摘要
随着拟南芥,水稻等模式植物基因组全序列测序工作的完成,植物科学界普遍认为其整体研究工作的重点应转为“功能基因组”的研究。借助已有的基因表达谱分析,发现拟南芥中一个功能未知的基因LWT1被低温和干旱诱导表达。然而,LWT1基因是否在低温和干旱胁迫中起重要的作用尚不清楚。
为了阐明LWT1基因的功能,从拟南芥种质资源中心获得了两个T-DNA插入LWT1基因的功能缺失型突变体lwt1-1和lwt1-2。本文研究了拟南芥野生型和突变体lwt1-1和lwt1-2对低温和干旱胁迫响应的特征。研究结果如下：
1.在低温胁迫条件下,lwt1-1和lwt1-2突变体的存活率都明显低于野生型；电导率的测定结果进一步证实lwt1-1和lwt1-2突变体在细胞水平上受到的低温伤害比野生型更轻。表明lwt1-1和lwt1-2突变体对低温胁迫表现敏感性。
2.在干旱胁迫条件下,lwt1-1和lwt1-2突变体的存活率显著低于野生型；lwt1-1和lwt1-2突变离体体叶片的失水率进一步证明了突变体的保水能力比野生型差。表明lwt1-1和lwt1-2突变体对干旱胁迫表现敏感性。
3.在低温和干旱胁迫条件下野生型和lwt1-1和lwt1-2突变体体内可溶性总糖无明显的差异,而脯氨酸的积累表现出较大差异性,这表明lwt1-1和lwt1-2突变体对低温和干旱的敏感性与其体内可溶性糖积累无关,而与脯氨酸含量的积累有关。
4.低温和干旱胁迫的相关基因表达分析结果也表明,lwt1-1和lwt1-2突变体在低温和干旱的胁迫下突变体与野生型中CBF上下游基因的表达量无明显差异,说明lwt1-1和lwt1-2突变体对低温和干旱的敏感性可能与CBF途径无关。
5.低温和干旱胁迫下脯氨酸合成基因P5CS和脯氨酸降解基因PDH分析表明,突变体lwt1-1和lwt1-2中P5CS基因的表达量与野生型无明显差异,而PDH基因在lwt1-1和lwt1-2突变体中的表达远远高于野生型。这表明,LWT1基因抑制了脯氨酸降解基因的表达,从而在低温和干旱胁迫下完成脯氨酸的积累,达到适应外界逆境环境胁迫。
6.lwt1-1和lwt1-2突变体对甘露醇,ABA,以及NaCL的胁迫表现出无明显的差异。因此推测,LWT1基因对脯氨酸的调控导致的细胞内积累的脯氨酸含量是突变体植株表现出对低温和干旱敏感的主要原因。
综上所述,本研究结果证明了LWT1基因调节植物对低温和干旱的响应,主要是通过对脯氨酸降解基因PDH的调控来实现的。
With the completion of genome sequence of arabidopsis, rice and other model plants, plant scientists generally felt that its overall research focus should face to "functional genomics" research. Using gene expression microarray analysis data, expression of the LWT1 gene encoding a unkown protein was found to be induced by low temperature and drought stresses. However, it is unclear whether the LWT1 gene plays important roles in the regulation of the responses of Arabidopsis plants to low temperature and drought stresses.
To elucidate the function of the LWT1 gene, two T-DNA insertion loss-of-function mutants lwt1-1 and lwt1-2 were obtained from the Arabidopsis Biological Resource Center. In this study, the responses of the lwt1-1 and lwt1-2 mutants to low temperature and drought stresses were investigated. The main results are as follows:
1. Under low temperature stress, survival rates of the lwt1-1 and lwt1-2 mutant plants were lower than those of the wild type, and the electrolyte leakages of the lwt1-1 and lwt1-2 mutants were higher than the wild type, suggesting that the injury of the lwt1-1 and lwt1-2 mutants to low temperature stress in cellular level is more serious than to the wild type, and that the lwt1-1 and lwt1-2 mutants are more sensitive to low temperature stress than the wild type.
2. Under drought stress, survival rates of the lwt1-1 and lwt1-2 mutant'plants were lower than those of the wild type, and the water loss rates of leaves in the lwt1-1 and lwt1-2 mutants were higher than the wild type, suggesting further evidence that mutant water-retention capacity is worse than wild-type and that the lwt1-1 and lwt1-2 mutants are more sensitive to drought stress than the wild type.
3. Under low temperature and drought stress conditions, no significant difference in the level of soluble sugar was detected between the wild type and the lwtl-1 and lwtl-2 mutants, while a higher accumulation of proline was found in the wild type than in the lwt1-1 and lwt1-2 mutants, suggesting that the lwtl-1 and lwtl-2 mutants were sensitive to low temperature and drought, which is related to the accumulation of proline,not to the accumulation of soluble sugar.
4. Analysis of expression of low temperature and drought stress-related genes showed that no significant difference in the pattern of CBFs gene expression was found between lwt1-1 and lwt1-2 mutants as well as the wild type, indicating that the sensitivity of the lwt1-1 and lwtl-2 mutants to low temperature and drought stresses was not related to CBFs signaling transduction pathway.
5. Analysis of expression of proline synthesis-related gene P5CS and proline degradation-related gene PDH showed that the P5CS gene expression in the mutant lwt1-1 and lwt1-2 had no significant difference compared with the wild-type, while the PDH gene expression in lwt1-1 and lwt1-2 mutants was significantly higher than in the wild type, suggseting that LWT1 gene inhibited the expression of proline degradation gene, resulting in the accumulation of proline to low temperatures and drought stresses to adapt to the adverse environmental stresses.
6. No significant differences in responses to mannitol, ABA, and NaCl treatments were found beween the lwt1-1 and lwt1-2 mutants and the wild-type. Our results suggusted that the intracellular accumulation of proline content which was regulated by LWT1 gene is the main reason for the sensitivity of lwt1-1 and lwtl-2 mutants to low temperature and drought stresses.
In summary, the results provided evidence that LWT1 gene regulated plant responses to low temperature and drought stresses, mainly by controling the exprssion of proline degradation-related PDH gene.
引文
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