【作者】 王芳；

【导师】 麻彤辉；

【作者基本信息】 东北师范大学 ， 细胞生物学， 2006， 博士

【摘要】 水分是影响植物生长发育和限制农作物产量提高的主要环境因子。按复合水分运输模型,水分在植物体内运输有三个途径,即质外体途径、共质体途径和跨细胞途径。水通道蛋白为水分跨细胞膜的运输提供了一条选择性通道。植物水通道蛋白是一个超家族,根据氨基酸序列同源性和亚细胞定位可以划分为四个家族:质膜内在蛋白PIP、液泡膜内在蛋白TIP、类Nodulin26膜内在蛋白NIP和小的基本膜内在蛋白SIP。PIP被分为两个亚家族:PIP1和PIP2。从序列水平看,与PIP2亚家族成员相比,PIP1亚家族成员有一个长的N末端和短的C末端。PIP1和PIP2亚家族成员在功能上也存在差异。在爪蟾卵母细胞中异源表达时,与PIP2蛋白相比,PIP1表现没有或具较低的水通道活性。植物水通道蛋白的活性可能受门控机制调控。影响门控行为的因子可能包括磷酸化、异源基因化、pH、Ca2+、渗透压、溶质梯度和温度。植物水通道蛋白在植物种子萌发、细胞伸长、气孔运动、受精等过程中调节水分的快速跨膜运输。有些水通道蛋白还在植物逆境应答中起重要作用,因此研究水通道蛋白与植物抗逆性的关系引起了广泛关注。植物主要通过调控细胞质膜水通道蛋白的表达来消除水分供应中的波动。本研究的主要目的是以荒漠植物甘草(Glycyrrhiza uralensis Fisch)为研究对象来分离、鉴定质膜水通道蛋白基因,研究其定位和表达模式,以探讨它们在植物抗逆应答中的作用。1.根据植物质膜水通道蛋白NPA保守区设计简并引物,利用RT-PCR和RACE技术,从甘草根中克隆到一个水通道蛋白基因,命名为GuPIP1。序列分析表明,基因全长cDNA序列为1.236kb,包含长度为71bp的5′非编码区、870bp的开放阅读框及295bp的3′非编码区。该基因编码一个由290个氨基酸残基组成的蛋白质(基因已登录GenBank,登录号为AY781788)。2 .生物信息学分析表明,甘草GuPIP1不但具有MIP家族的信号序列(SGXHXNPAVT),而且具有质膜水通道蛋白的特征信号序列GGGANXXXXGY和TGI/TNPARSI/FGAAI/VI/VF/YN。同时甘草GuPIP1具有水通道蛋白典型的6个跨膜区和两个NPA单元,在第1和第4跨膜区均具有与水通道形成有关的高度保守的EXXXTXXF/L单元,与已经证明具有水通道蛋白功能的AtPIP1;4和ZmPIP1;5功能区的氨基酸序列高度一致,与其同源性高的其它植物水通道蛋白均为PIP1亚家族的成员,而其本身的N-末端比C-末端长46个氨基酸,符合植物水通道蛋白PIP1亚家族成员的N-末端长的特点,因此,甘草GuPIP1应属于PIP1亚家族成员。3.Western blot分析表明,甘草GuPIP1基因在幼苗的根、茎、叶中均有表达,不具有明显的组织特异性。甘草GuPIP1可能在整株植物的水分运输中发挥作用。更多还原

【Abstract】 Water is an ubiquitous and indispensable molecule for plant growth and development.According to the composite water flux model, water passes plant organs by three pathways:apoplastic, symplastic, and transcellular. The latter two routes can not be experimentallydistinguished and are combined as the cell-to-cell pathway. Aquaporins represent an importantselective pathway for water to move across cellular membranes. Genome studies have revealedthat aquaporins constitute a superfamily in plants. Arabidopsis. thaliana, Zea mays and Oryza sativahave 38, 35 and 33 genes, respectively. Based on amino-acid sequence homology andsubcellular location, plant aquaporins are classified into four subfamilies: PIPs for plasmamembrane intrinsic proteins;TIPs for tonoplast intrinsic proteins;NIPs for NOD26-likeintrinsic proteins;SIPs for small basic intrinsic proteins. The PIP subfamily has been furtherdivided into two major groups: PIP1 and PIP2. At the sequence level, PIP1s can bedistinguished from PIP2s proteins by a longer N-terminal extension and a shorter C-terminalend. PIP1s proteins exhibit no or very low water channel activity compared with PIP1sproteins, when expressed in the X. laevis oocyte heterologous system. Plant aquaporinactivity might be regulated by gating mechanisms. The factors affecting the gating behaviourpossibly involve phosphorylation, heteromerization, pH, Ca2+, osmotic pressure, solutegradients and temperature. Plant aquaporins are involved in seed germination, cellelongation, stomatal movement, fertilization and so on. Some plant aquaporins also play animportant role in response to stress. Many studies have focused on the relationship betweenplant aquaporins and stress resistance. Plants counteract fluctuations in water supply byregulating all aquaporins in the cell plasma membrane. An endemic drought-tolerant plant,Glycyrrhiza uralensis F., growing in the sandy desert at the west of China was collected andcultured for the molecular cloning of aquaporin genes and for monitoring the distribution andexpression patterns of aquaporins in order to further elucidate their involvement in eremicplant water transport.1. Two degenerate oligonucleotide primers were designed based on two conserved domainNPA boxes in the plant aquaporin amino acid sequence. Using RT-PCR, 5′RACE and3′RACE, a full length cDNA of aquaporin-like gene was isolated from cDNA preparedfrom Glycyrrhiza uralensis F. roots, named GuPIP1. The analysis of the sequence showsthat the GuPIP1 cDNA consists of 1,236-bp upstream of the poly (A) tail, which includes a71-bp 5′-noncoding region , followed by 870 bp of open reading frame encoding 290amino acids, and, finally, a 295-bp 3′-noncoding region (accession no. AY781788).2. The analysis of the amino acid sequence reveals that GuPIP1 contains the MIP familysequence SGXHXNPAVT, the PIP family signature sequence GGGANXXXXGY andTGI /TNPARSI/FGAAI/VI/VF/YN. GuPIP1 exhibits six predicted transmembrane helicesand possesses two sets of conserved NPA motifs as well as the highly conserved motifs onM1 and M4 as EXXXTXXF/L which may be involved in the recognition and transport ofwater molecules across the water channel. As compared to PIPs identified from the otherplant species AQPs, GuPIP1 has the highly sequence identity at the amino acid levelwith PIP1s among all plant AQPs compared, including AtPIP1;4 and ZmPIP1;5 testifiedas functional aquaporins. This aquaporin has a longer N-terminal extension thanC-terminal extension by 46 amino acids, which is consistent with the special PIP1signature sequence. So GuPIP1 should be a member of PIP1 subfamily.3. GuPIP1 protein was confirmed to be localized in the PM by immunochemical reaction withpeptide-specific antibodies. GuPIP1 was localized in epidermis cells and calyptrogen cells ofGlycyrrhiza uralensis F. radicles through immunohistochemical method. Western blotanalysis indicated that the expression of GuPIP1 was not tissue-specific but in roots, stems andleaves. So, GuPIP1 plays an important role in whole plant water transport.4. Quantitative real-time PCR analysis showed that the expression of GuPIP1 was up-regulated bydrought, ABA and salt stress. GuPIP1 may play a putative role as osmotic or turgor sensors.更多还原