rainfall conditions by a functional regulation of the water channels. For a similar reason, the low-water-potential-induced Pos of Ritmo may also correspond to a down-regulation of the aquaporins, reflecting adaptation of this wheat to water-deficit conditions.     

The arbuscular mycorrhizal symbiosis regulates aquaporins activity and improves root cell water permeability in maize plants subjected to water stress

Studies have suggested that increased root hydraulic conductivity in mycorrhizal roots could be the result of increased cell‐to‐cell water flux via aquaporins. This study aimed to elucidate if the key effect of the regulation of maize aquaporins by the arbuscular mycorrhizal (AM) symbiosis is the enhancement of root cell water transport capacity. Thus, water permeability coefficient (P_f) and cell hydraulic conductivity (L_pc) were measured in root protoplast and intact cortex cells of AM and non‐AM plants subjected or not to water stress. Results showed that cells from droughted‐AM roots maintained P_f and L_pc values of nonstressed plants, whereas in non‐AM roots, these values declined drastically as a consequence of water deficit. Interestingly, the phosphorylation status of PIP2 aquaporins increased in AM plants subjected to water deficit, and P_f values higher than 12 μm s^?1 were found only in protoplasts from AM roots, revealing the higher water permeability of AM root cells. In parallel, the AM symbiosis increased stomatal conductance, net photosynthesis, and related parameters, showing a higher photosynthetic capacity in these plants. This study demonstrates a better performance of AM root cells in water transport under water deficit, which is connected to the shoot physiological performance in terms of photosynthetic capacity.