玉米ZmLTPL63基因的分离及功能鉴定
发布时间：2018-08-24 17:47
【摘要】：植物在生长发育过程中经常受到高温、干旱、盐等多种非生物胁迫的影响。这些逆境可以造成植物体内生理代谢障碍,严重影响作物的产量和品质。为了应对胁迫危害,植物在长期进化中形成了一套复杂而精密的防御机制,包括多重信号通路、各种转录因子及功能基因等。近年来研究发现,脂质转运蛋白(Lipid Transfer Proteins,LTPs)在植物的生长发育调节及非生物胁迫响应中起着重要作用,但大多数LTPs的具体作用机理尚不清楚。本研究根据玉米热带种质POB21自选系响应高温和干旱的转录组学差异,从中筛选到一个胁迫响应上调表达的功能未知LTPs-like基因。根据玉米基因组数据库Maize GDB中B73基因组中参考基因DRMZM2G05208的序列,通过RT-PCR方法获得同源克隆,命名为Zm LTPL63。对该基因的结构、进化关系、表达模式及生物学功能进行系统分析,主要研究结果如下:(1)基因序列分析表明,该基因CDS全长759bp,编码长252个氨基酸的肽链。预测Zm LTPL63蛋白的分子量约25.9k D,在N末端存在长度为23个氨基酸的信号肽。蛋白具有典型的LTPs结构特征,肽链中8个位置特异的半胱氨酸残基(8CM)可以配对产生4个二硫键,4个α螺旋折叠形成一个立体的疏水腔,可用来结合脂质分子。但与典型LTPs相比,Zm LTPL63编码的氨基酸序列更长,并且在N端信号肽和8CM基序之间富含脯氨酸。蛋白种间同源比对及进化分析表明,Zm LTPL63在多种植物中存在同源蛋白,与粟XP_012699184.1、高粱XP_002439994.1、渐尖二型花属OEL21002.1、粗山羊草EMT12664.1、二穗短柄草KQK15914.1及水稻BAC65920.1等蛋白存在较高同源性,而与拟南芥AT3G43720同源性较低。(2)通过q RT-PCR方法分析玉米Zm LTPL63基因的表达模式,结果表明其可以响应多种胁迫、植物激素和信号分子。Zm LTPL63基因的表达具有一定的组织特异性,在幼苗中的表达水平表现为根茎叶。Zm LTPL63可以受高温、PEG模拟干旱、盐(Na Cl)诱导表达。脱落酸(ABA)、乙烯(ET)、茉莉酸甲酯(Me JA)和H_2O_2能显著上调Zm LTPL63基因的表达水平,赤霉素(GA)处理后其表达水平出现下调。(3)为确定Zm LTPL63蛋白的亚细胞定位,构建了p ROKII-35S::Zm LTPL63-GFP融合表达载体并通过农杆菌瞬时侵染本生烟草叶片。利用激光共聚焦显微镜观察GFP荧光,发现Zm LTPL63-GFP的荧光只出现在细胞外周。(4)根据B73参考基因组序列克隆Zm LTPL63基因编码区上游长1500bp的DNA序列。序列分析软件预测表明启动子存在真菌、高温、干旱、低温等多种(非)生物胁迫响应元件和Me JA等激素响应元件。p BI121-PZm LTPL63::GUS转基因拟南芥幼苗的X-Gluc染色表明,基因在植株各器官中广泛表达,尤其在茎尖和和根尖表达较高。ABA和H_2O_2处理能够增强GUS表达,说明启动子表达调控特性与基因表达特性一致。(5)组成型超表达Zm LTPL63基因能增强转基因植株对干旱和盐的非生物胁迫抗性。在胁迫条件下,超表达Zm LTPL63转基因拟南芥比野生型幼苗根生长更快,成苗的存活率更高;转基因拟南芥离体叶盘在渗透胁迫下的褪绿黄化明显慢于野生型;同时,转基因拟南芥离体叶片失水速率也低于野生型。与野生型相比,转基因植株在干旱和高盐胁迫下ROS的积累量较低,抗氧化酶的活性更高,丙二醛含量较少,对H_2O_2的敏感性降低。以上结果表明超表达Zm LTPL63可以通过提高植株抗氧化能力来增加对干旱和盐胁迫的耐受性。(6)在本生烟叶片中瞬时表达Zm LTPL63,气相色谱检测表明叶片中不饱和脂肪酸如亚油酸、亚麻酸较对照明显上升。说明Zm LTPL63参与脂肪酸代谢的调节。(7)表达Zm LTPL63影响植株的生长发育进程。转基因拟南芥在生长发育后期表现为抽苔和开花延迟。q RT-PCR结果发现,参与开花正向调控的关键基因的表达量明显降低,推测Zm LTPL63可能通过抑制关键成花基因的表达导致开花延迟。
[Abstract]:Plants are often affected by abiotic stresses such as high temperature, drought and salt during their growth and development. These stresses can cause physiological and metabolic disorders in plants and seriously affect the yield and quality of crops. Recent studies have shown that Lipid Transfer Proteins (LTPs) play an important role in plant growth and development regulation and response to abiotic stress, but the specific mechanism of most LTPs is still unclear. According to the sequence of the reference gene DRMZM2G05208 in the B73 genome of Maize GDB, a homologous clone named Zm LTPL63 was obtained by RT-PCR and named Zm LTPL63. The main results were as follows: (1) The CDS gene was 759 BP in length and encoded a peptide chain of 252 amino acids. It was predicted that the molecular weight of Zm LTPL63 protein was about 25.9 kD, and there were signal peptides with 23 amino acids at the N-terminal. The protein had typical LTPs structure and 8 peptides in the peptide chain. Location-specific cysteine residues (8CM) can be paired to produce four disulfide bonds, and four alpha helixes fold into a three-dimensional hydrophobic cavity to bind lipid molecules. However, compared with typical LTPs, Zm LTPL63 encodes longer amino acid sequences and is rich in proline between N-terminal signal peptides and 8CM motifs. The results showed that Zm LTPL63 had high homology with millet XP_012699184.1, sorghum XP_002439994.1, oligomeric dimorphism OEL21002.1, Aegilops EMT12664.1, two-spike short-stalk grass KQK15914.1 and rice BAC65920.1, but low homology with Arabidopsis AT3G43720. The expression pattern of Zm LTPL63 gene in maize showed that it could respond to various stresses, plant hormones and signaling molecules. The expression of Zm LTPL63 gene had tissue specificity, and its expression level in seedlings showed rhizome and leaf. Zm LTPL63 could be induced by high temperature, PEG simulated drought, salt (Na Cl). Abscisic acid (ABA), ethylene (ET), jasmine. Me JA and H_2O_2 could significantly up-regulate the expression of Zm LTPL63 gene and down-regulate the expression level of Zm LTPL63 gene after treatment with GA. (3) To determine the subcellular localization of Zm LTPL63 protein, a fusion expression vector p ROKII-35S:: Zm LTPL63-GFP was constructed and transiently infected tobacco leaves by Agrobacterium tumefaciens. Laser confocal microscopy was used. The fluorescence of Zm LTPL63-GFP was observed under microscope, and it was found that the fluorescence of Zm LTPL63-GFP only appeared in the periphery of the cells. (4) A 1500 BP DNA sequence was cloned from the upstream coding region of Zm LTPL63 gene according to the B73 reference genome sequence. X-Gluc staining of transgenic Arabidopsis thaliana seedlings with P BI121-PZm LTPL63:: GUS showed that the gene was widely expressed in various organs of plants, especially in shoot and root tips. ABA and H_2O_2 treatment could enhance GUS expression, indicating that promoter expression regulation characteristics were consistent with gene expression characteristics. (5) Constitutive overexpression of Zm LTPL63 gene could be enhanced. Transgenic plants were resistant to abiotic stresses of drought and salt. Under stress conditions, the roots of transgenic Arabidopsis thaliana overexpressing Zm LTPL63 grew faster and the survival rate of seedlings was higher than that of wild-type seedlings; chlorosis and yellowing of in vitro leaf discs of transgenic Arabidopsis thaliana was slower than that of wild-type seedlings under osmotic stress. Compared with wild type, transgenic plants had lower ROS accumulation, higher antioxidant enzyme activity, lower MDA content and lower sensitivity to H_2O_2 under drought and high salt stress. Receptivity. (6) Transient expression of Zm LTPL63 in native tobacco leaves showed that unsaturated fatty acids such as linoleic acid and linolenic acid in leaves were significantly increased compared with the control. It was suggested that Zm LTPL63 was involved in the regulation of fatty acid metabolism. (7) Expression of Zm LTPL63 affected the growth and development of plants. Transgenic Arabidopsis showed smoking in the late growth and development stage. The results of Q RT-PCR showed that the expression of key genes involved in the positive regulation of flowering was significantly decreased, suggesting that Zm LTPL63 might cause delayed flowering by inhibiting the expression of key flowering genes.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;S513
[Abstract]:Plants are often affected by abiotic stresses such as high temperature, drought and salt during their growth and development. These stresses can cause physiological and metabolic disorders in plants and seriously affect the yield and quality of crops. Recent studies have shown that Lipid Transfer Proteins (LTPs) play an important role in plant growth and development regulation and response to abiotic stress, but the specific mechanism of most LTPs is still unclear. According to the sequence of the reference gene DRMZM2G05208 in the B73 genome of Maize GDB, a homologous clone named Zm LTPL63 was obtained by RT-PCR and named Zm LTPL63. The main results were as follows: (1) The CDS gene was 759 BP in length and encoded a peptide chain of 252 amino acids. It was predicted that the molecular weight of Zm LTPL63 protein was about 25.9 kD, and there were signal peptides with 23 amino acids at the N-terminal. The protein had typical LTPs structure and 8 peptides in the peptide chain. Location-specific cysteine residues (8CM) can be paired to produce four disulfide bonds, and four alpha helixes fold into a three-dimensional hydrophobic cavity to bind lipid molecules. However, compared with typical LTPs, Zm LTPL63 encodes longer amino acid sequences and is rich in proline between N-terminal signal peptides and 8CM motifs. The results showed that Zm LTPL63 had high homology with millet XP_012699184.1, sorghum XP_002439994.1, oligomeric dimorphism OEL21002.1, Aegilops EMT12664.1, two-spike short-stalk grass KQK15914.1 and rice BAC65920.1, but low homology with Arabidopsis AT3G43720. The expression pattern of Zm LTPL63 gene in maize showed that it could respond to various stresses, plant hormones and signaling molecules. The expression of Zm LTPL63 gene had tissue specificity, and its expression level in seedlings showed rhizome and leaf. Zm LTPL63 could be induced by high temperature, PEG simulated drought, salt (Na Cl). Abscisic acid (ABA), ethylene (ET), jasmine. Me JA and H_2O_2 could significantly up-regulate the expression of Zm LTPL63 gene and down-regulate the expression level of Zm LTPL63 gene after treatment with GA. (3) To determine the subcellular localization of Zm LTPL63 protein, a fusion expression vector p ROKII-35S:: Zm LTPL63-GFP was constructed and transiently infected tobacco leaves by Agrobacterium tumefaciens. Laser confocal microscopy was used. The fluorescence of Zm LTPL63-GFP was observed under microscope, and it was found that the fluorescence of Zm LTPL63-GFP only appeared in the periphery of the cells. (4) A 1500 BP DNA sequence was cloned from the upstream coding region of Zm LTPL63 gene according to the B73 reference genome sequence. X-Gluc staining of transgenic Arabidopsis thaliana seedlings with P BI121-PZm LTPL63:: GUS showed that the gene was widely expressed in various organs of plants, especially in shoot and root tips. ABA and H_2O_2 treatment could enhance GUS expression, indicating that promoter expression regulation characteristics were consistent with gene expression characteristics. (5) Constitutive overexpression of Zm LTPL63 gene could be enhanced. Transgenic plants were resistant to abiotic stresses of drought and salt. Under stress conditions, the roots of transgenic Arabidopsis thaliana overexpressing Zm LTPL63 grew faster and the survival rate of seedlings was higher than that of wild-type seedlings; chlorosis and yellowing of in vitro leaf discs of transgenic Arabidopsis thaliana was slower than that of wild-type seedlings under osmotic stress. Compared with wild type, transgenic plants had lower ROS accumulation, higher antioxidant enzyme activity, lower MDA content and lower sensitivity to H_2O_2 under drought and high salt stress. Receptivity. (6) Transient expression of Zm LTPL63 in native tobacco leaves showed that unsaturated fatty acids such as linoleic acid and linolenic acid in leaves were significantly increased compared with the control. It was suggested that Zm LTPL63 was involved in the regulation of fatty acid metabolism. (7) Expression of Zm LTPL63 affected the growth and development of plants. Transgenic Arabidopsis showed smoking in the late growth and development stage. The results of Q RT-PCR showed that the expression of key genes involved in the positive regulation of flowering was significantly decreased, suggesting that Zm LTPL63 might cause delayed flowering by inhibiting the expression of key flowering genes.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;S513
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本文编号：2201570
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