【作者】 杨靓；

【导师】 朱延明；

【作者基本信息】 东北农业大学 ， 植物学， 2010， 博士

【摘要】 高盐、干旱和低温等逆境条件是影响植物生长、发育和地理分布的重要环境限制因子,严重制约我国乃至世界的农业生产。随着分子生物学和基因工程技术的不断发展与日趋成熟,应用基因工程技术培育耐逆作物新品种已成为现代农业生产的一个重要途径。然而植物对胁迫的耐受是一个系统的调控过程,需要众多基因和蛋白的参与,导入单个功能基因对作物抗逆性的改良效果十分有限,难以满足实际农业生产的要求。而一些在信号转导网络中起调控作用的蛋白质因子,尤其是激酶蛋白,些许单基因的表达就可以启动信号传导网络,激活下游众多功能基因的转录、表达和功能蛋白的活化,从而达到综合改良作物抗逆性的效果。因此,挖掘抗逆蛋白激酶基因,将为作物转基因育种提供功能更加显著的基因资源。野生大豆是我国宝贵的野生种质资源,具有很强的抗逆性和适应能力,是抗逆基因克隆的理想材料。本研究以耐盐东北野生大豆为试材,结合本研究室已构建的耐盐东北野生大豆高盐、干旱、低温胁迫反应的基因表达谱,高通量筛选出胁迫早期应答蛋白激酶基因。并选取了其中SnRK类蛋白激酶基因GsAPK、钙/钙调素调控的受体类蛋白激酶基因GsCBRLK和LRR受体类蛋白激酶基因GsLRPK进行全长基因的克隆;通过半定量RT-PCR分析,揭示了靶基因在不同胁迫处理下的表达特性;通过亚细胞定位、结合特异性分析及酶学特性分析,初步研究了靶基因在非生物胁迫信号传导通路中的应答机制;最后通过超量表达技术分析了靶基因的抗逆功能。通过本研究,将加深对植物抗逆分子机理的认识,同时得到具有独立知识产权的、对植物渗透胁迫抗性起重要作用的新基因,为作物抗逆分子育种提供基因资源和奠定理论基础。本研究获得的主要研究结果如下:1.野生大豆渗透胁迫早期应答蛋白激酶ESTs的筛选结合野生大豆高盐、干旱和低温胁迫基因表达谱,利用Linux序列分析平台,预测了野生大豆ESTs数据库中所有ESTs在渗透胁迫早期的表达情况。在野生大豆ESTs数据库所包含的9983条Contigs序列中,7214条可在芯片上找到对应探针组。筛选出其中在低温胁迫(4oC)处理下表达量上调的非冗余蛋白激酶ESTs 18条,在NaCl胁迫(200mM)处理下表达量上调的非冗余蛋白激酶ESTs 20条,在干旱(30%PEG模拟)胁迫下表达量上调的非冗余蛋白激酶ESTs 27条。进一步通过蛋白数据库UniProt和Go注释数据库预测蛋白激酶ESTs的功能,选取在胁迫信号转导过程中起关键作用的3个蛋白激酶EST(sBM521010、BG044348和CL1238Contig1)进行全长基因克隆和生物学功能的研究。2.蛋白激酶全长基因的克隆及序列分析利用电子克隆、RACE技术克隆得到了3个野生大豆胁迫早期应答全长蛋白激酶基因:GsAPK(CDS区共1020bp,编码399个氨基酸)、GsCBRLK(CDS区共1362bp,编码453个氨基酸)和GsLRPK(CDS区共2145bp,编码714个氨基酸)。对3个基因进行序列比对、蛋白结构预测,结果表明:GsAPK基因产物N端不含有信号肽序列,但含有1个肉豆蔻酰基化位点,可能受翻译后修饰的调控。C端具有SnRK蛋白激酶家族的特征性催化结构域。此外,GsAPK基因产物可形成1个强跨膜结构域(184-201aa)。GsCBRLK基因产物N端不含有信号肽序列,不能形成跨膜结构域,但含有1个钙调素结合结构域(147-169aa),可与钙调素结合传导胁迫信号;1个肉豆蔻酰基化位点,可能受翻译后修饰的调控。C端具有丝/苏氨酸蛋白激酶家族的保守催化结构域。GsLRPK蛋白具有胞外串联排列的LRR基序、跨膜结构域和胞内蛋白激酶催化结构域,符合LRR-RLK家族的特征性结构。此外,N端含有1个信号肽序列(1-22aa)。3.蛋白激酶基因在不同胁迫处理下的表达特性分析利用半定量RT-PCR分析蛋白激酶基因在不同胁迫处理下的表达特性,结果表明: GsAPK、GsCBRLK和GsLRPK基因的转录受低温、干旱、高盐和ABA处理的诱导,可能在信号传导通路中参与ABA依赖的途径。但根中的变化趋势与叶中的变化趋势明显不同。在胁迫处理时间点上的分析表明,根中发生胁迫响应的时间明显的早于叶,存在组织特异性表达。4.蛋白激酶基因的亚细胞定位分析利用GFP依赖的瞬时表达系统分析蛋白激酶基因的亚细胞定位情况。构建了3个激酶蛋白与GFP融合的瞬时表达载体,采用农杆菌浸润法,使本氏烟叶片的表皮细胞瞬时表达重组蛋白,激光共聚焦显微镜的观察结果表明GsAPK、GsCBRLK和GsLRPK蛋白均定位于植物细胞的质膜上,可能参与胁迫信号的感受和传导。5.激酶蛋白GsCBRLK的结合特异性分析利用缺失突变法分析了GsCBRLK蛋白与GsCaM的体外结合特异性。构建了GsCBRLK蛋白全长、3个缺失突变和GsCaM蛋白的原核表达载体,并利用大肠杆菌BL21表达了重组蛋白。体外的结合实验表明GsCBRLK蛋白N端含有钙调素的结合位点(147-169aa),并且这种结合特异性是Ca2+依赖的。利用酵母双杂交分析了GsCBRLK蛋白与GsCaM的体内结合特异性。构建了GsCBRLK蛋白全长、3个缺失突变和GsCaM蛋白的酵母表达载体,并进行了共转化。报告基因β-半乳糖苷酶的转录和表达表明GsCBRLK蛋白可以和GsCaM在体内特异性的结合。6.蛋白激酶的酶学特性分析构建了GsAPK和GsLRPK蛋白的植物表达载体,并利用大肠杆菌BL21表达了重组蛋白。采用体外磷酸化分析纯化蛋白的酶学特性。结果表明:GsAPK蛋白的磷酸化活性需要ABA的激活,可以不依赖于Ca2+而存在。GsCBRLK蛋白的自磷酸化活性不受Mn2+激活,Mg2+有微弱的激活作用,而Ca2+的存在可以强烈的激活其自磷酸化活性。同时CaM可以通过直接与GsCBRLK蛋白的CaMBD区结合来正调控GsCBRLK蛋白自磷酸化和底物磷酸化活性。GsLRPK蛋白的磷酸化活性需要低温和干燥的激活。7.激酶基因的超量表达及抗逆功能分析构建了2个融合有His6-Tag和GsAPK、GsCBRLK激酶蛋白的植物超量表达载体,采用农杆菌介导的花序浸蘸法对模式植物拟南芥进行遗传转化。通过Southern blot和RT-PCR对抗性植株进行了分子生物学检测,每个激酶基因得到T4代转基因植株2株。通过分析转基因及野生型植株在胁迫处理下的表型,确定激酶基因的抗逆功能。结果表明:GsAPK提高了转基因植株对ABA胁迫的抗性,却降低了转基因植株对NaCl胁迫的抗性;GsCBRLK提高转基因植株对NaCl和ABA胁迫的抗性。构建了GsLRPK蛋白的酵母超量表达载体,采用LiAc法对酵母进行转化。通过分析转GsLRPK基因酵母和转空载体酵母在胁迫处理下的表型,确定激酶基因GsLRPK的抗逆功能。结果表明:GsLRPK能够提高转基因酵母对低温胁迫的抗性,而不能改变对NaCl和高渗的抗逆。更多还原

【Abstract】 High salinity, drought and low temperature are the major environmental factors that limit plant growth, development, geographical distribution and adversely affect crop production in the world. With the rapid development of molecular biology and bioengineering, using the bioengineering strategies to improve stress tolerance in crops has been an important aspect of modern agricultural research. However, stress tolerance is a complex physiological process in which many genes are involved. Using the bioengineering strategies to transfer a key controlling gene (eg: kinase gene, responding and transducting the stress signal) is a more efficient way to slove the problem. If the key kinase gene regulating signal transduction pathways in the plant resistance to stress could be gained, it would provide important gene resource for molecular breeding and theoretic support to the study of plant molecular mechanism of osmotic stress resistance.Wild soybean (Glycine soja) is characteristic of better stress-resistance and adaptive capacity. It is an important gene resource in molecular breeding by means of transgenic technology. In this study, the stress-responsive kinase genes of wild soybean were selected according to gene expression profiles under salinity, drought and cold stresses, which had been established in our laboratory previously. Full-length sequences of three kinase genes were obtained by in silico cloning and SMART-RACE technique. We analyzed the expression patterns of target genes under different stresses. Subcellular localization, kinase activities and physiological function were also detected. This research will provide key gene for plant genetic engineering of osmotic stress and important information for stress tolerance researches. The main results were summarized as follows:1. Selection of kinase ESTs early responding to osmotic stresses from G.sojaESTs of Glycine soja downloaded from dbEST database were aligned with more than 60 000 probe-sets of the Affymetrix soybean chip, and the expression patterns of ESTs were infered using gene expression profiles of wild soybean under salinity, drought and cold stress. The initial result showed that there were 18 kinase ESTs up-regulated under the 4oC treatment, 20 kinase ESTs up-regulated under the NaCl treatment and 27 kinase ESTs up-regulated under the PEG treatment. According to the annotations of UniProt and Go database, we choose the ESTs of GsAPK, GsCBRLK and GsLRPK for the full-length genes isolation.2. Isolation of protein kinase genes early responding to osmotic stresses from G. soja The full-length cDNA of the three kinase genes were isolated by in silico cloning method and the modified SMART-RACE technique. The full-length cDNA of GsAPK is 1020bp, encoding a 399 amino acid protein. The full-length cDNA of GsCBRLK gene is 1362bp, encoding a 453 amino acid protein. The full-length cDNA of GsLRPK gene is 2145bp, encoding a 714 amino acid protein. The bioinformatical analysis revealed as follows:GsAPK protein contains the catalytic domain of protein kinase family in its C terminal; myristyl-sitesbut without signal peptide in its N terminal, which suggests this protein might be regulated or activated by post-translation modifications. In addition, it has a transmembrane region from residue 184 to 201GsCBRLK protein has a CaM binding domain, a myristyl-sites, no signal peptide in its N terminal and the catalytic domain of protein kinase family in its C terminal.GsLRPK protein contains the feature of LRR-RLK family, LRRs motifs, transmembrane region, protein kinase catalytic domain and a signal peptide which can introduce the protein to the secretory vacuole.3. Expression patterns analysis of kinase genes under various stress treatmentsSemi-quantitative RT-PCR was used to analyze the expression patterns of kinase genes under different stresses. GsAPK, GsCBRLK and GsLRPK genes were induced by low-temperature, drought, high salinity and ABA stress treatments which demonstrate that they are likely involved in ABA-dependent signaling pathways and might serve as a master regulator in plant abiotic stress response. The tissue specific expression pattern in leaves and roots likely indicate the existence of different regulatory mechanisms in these two tissue types.4. Subcellular localization of kinase proteinsThree subcelluar localization vectors were constructed and the GFP-fusion kinase proteins were transiently expressed in tobacco leaf cells via the Agro-bacterial infiltration. GFP fusion kinase proteins were observed solely to be localized to the plasma membrane using a confocal laser scanning microscope.5. Protein GsCBRLK binding assayThe full-length GsCBRLK, three truncated forms and GsCaM were cloned into the downstream of HIS6 tag into the pET-32b expression vector. The constructs were then transformed into E. coli strain BL21(DE3) pLysS and fusion proteins were induced by IPTG. Using the CaM binding assay, we found that the CaM-binding property of GsCBRLK is Ca2+-dependent and the CaM-binding site is located at amino acids 147-169 in GsCBRLK.The full-length GsCBRLK, three truncated forms and GsCaM were cloned into the Y2H vectors and introduced into the yeast strain Y187. The activity ofβ-galactosidase indicated that GsCaM and GsCBRLK could interact in vivo. 6. Enzymatic activity assayIn vitro phosphorylation assays were performed to detect the enzymatic activity of target kinase proteins. The results showed that GsAPK is an ABA-activated Ca2+-independent protein kinase. GsCBRLK kinase activity is Ca2+ dependent and is possibly up-regulated through the direct interaction between CaMBD and CaM. In addition, Mn2+ did not activate GsCBRLK autophosphorylation while Mg2+ only had a weak effect. Interestingly, strong activity was observed in the presence of Ca2+. However, autophosphorylation of GsCBRLK is Mn2+-activated, not Mg2+ or Ca2+. GsLRPK is a cold and drought activated protein kinase.7. Overexpression of target kinase genes and the phenotypic analysis of transgenic plantsTwo binary vectors harboring the HIS6-tag-fused-kinase proteins were constructed and introduced into Arabidopsis plants by Agrobacterium tumefaciens-mediated transformation. DNA gel blot and the semi-quantitative RT-PCR showed the presence of kinase genes in the two independent T4 generation transgenic lines.Root growth assays were performed to detect the seedlings response to various stresses. The results showed that the growth was less significantly inhibited in the wild-type plants compared to that of GsAPK over-expressor lines under NaCl stress. Conversely, in ABA treatment, growth of transgenic Arabidopsis plants is better than wild-type plants. Statistical analysis confirmed that over-expressing GsAPK can significantly alter plant tolerance to salinity and ABA stress. GsCBRLK gene can significantly improve plant tolerance to salinity and ABA stress. GsLRPK gene functions in cold stress response of yeast cells.更多还原