摘要
干旱是影响小麦(Triticum aestivum L.)产量的主要因素之一。利用分子生物技术,发掘利用优异的抗旱基因资源提高小麦的抗旱性,是保障小麦生产可持续发展的最有效途径。蛋白质可逆磷酸化是细胞信号传递过程中几乎所有信号传递途径的中心环节,蛋白激酶在渗透胁迫等非生物胁迫下的信号转导过程中起关键作用。前人研究表明蔗糖非发酵相关蛋白激酶家族2(SnRK2)受渗透胁迫诱导表达,小麦TaSnRK2.3基因是SnRK2基因家族成员之一。
本研究以强抗旱小麦品种旱选10号为试验材料,首次从中分离到小麦TaSnRK2.3基因,对其进化关系、表达特性及功能进行了分析。主要结果如下：
1.克隆得到TaSnRK2.3全长cDNA,开放阅读框为1029bp,共编码342个氨基酸。等电点为5.55,分子量为38KD,存在丝氨酸／苏氨酸蛋白激酶催化域。与水稻OsSAPK3亲缘关系最近,与水稻、玉米、拟南芥中的SnRK2.3基因同源性依次为84.7%,73.1%,63.2%。
2.TaSnRK2.3在小麦拔节期叶片中表达量最高,在抽穗期幼穗中表达量最低,表达量次序依次为：拔节期叶>幼苗根>幼苗叶>抽穗期幼穗。TaSnRK2.3响应外源ABA和低温、高盐、高渗胁迫诱导,对这4种不同处理的敏感性次序依次为：高渗>高盐>ABA>低温；TaSnRK2.3在高盐和干旱胁迫下迅速大量表达且表达峰值均为对照的25倍以上,而在外源ABA诱导下表达高峰出现在24小时后且峰值仅为对照的5倍,由此推测TaSnRK2.3属于植物信号传导过程中非依赖ABA的基因一类。亚细胞定位发现TaSnRK2.3在细胞核、细胞质和细胞膜上均有分布。
3.过量表达TaSnRK2.3的拟南芥植株在幼苗期主根长度和侧根数目明显高于野生型和空载体植株。在高盐、高渗和外源ABA胁迫的培养基上,转基因植株种子萌发受到抑制的程度明显低于对照。生理指标检测表明,转基因植株在成株期间的叶绿素含量、叶绿素荧光(光系统Ⅱ最大光化学效率)、保水能力及水分利用能力均高于对照。逆境胁迫条件下,转基因植株的脯氨酸含量、细胞膜稳定性等抗性指标和表型数据表明,转基因植株对高盐和干旱胁迫的忍受力明显优于对照植株。对转基因拟南芥的不同株系进行目标基因表达量检测,其表达量越高,转基因植株耐盐耐旱能力越强。
综上所述,本研究初步证明了小麦TaSnRK2.3基因参与了渗透和干旱胁迫的应答反应过程,过量表达该基因能提高拟南芥的耐盐和耐旱性。
Drought is one of the main factors to affect wheat yield (Triticum aestivum L).It adopts molecular biotechnology to utilize genetic resources of high-quality wheat and improve wheat drought resistance, which is the most effective shortcut to improve wheat drought-resistance and steady yield potenttial. Protein reversible phosphorylation is the key link of cell single transduction in almost all signaling pathways.Protein kinase (PK) plays an important role in signal transduction under abiotic stresses, such as osmosis, etc.The previous studies indicated that the expression of sucrose non-fermentation associated PK family (SnRK2) was induced by osmotic stress,and the wheat gene TaSnRK2.3 is one of the members in SnRK2 gene family.
In this study, Hanxuan 10, a prominent drought resistance wheat variety, was used to isolate TaSnRK2.3 and characterize gene expression patterns under diverse stress conditions.The main results are following.
1.The TaSnRK2.3 full-length CDNA was obtained by cloning, with open reading frame (ORF) is 1029bp,encoding a protein with-342 amino acids.The isoelectric point is 5.55,and molecular weight is 38KD.There exists the active site of serine/threonine protein kinase (PK).It has the closest genetic relationship with rice OsSAPK3,and the gene homology of SnRK2.3 in rice, corn and Arabidopsis thaliana is 84.7%,73,1%, 63.2% respectively.
2.The TaSnRK2.3 have the highest expression level in leaf blade at jointing stage, and the lowest expression level in young ears at heading stage.The expression level is as follow:Spindle leaf at jointing stage> root of seedling> leaf of seedling> young ear at heading stage.The TaSnRK2.3 have different expression patterns under induction of response to exogenous gene ABA, low temperature, high salt and high-osmotic stress. In these four different processing modes,the sensitivity is as follow:High-osmosis> high-salt> ABA> low temperature.The expression levels of TaSnRK2.3 peaked for-PEG and NaCl quickly,and 24h fei ABA,and (?)e conespon(?)ing maxima were 25,25 and 5 times the check, suggesting that TaSnRK2.3 might participate in non ABA-dependent signal transduction pathways.The TaSnRK2.3 distribute on cell nuclei, cell plasma and cell membrane.
3.During young seedling, the number of taproots and adventitious roots of transgenic TaSnRK2.3 in Arabidopsis thaliana plants is obviously more than in wild-type plants. In stress media, the seed germination of transgenic plants is obviously less inhibited than check plants.the chlorophyll content, maximal quantum yield of PS II photochemistry, water retention ability and osmotic potential of the transgenic plants have obviously stronger resistance than check plants.In adult period, the related resilience physiological such as free proline and cell membrane stability, and biochemical indexes and phenotypic data indicates the plants of transgenic target genes have obviously stronger resistance on high salt and drought stress than wild-type plants and empty-vector plants.Through detecting expression level of target genes in different transgenic strains, the higher expression level, the stronger capacity of salt and drought resistance in transgenic plants.
The studies in this paper indicate that wheat genes TaSnRK2.3 participated response to osmosis and drought stress, and over expression of the genes can improve the salt and drought resistance for plants.
引文
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