煙草NtLTP4基因的克隆及功能分析
發(fā)布時間：2019-05-16 18:07
【摘要】：煙草產(chǎn)業(yè)是我國財政稅收收入的重要來源。但是,干旱、鹽堿、病蟲害等逆境脅迫嚴(yán)重影響了煙草的產(chǎn)量和品質(zhì),從而制約了煙草的產(chǎn)業(yè)發(fā)展。如何提高煙草的生物及非生物脅迫抗性已成為當(dāng)今研究的重要課題。為了應(yīng)對這種逆境脅迫,植物本身形成了一套精密而復(fù)雜的防御機制。其中各種脅迫相關(guān)基因的表達(dá)調(diào)控在植物應(yīng)答環(huán)境信號刺激反應(yīng)過程中起著重要的作用。非特異性脂轉(zhuǎn)移蛋白(non-specific lipid transfer protein,nsLTPs)在植物抗逆過程中就起到非常重要的作用。本研究以普通煙(Nicotiana tabacum,NC89)為材料,克隆得到NtLTP4基因,并對其進行生物學(xué)功能分析,具體研究結(jié)果如下:(1)基因序列分析表明,該基因cDNA全長為354bp,編碼117個氨基酸的多肽,預(yù)測分子量大約為9kD,其中含有保守的五肽結(jié)構(gòu)域:T/S-X-X-D-R/K和P-Y-X-I-S,且有8個位置保守的半胱氨酸。NtLTP4具有nsLTP蛋白典型的三級結(jié)構(gòu),即4個α-螺旋,4對二硫鍵,1個可結(jié)合和容納脂質(zhì)分子的疏水腔,對結(jié)合脂質(zhì)分子具有重要的作用。聚類分析表明,煙草NtLTP4與煙樹NgLTP1,擬南芥AtLTP7及AtLTP5等同源性較高,而這些基因均屬于LTP家族中的Type I類。以上結(jié)果表明,NtLTP4屬于LTP家族中的Type I類。(2)構(gòu)建35S-NtLTP4::GFP融和表達(dá)載體,瞬時侵染本生煙葉片,利用激光共聚焦顯微鏡觀察GFP熒光,發(fā)現(xiàn)35s-GFP在細(xì)胞質(zhì)和細(xì)胞核中均有熒光,但是35s-NtLTP4::GFP只在細(xì)胞壁周圍有熒光,說明NtLTP4可能定位在細(xì)胞壁,為NtLTP4的功能研究提供了理論依據(jù)。(3)采用qRT-PCR的方法,對NtLTP4基因在煙草受到不同脅迫時的表達(dá)特性進行了研究。結(jié)果表明,NtLTP4存在組織特異性,且轉(zhuǎn)錄受機械損傷、高鹽和聚乙二醇(PEG)等非生物脅迫誘導(dǎo);青枯病菌和馬鈴薯Y病毒等生物脅迫也不同程度的誘導(dǎo)該基因的表達(dá);激素信號分子MeJA、ABA和SA可強烈誘導(dǎo)NtLTP4基因的表達(dá)。以上結(jié)果表明NtLTP4可能參與不同的信號途徑,在植物生長發(fā)育及脅迫應(yīng)答過程中發(fā)揮重要作用。(4)為研究NtLTP4的功能,構(gòu)建NtLTP4與正義植物表達(dá)載體pROKII的重組載體pROKII-NtLTP4,采用農(nóng)桿菌介導(dǎo)法轉(zhuǎn)化普通煙。通過擴增部分表達(dá)載體序列鑒定陽性植株。對部分轉(zhuǎn)基因植株進行qRT-PCR分析,選取NtLTP4表達(dá)水平高、中、低的3個株系用于功能分析實驗。(5)鹽脅迫處理后,與野生型普通煙相比,轉(zhuǎn)基因植株提高了對鹽脅迫的抗性。在含不同濃度NaCl(50mM、100mM和200mM)的MS培養(yǎng)基上,轉(zhuǎn)基因煙草種子比野生型種子的萌發(fā)率要高;轉(zhuǎn)基因煙草幼苗比野生型煙草幼苗的根長較長,生長勢好。鹽脅迫條件下,轉(zhuǎn)基因植株中丙二醛含量較野生型低43%左右,說明轉(zhuǎn)基因植物細(xì)胞膜脂過氧化程度較低,植物細(xì)胞受損害程度較輕;鹽脅迫條件下,轉(zhuǎn)基因植株中ROS產(chǎn)生相關(guān)的基因的表達(dá)量降低,ROS清除相關(guān)基因的表達(dá)量增加,各種抗氧化酶的活性升高,H_2O_2和O~(2-)含量較野生型分別低29%和18%左右,表明NtLTP4基因通過提高ROS清除相關(guān)基因,提高抗氧化酶活性,從而降低了植株中的ROS含量。以上結(jié)果表明,超表達(dá)NtLTP4通過提高抗氧化能力進而增加了鹽脅迫抗性。(6)干旱脅迫處理后,超表達(dá)NtLTP4的轉(zhuǎn)基因植株提高了對干旱脅迫的抗性。在含不同濃度的甘露醇MS培養(yǎng)基上,轉(zhuǎn)基因煙草比野生型幼苗長勢更好;H_2O_2和O~(2-)含量較野生型分別低25%和21%;且與ROS相關(guān)的基因的表達(dá)量均有所改變,各種抗氧化酶的活性也有所升高。以上結(jié)果表明,超表達(dá)NtLTP4可能正調(diào)控干旱脅迫抗性。(7)超表達(dá)NtLTP4的轉(zhuǎn)基因植株提高了對青枯病菌(Ralstonia solanacearum)的抗性。與野生型植株相比,青枯菌處理后轉(zhuǎn)基因植株中與SA信號途徑相關(guān)的PR基因(PR1a、PR5)和與MeJA相關(guān)的PR基因(PR4)的表達(dá)量有所增加,推測NtLTP4可能通過SA-和JA-介導(dǎo)的信號途徑的綜合作用提高了對青枯菌的抗性。(8)超表達(dá)NtLTP4的轉(zhuǎn)基因植株提高了對病毒的抗性。馬鈴薯Y病毒普通株系(Potato virus Y Ordinary strain,PVY-O)處理后轉(zhuǎn)基因植株中PR基因(PR1a、PR5)的表達(dá)量增加,推測NtLTP4可能通過SA-介導(dǎo)的信號途徑來提高對病毒的抗性。綜上所述,NtLTP4提高了轉(zhuǎn)基因植株對生物和非生物脅迫的抗性,這些研究結(jié)果對于進一步了解I類LTP的功能提供了理論基礎(chǔ),同時也為煙草抗逆及抗病新品種提供了依據(jù)。
[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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q943.2;S572
本文編號：2478466
[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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q943.2;S572
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