摘要
SnRK2 (Sucrose non-fermenting 1-related protein kinases 2)是一类仅存在于植物中的蛋白激酶,是SnRK家族三个亚家族(SnRK1, SnRK2, SnRK3)的成员之一,已在众多植物中被克隆出来。该家族成员,编码Ser/Thr类蛋白激酶,可以对不同逆境信号作出应答,在植物抗逆生理中发挥重要的作用。
本研究中,我们从碱茅NaHCO3处理的cDNA文库中克隆得到了碱茅SnRK2基因(PutSnRK2)。该基因cDNA全长1726bp,1077bp的ORF编码了分子量41.1kDa,等电点为5.63的蛋白。通过BLAST比较,碱茅PutSnRK2基因编码的氨基酸序列与拟南芥、水稻、玉米等植物SnRK2序列有很高的同源性,PutSnRK2与水稻SAPK7和玉米ZmSnRK2.7的氨基酸序列有91%的相似性。SnRK2成员在酶活结构域上相对保守,而在C-端表现出多样性。
实验利用半定量RT-PCR方法,对PutSnRK2基因mRNA水平上的转录特性进行了研究。结果表明,PutSnRK2基因在各个器官均有表达,且在转录水平上不同程度的受到盐(NaCl、NaHCO3、Na2CO3)、干旱(ABA、PEG6000)等逆境的诱导,并随时间延长而发生变化。结果说明,PutSnRK2基因与许多逆境都存在一定的应答关系。
以pYES2为表达载体,将pYES2-PutSnRK2重组质粒转化到酵母(Saccharomyces cerevisiae) INVSC1中。实验结果表明,在Na2C03, NaHCO3及H202逆境下,特别是NaCl及Mannitol逆境下,表达PutSnRK2基因的酵母的长势要好于对照组。这一结果说明在环境逆境下,PutSnRK2基因的表达对提高酵母的抗性有着重要的作用。另外,利用GFP-PutSnRK2融合蛋白对PutSnRK2基因编码的蛋白质进行了定位,结果表明GFP-PutSnRK2融合蛋白定位于细胞核及细胞质中。
将碱茅PutSnRK2基因克隆到二元植物表达载体pBI121上,并利用农杆菌介导的浸润法将基因转入拟南芥中。通过PCR,Northern杂交等分子手段进行鉴定,实验共获得了16个相互独立并稳定表达PutSnRK2-GFP的转基因拟南芥植株。从中选取3个株系,进行了进一步的耐盐性分析,结果表明在NaCl逆境下,转基因拟南芥的生长状态要明显好于野生型,这些结果说明碱茅PutSnRK2基因对拟南芥在盐逆境中的生存发挥着重要的作用。
实验选取了PutSnRK2基因表达量较大的转基因拟南芥株系,对PutSnRK2基因相关基因的表达进行了初步的解析,结果表明PutSnRK2基因可以通过作用于AREB转录因子,调控AREB下游基因的表达,我们还发现在NaCl胁迫下PutSnRK2基因也可作用于AREB转录因子的某种上游基因,调控AREB的表达。
SnRK2 (Sucrose non-fermenting 1-related protein kinases 2) is a kind of protein kinase which exists only in plant. As a member of family of SnRK family (SnRK1, SnRK2, SnRK3), SnRK2s had been cloned in many plants.The members of SnRK family, encoding Ser/Thr like protein kinase,can response to different stress signals in plant, which play an improtant role in plant stress resistance physiological.
In this study, we cloned a SnRK2 gene from Puccinellia tenuifolra cDNA library dealted with NaHCO3. The full-length of PutSnRK2 gene was 1726bp, with an open reading frame (ORF) of 1077bp. This gene encoded a protein of 358 amino acids, with a predicted molecular of 41.1 KDa, and an isoelectric point of 5.63. BLAST at NCBI, we found PutSnRK2 has high homology to SnRK2 genes in Arabidopsis, rice and maize. The protein sequence of PutSnRK2 shared 91% identities with rice SAPK7 and maize ZmSnRK2.7. The acticity domain of SnRK2 members were relatively conservative, but their C-terminal showed high diversity.
Semi-quantitative RT-PCR was conducted to analysis of the expression of PutSnRK2 gene at mRNA level. The results showed that PutSnRK2 gene expressed in each tissue. The transcriptional levels of the PutSnRK2 genes were effected by salt (NaCl, NaHCO3, Na2CO3) and drought (ABA, PEG6000), and changed with the time course, which suggested that there existed a relationship between the expression of PutSnRK2 gene and enrironment stress.
The experiment used pYES2 as expression vector, and pYES2-PutSnRK2 was transformed into yeast (Saccharomyces cerevisiae) stain INVScl. The result showed that the growth of cells expressing PutSnRK2 was better than control under Na2CO3, NaHC03 and H2O2 stress, especially under NaCl and Mannitol. These results suggested that the expression of PutSnRK2 in yeast increased the cells' tolerance to various environmental stress. Additionally, in order to understand the sublocation of PutSnRK2, we expressed the GFP-PutSnRK2 fusion protein in yeast. The results showed that the GFP-PutSnRK2 fusion protein located in the nucleus and the cytoplasm.
The gene was cloned into pBI121, and introduced into A.thaliana by Agrobacterium tumefaciens-mediated transformation using the vacuum infiltration method. The 16 independent and stable expression lines of over-expressing PutSnRK2-GFP gene were confirmed by PCR and Northern blot analysis. Salt tolerance of the three independent transgenic lines were examined, and the transgenic plants grew better than wild-type plants. These results indicated that PutSnRK2 played an important role in plant response to salt.
We chose the transgenic lines with high expression, in order to identify the expression of PutSnRK2 interacted genes. The result showed that PutSnRK2 can regulate the expression of AREB downstream genes, and may be have interact with some AREB upstream genes to regulate the expression of AREB.
引文
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