摘要
植物凝集素蛋白激酶(LecRK)是植物体内广泛存在的一类类受体蛋白激酶,一般认为它们参与了多种信号转导和糖信号的识别,但是迄今为止对其具体基因功能的研究非常少,大部分基因功能未知。因此,本文对植物凝集素类受体蛋白激酶LecRK-b2开展了生物信息学分析及基因功能研究,获得了如下主要研究结果：
(1)利用生物信息学方法分析了LecRK-b2的核酸及其编码氨基酸序列,利用同源建模方法构建了LecRK-b2蛋白N端凝集素受体结构域的三维结构模型。研究发现LecRK-b2蛋白包含三个功能域：N端为大豆凝集素类似的配体识别功能域,中间为跨膜结构域,C端为丝／苏氨酸激酶功能域。疏水性分析表明LecRK-b2蛋白属于亲水性蛋白。三维结构模型显示LecRK-b2蛋白的N端Lectin功能域与现已测定结构的植物凝集素有非常相似的β-折叠和与碳水化合物结合的位点。plantCARE分析表明LecRK-b2基因的调控区域存在大量激素响应相关的元件以及与胁迫诱导相关的元件。PROSCAN预测发现LecRK-b2蛋白可能含有19个磷酸化位点,1个丝苏氨酸蛋白磷酸化活性位点,2个ATP结合位点；初步判断LecRK-b2蛋白属于丝苏氨酸蛋白激酶。
(2)阐明了LecRK-b2基因的时空表达特性及其编码蛋白的亚细胞定位,并初步了解了LecRK-b2基因在胁迫应答过程中所发挥的作用。RT-PCR分析结果显示：LecRK-b2基因在野生型拟南芥的各个组织器官的表达量都非常低,但是在种子萌发的初期其表达量明显上升。ABA、NaCl、甘露醇、水杨酸、机械损伤、衰老都能使LecRK-b2基因的转录水平明显升高,说明LecRK-b2基因的转录受环境胁迫的诱导。GFP融合蛋白的分析显示LecRK-b2蛋白定位在细胞膜上。Promoter::GUS试验结果表明所克隆的LecRK-b2基因上游的904bp的片段具有完整启动子的活性,具有时空表达的特异性,它控制LecRK-b2基因在种子萌发过程中高水平表达,而在成年植株中则基本不表达。
比较LecRK-b2基因的T-DNA插入突变体(lecrk-b2)和野生型种子在不同浓度ABA培养基上的萌发率后,发现lecrk-b2种子的萌发对ABA不敏感。此外,研究发现lecrk-b2和野生型种子中ABA含量没有明显差异,lecrk-b2的种子萌发和幼苗的根伸长对盐胁迫、渗透胁迫也呈现出不敏感表型。
构建了35S::LecRK-b2过表达载体,并用浸花蕾法转化SALK_020262的纯合子株系lecrk-b2,获得了转基因的植株。萌发实验结果显示：转基因株系与野生型的种子在不同浓度ABA培养基上的萌发没有明显差异,表明在T-DNA插入突变体lecrk-b2中过表达LecRK-b2基因能够恢复到野生型表型,从而证实是LecRK-b2基因的缺失导致了lecrk-b2的种子萌发对ABA不敏感。
(3)发现ABI3正调控LecRK-b2基因的转录。双亮荧光分析结果显示,瞬时过量表达的ABI3蛋白能够在拟南芥体内激活与LecRK-b2基因的启动子序列结合,并启动与之相连接的LUC的表达。通过对LecRK-b2基因的启动子序列调控元件分析,发现LecRK-b2基因的启动子序列中存在一个ABI3的结合元件RY/G基序。此外,定量PCR结果显示,在种子萌发过程中abi3突变体中LecRK-b2基因的转录水平明显降低。上述结果表明ABI3是LecRK-b2基因转录的调控因子之一。
(4)利用体外重组蛋白证实了LecRK-b2蛋白的丝／苏氨酸激酶活性。利用pColdTF载体对LecRK-b2基因编码蛋白进行了大肠杆菌表达,纯化得到了大量可溶的重组蛋白。利用同位素磷32标记的三磷酸腺苷([γ-32P]ATP)鉴定了重组蛋白的自激活活性,发现LecRK-b2的重组蛋白具有二价锰离子依赖的自激活激酶活性。将磷32标记了的LecRK-b2重组蛋白完全水解,通过薄层色谱(TLC)分析,发现重组蛋白的丝氨酸和苏氨酸在自我磷酸化过程中被磷酸化,表明LecRK-b2属于丝／苏氨酸激酶。
(5)体内和体外实验表明LecRK-b2蛋白重组蛋白以二聚体的形式存在。体外pulldown实验显示LecRK-b2蛋白的Lectin结构域和全长蛋白自身及其两者之间均能相互作用,而C端激酶功能域自身则不能相互作用。酵母双杂交结果显示Lectin结构域能够相互作用,而C端激酶功能域也不能相互作用,这与体外Pulldown的结果一致。进一步通过裂解荧光素酶互补分析,发现在体内LecRK-b2蛋白的N端及其全长存在相互作用。这些结果表明：LecRK-b2受体蛋白的N端及全长蛋白在拟南芥活体细胞内存在相互作用并以二聚体的形式存在。
Lectin protein kinase (LecRK) belongs to receptor-like protein kinase which is widespread in plants. It is generally believed that they participate in varieties of signal transduction and the identification of sugar signals. But to date, little is known about their gene function. In this study, Arabidopsis lectin receptor-like kinase LecRK-b2 was analyzed with bioinformatics methods and its gene functions were investigated. The results of detailed research are as follows:
(1) Using the bioinformatics methods, the amino acid sequence and functional domains of LecRK-b2 were analyzed. Applying the homology modeling, the three-dimensional structure model of lectin receptor domain of LecRK-b2 protein was constructed. The results showed that LecRK-b2 contained three functional domains: N-terminal was the soybean-agglutinin-similar ligand recognition domain, the middle part was a transmembrane domain, C-terminal contained a serine/threonine kinase domain. The 24 amino acids nearby the N-terminal were predicted by the software for the signal peptide sequence. Hydrophobicity analysis results showed that, except for an obviously hydrophobic areas which formed a transmembrane structure, there were no other significantly hydrophobic domains. The total number of hydrophilic amino acid was slightly more than hydrophobic amino acids, so LecRK-b2 was a hydrophilic protein. Three-dimensional structure model showed that the N-terminal domain of LecRK-b2 protein was very similar to the plant lectins whose crystal structure was determined inβ-sheet and carbohydrate binding site. plantCARE analysis showed that there existed a large number of hormone-related boxes, as well as stress-induced associated boxes in the regulatory region of LecRK-b2 gene. The predicted results of PROSCAN program showed that LecRK-b2 protein might contain 19 phosphorylation site, a serine/threonine protein phosphorylation active site, two ATP-binding site. It seemed that LecRK-b2 was a serine/threonine protein kinase.
(2) RT-PCR analysis revealed that LecRK-b2 gene had very low expression levels in various tissues and organs in wild-type Arabidopsis, but its expression level significantly increased in the first 24 hours during germination process, and its declined to a very low level after the completion of germination. ABA, NaCl, mannitol, salicylic acid, wounding, and senescence could induce the transcription of LecRK-b2 gene. GFP fusion protein analysis revealed that the LecRK-b2 localized in the plasmid membrane. Promoter::GUS test results showed that the 904bp sequence in the upstream of LecRK-b2 gene had a completed promoter activity, temporal and spatial specificity expression patterns, and controled LecRK-b2 gene kept in a high level of expression during germination. While the transcription of LecRK-b2 gene was almost unable to be detectedin adult plants..
The seeds germination of T-DNA insertion mutants (lecrk-b2) was insensitive to ABA. Analysis results indicated that there was no significant difference in ABA content between lecrk-b2 and wild-type seeds. It illustrated that the ABA-insensitive germination of lecrk-b2 seed was not caused by the different endogenous ABA content, but had relationship with the ABA-related signal transduction. In addition, germination and seedling root elongation of lecrk-b2 was insensitive to salt stress and osmotic stress.
Transgenic plants were obtained by constructing 35S::LecRK-b2 plasmid and transforming lecrk-b2. The results showed that there was no significant difference in germination between overexpression transgenic lines and wild-type on different concentrations of ABA containing medium. This revealed that the T-DNA insertion mutant SALK_020262 could be restored to the wild-type phenotype by 35S::LecRK-b2. It indicated that LecRK-b2 gene deletion led to ABA-insensitive phenotype of lecrk-b2 seeds.
(3) Quantitative PCR results showed that, during the process of seed germination, transcription levels of LecRK-b2 gene were significantly declined in abi3 mutant. It indicated that LecRK-b2 gene transcription was positively regulated by ABI3. Dual-LUC analysis showed that the transiently over-expressed ABI3 protein could active the expression of the LUC, which was controlled by LecRK-b2 gene promoter in vivo in Arabidopsis thaliana. LecRK-b2 gene promoter sequence analysis displayed that there was a binding site of ABI3 named RY/G motif. Combining these results, we concluded that LecRK-b2 gene transcription was regulated by ABI3 protein in Arabidopsis in vivo.
(4) By applying pColdTF vector, we expressed LecRK-b2 protein in Escherichia coli and obtained purified soluble recombinant protein. By using 32P-isotope labeled adenosine triphosphate ([γ-32P] ATP) to provide phosphate, we confirmed that the recombinant protein was autophosphorylated and the autophosphorylation activity of recombinant LecRK-b2 was divalent manganese ion dependent. After the recombinant LecRK-b2 protein labeled with 32P hydrolyzed completely in 6N of HCl, the thin-layer chromatography (TLC) was used for the analysis. It was found that the serine and threonine of recombinant protein was phosphorylated in the self-phosphorylation process. It indicated that the LecRK-b2 belonged to serine/threonine kinase.
(5) The in vitro pull-down assay showed that recombinant LecRK-b2 protein and the lectin domain could interacted with itself respectively, but the C-terminal kinase domain could not self-interact. Yeast two-hybrid results showed that the lectin domain had self-interaction, and the C-terminal kinase domain did not interact with itself. This result was consistent with that of pull-down assay. Finally, split luciferase complementary assays displayed that the N-terminal and full-length LecRK-b2 protein had self-interaction in vivo. It indicated that LecRK-b2 protein could form dimer in living cells of Arabidopsis thaliana, and the lectin domain of N-terminal played a major role for the formation of protein dimers; and a separate C-terminal kinase domain could not interact with itself, it did not contribute to the formation of dimer structure of the entire receptor protein.
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