烟草NtLTP4基因的克隆及功能分析

发布时间：2019-05-16 18:07

【摘要】：烟草产业是我国财政税收收入的重要来源。但是,干旱、盐碱、病虫害等逆境胁迫严重影响了烟草的产量和品质,从而制约了烟草的产业发展。如何提高烟草的生物及非生物胁迫抗性已成为当今研究的重要课题。为了应对这种逆境胁迫,植物本身形成了一套精密而复杂的防御机制。其中各种胁迫相关基因的表达调控在植物应答环境信号刺激反应过程中起着重要的作用。非特异性脂转移蛋白(non-specific lipid transfer protein,nsLTPs)在植物抗逆过程中就起到非常重要的作用。本研究以普通烟(Nicotiana tabacum,NC89)为材料,克隆得到NtLTP4基因,并对其进行生物学功能分析,具体研究结果如下:(1)基因序列分析表明,该基因cDNA全长为354bp,编码117个氨基酸的多肽,预测分子量大约为9kD,其中含有保守的五肽结构域:T/S-X-X-D-R/K和P-Y-X-I-S,且有8个位置保守的半胱氨酸。NtLTP4具有nsLTP蛋白典型的三级结构,即4个α-螺旋,4对二硫键,1个可结合和容纳脂质分子的疏水腔,对结合脂质分子具有重要的作用。聚类分析表明,烟草NtLTP4与烟树NgLTP1,拟南芥AtLTP7及AtLTP5等同源性较高,而这些基因均属于LTP家族中的Type I类。以上结果表明,NtLTP4属于LTP家族中的Type I类。(2)构建35S-NtLTP4::GFP融和表达载体,瞬时侵染本生烟叶片,利用激光共聚焦显微镜观察GFP荧光,发现35s-GFP在细胞质和细胞核中均有荧光,但是35s-NtLTP4::GFP只在细胞壁周围有荧光,说明NtLTP4可能定位在细胞壁,为NtLTP4的功能研究提供了理论依据。(3)采用qRT-PCR的方法,对NtLTP4基因在烟草受到不同胁迫时的表达特性进行了研究。结果表明,NtLTP4存在组织特异性,且转录受机械损伤、高盐和聚乙二醇(PEG)等非生物胁迫诱导;青枯病菌和马铃薯Y病毒等生物胁迫也不同程度的诱导该基因的表达;激素信号分子MeJA、ABA和SA可强烈诱导NtLTP4基因的表达。以上结果表明NtLTP4可能参与不同的信号途径,在植物生长发育及胁迫应答过程中发挥重要作用。(4)为研究NtLTP4的功能,构建NtLTP4与正义植物表达载体pROKII的重组载体pROKII-NtLTP4,采用农杆菌介导法转化普通烟。通过扩增部分表达载体序列鉴定阳性植株。对部分转基因植株进行qRT-PCR分析,选取NtLTP4表达水平高、中、低的3个株系用于功能分析实验。(5)盐胁迫处理后,与野生型普通烟相比,转基因植株提高了对盐胁迫的抗性。在含不同浓度NaCl(50mM、100mM和200mM)的MS培养基上,转基因烟草种子比野生型种子的萌发率要高;转基因烟草幼苗比野生型烟草幼苗的根长较长,生长势好。盐胁迫条件下,转基因植株中丙二醛含量较野生型低43%左右,说明转基因植物细胞膜脂过氧化程度较低,植物细胞受损害程度较轻;盐胁迫条件下,转基因植株中ROS产生相关的基因的表达量降低,ROS清除相关基因的表达量增加,各种抗氧化酶的活性升高,H_2O_2和O~(2-)含量较野生型分别低29%和18%左右,表明NtLTP4基因通过提高ROS清除相关基因,提高抗氧化酶活性,从而降低了植株中的ROS含量。以上结果表明,超表达NtLTP4通过提高抗氧化能力进而增加了盐胁迫抗性。(6)干旱胁迫处理后,超表达NtLTP4的转基因植株提高了对干旱胁迫的抗性。在含不同浓度的甘露醇MS培养基上,转基因烟草比野生型幼苗长势更好;H_2O_2和O~(2-)含量较野生型分别低25%和21%;且与ROS相关的基因的表达量均有所改变,各种抗氧化酶的活性也有所升高。以上结果表明,超表达NtLTP4可能正调控干旱胁迫抗性。(7)超表达NtLTP4的转基因植株提高了对青枯病菌(Ralstonia solanacearum)的抗性。与野生型植株相比,青枯菌处理后转基因植株中与SA信号途径相关的PR基因(PR1a、PR5)和与MeJA相关的PR基因(PR4)的表达量有所增加,推测NtLTP4可能通过SA-和JA-介导的信号途径的综合作用提高了对青枯菌的抗性。(8)超表达NtLTP4的转基因植株提高了对病毒的抗性。马铃薯Y病毒普通株系(Potato virus Y Ordinary strain,PVY-O)处理后转基因植株中PR基因(PR1a、PR5)的表达量增加,推测NtLTP4可能通过SA-介导的信号途径来提高对病毒的抗性。综上所述,NtLTP4提高了转基因植株对生物和非生物胁迫的抗性,这些研究结果对于进一步了解I类LTP的功能提供了理论基础,同时也为烟草抗逆及抗病新品种提供了依据。
[Abstract]:The tobacco industry is an important source of the tax revenue of our country. However, stress stress, such as drought, saline-alkali and plant diseases and insect pests, has seriously affected the yield and quality of the tobacco, thus restricting the industrial development of the tobacco. How to improve the biological and abiotic stress resistance of tobacco has become an important subject of today's research. In order to cope with such stress, the plant itself forms a sophisticated and complex defense mechanism. The expression regulation of various stress-related genes plays an important role in the response of plant response to signal stimulation. The non-specific lipid transfer protein (ntLPs) plays a very important role in the process of plant stress resistance. The results of this study were as follows: (1) The gene sequence analysis showed that the full length of the cDNA was 354 bp, and the predicted molecular weight was about 9 kD. There are conserved pentapeptide domains: T/ S-X-X-D-R/ K and P-Y-X-I-S, and there are 8 positions of conserved cysteine. NtLtP4 has a typical three-level structure of the nsLTP protein, i.e.,4 p-helix,4 pairs of disulfide bonds, one hydrophobic cavity which can bind and contain lipid molecules, and has an important effect on the binding of lipid molecules. The cluster analysis showed that the homologies of NtLP4 and NgLTP1, Arabidopsis AtLTP7 and AtLP5 were high, and all of these genes belonged to the Type I in the LTP family. The above results indicate that NtLTP4 belongs to the Type I class in the LTP family. (2) Construction of 35S-NtLtP4:: GFP fusion and expression vector, transient infection of the green tobacco leaf, observed GFP fluorescence by using a laser confocal microscope, and found that 35 s-GFP had fluorescence in the cytoplasm and the nucleus, but 35 s-NtLtP4:: GFP only had fluorescence around the cell wall, indicating that NtLtP4 could be located in the cell wall, And provides a theoretical basis for the function research of the NtLtP4. (3) The expression of NtLP4 gene in tobacco was studied by qRT-PCR. The results showed that NtLP4 had tissue specificity, and the transcription was induced by non-abiotic stress such as mechanical injury, high salt and polyethylene glycol (PEG), and the expression of the gene was also induced by biological stress such as bacterial wilt and potato Y virus. ABA and SA can strongly induce the expression of NtLP4 gene. The results show that NtLtP4 may take part in different signal paths and play an important role in the process of plant growth and stress response. (4) To study the function of NtLtP4, construct the recombinant vector pROKII-NtLP4 of NtLTP4 and the expression vector pROKII of the justice plant, and transform the normal cigarette by Agrobacterium-mediated method. The positive plant was identified by the amplification of the partial expression vector sequence. Some transgenic plants were analyzed by qRT-PCR, and the expression of NtLP4 was selected to be high, medium and low. (5) After salt stress treatment, the transgenic plants increased the resistance to salt stress as compared to wild-type normal tobacco. On MS medium containing different concentration of NaCl (50 mM,100 mM and 200 mM), the germination rate of transgenic tobacco seeds was higher than that of wild-type seeds, and the growth potential of transgenic tobacco seedlings was longer than that of wild-type tobacco seedlings. Under the condition of salt stress, the content of MDA in the transgenic plants is about 43% lower than that of the wild type, indicating that the lipid peroxidation degree of the transgenic plant cell membrane is low and the damage degree of the plant cells is light; under the condition of salt stress, the expression amount of the gene related to the ROS production in the transgenic plant is reduced, The expression of the related genes of ROS was increased, the activity of various antioxidant enzymes increased, and the content of H _ 2O _ 2 and O ~ (2-) was 29% and 18% lower than that of wild-type, indicating that the NtLtP4 gene could improve the activity of antioxidant enzymes by increasing ROS, thus reducing the ROS content in the plants. The results showed that the super-expression of NtLP4 increased the resistance of salt stress by increasing the anti-oxidation ability. (6) After drought stress treatment, the super-expression of NtLtP4 transgenic plants increased the resistance to drought stress. In MS medium containing different concentration of mannitol, the growth potential of transgenic tobacco was better than that of wild-type seedling, and the content of H _ 2O _ 2 and O ~ (2-) was 25% and 21% lower than that of wild-type. The above results show that overexpression of NtLP4 may be regulating the resistance of drought stress. (7) The transgenic plants with super-expression of NtLP4 increased the resistance to Ralstonia solanacearum. The expression levels of the PR genes (PR1a, PR5) associated with the SA signal pathway and the PR genes (PR4) associated with the MeJA were increased in the transgenic plants after the treatment of the Ralstonia, as compared to the wild-type plants, It is suggested that NtLtP4 may increase the resistance of NtLP4 to the Ralstonia solani by the combination of SA-and JA-mediated signaling pathways. (8) the super-expression of the transgenic plant of the NtLtP4 improves the resistance to the virus. The expression of PR gene (PR1a, PR5) in the transgenic plant was increased after the treatment of the virus Y-Ordinary strain (PVY-O), and it was suggested that NtLtP4 might increase the resistance to the virus by SA-mediated signaling pathway. In conclusion, NtLt4 increased the resistance of transgenic plants to biological and abiotic stress, which provided a theoretical basis for further understanding of the function of class I LTP, and also provided the basis for tobacco resistance and new variety of disease resistance.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2;S572

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