【摘要】：植物在生長(zhǎng)發(fā)育過程中經(jīng)常受到高溫、干旱、鹽等多種非生物脅迫的影響。這些逆境可以造成植物體內(nèi)生理代謝障礙,嚴(yán)重影響作物的產(chǎn)量和品質(zhì)。為了應(yīng)對(duì)脅迫危害,植物在長(zhǎng)期進(jìn)化中形成了一套復(fù)雜而精密的防御機(jī)制,包括多重信號(hào)通路、各種轉(zhuǎn)錄因子及功能基因等。近年來(lái)研究發(fā)現(xiàn),脂質(zhì)轉(zhuǎn)運(yùn)蛋白(Lipid Transfer Proteins,LTPs)在植物的生長(zhǎng)發(fā)育調(diào)節(jié)及非生物脅迫響應(yīng)中起著重要作用,但大多數(shù)LTPs的具體作用機(jī)理尚不清楚。本研究根據(jù)玉米熱帶種質(zhì)POB21自選系響應(yīng)高溫和干旱的轉(zhuǎn)錄組學(xué)差異,從中篩選到一個(gè)脅迫響應(yīng)上調(diào)表達(dá)的功能未知LTPs-like基因。根據(jù)玉米基因組數(shù)據(jù)庫(kù)Maize GDB中B73基因組中參考基因DRMZM2G05208的序列,通過RT-PCR方法獲得同源克隆,命名為Zm LTPL63。對(duì)該基因的結(jié)構(gòu)、進(jìn)化關(guān)系、表達(dá)模式及生物學(xué)功能進(jìn)行系統(tǒng)分析,主要研究結(jié)果如下:(1)基因序列分析表明,該基因CDS全長(zhǎng)759bp,編碼長(zhǎng)252個(gè)氨基酸的肽鏈。預(yù)測(cè)Zm LTPL63蛋白的分子量約25.9k D,在N末端存在長(zhǎng)度為23個(gè)氨基酸的信號(hào)肽。蛋白具有典型的LTPs結(jié)構(gòu)特征,肽鏈中8個(gè)位置特異的半胱氨酸殘基(8CM)可以配對(duì)產(chǎn)生4個(gè)二硫鍵,4個(gè)α螺旋折疊形成一個(gè)立體的疏水腔,可用來(lái)結(jié)合脂質(zhì)分子。但與典型LTPs相比,Zm LTPL63編碼的氨基酸序列更長(zhǎng),并且在N端信號(hào)肽和8CM基序之間富含脯氨酸。蛋白種間同源比對(duì)及進(jìn)化分析表明,Zm LTPL63在多種植物中存在同源蛋白,與粟XP_012699184.1、高粱XP_002439994.1、漸尖二型花屬OEL21002.1、粗山羊草EMT12664.1、二穗短柄草KQK15914.1及水稻BAC65920.1等蛋白存在較高同源性,而與擬南芥AT3G43720同源性較低。(2)通過q RT-PCR方法分析玉米Zm LTPL63基因的表達(dá)模式,結(jié)果表明其可以響應(yīng)多種脅迫、植物激素和信號(hào)分子。Zm LTPL63基因的表達(dá)具有一定的組織特異性,在幼苗中的表達(dá)水平表現(xiàn)為根莖葉。Zm LTPL63可以受高溫、PEG模擬干旱、鹽(Na Cl)誘導(dǎo)表達(dá)。脫落酸(ABA)、乙烯(ET)、茉莉酸甲酯(Me JA)和H_2O_2能顯著上調(diào)Zm LTPL63基因的表達(dá)水平,赤霉素(GA)處理后其表達(dá)水平出現(xiàn)下調(diào)。(3)為確定Zm LTPL63蛋白的亞細(xì)胞定位,構(gòu)建了p ROKII-35S::Zm LTPL63-GFP融合表達(dá)載體并通過農(nóng)桿菌瞬時(shí)侵染本生煙草葉片。利用激光共聚焦顯微鏡觀察GFP熒光,發(fā)現(xiàn)Zm LTPL63-GFP的熒光只出現(xiàn)在細(xì)胞外周。(4)根據(jù)B73參考基因組序列克隆Zm LTPL63基因編碼區(qū)上游長(zhǎng)1500bp的DNA序列。序列分析軟件預(yù)測(cè)表明啟動(dòng)子存在真菌、高溫、干旱、低溫等多種(非)生物脅迫響應(yīng)元件和Me JA等激素響應(yīng)元件。p BI121-PZm LTPL63::GUS轉(zhuǎn)基因擬南芥幼苗的X-Gluc染色表明,基因在植株各器官中廣泛表達(dá),尤其在莖尖和和根尖表達(dá)較高。ABA和H_2O_2處理能夠增強(qiáng)GUS表達(dá),說明啟動(dòng)子表達(dá)調(diào)控特性與基因表達(dá)特性一致。(5)組成型超表達(dá)Zm LTPL63基因能增強(qiáng)轉(zhuǎn)基因植株對(duì)干旱和鹽的非生物脅迫抗性。在脅迫條件下,超表達(dá)Zm LTPL63轉(zhuǎn)基因擬南芥比野生型幼苗根生長(zhǎng)更快,成苗的存活率更高;轉(zhuǎn)基因擬南芥離體葉盤在滲透脅迫下的褪綠黃化明顯慢于野生型;同時(shí),轉(zhuǎn)基因擬南芥離體葉片失水速率也低于野生型。與野生型相比,轉(zhuǎn)基因植株在干旱和高鹽脅迫下ROS的積累量較低,抗氧化酶的活性更高,丙二醛含量較少,對(duì)H_2O_2的敏感性降低。以上結(jié)果表明超表達(dá)Zm LTPL63可以通過提高植株抗氧化能力來(lái)增加對(duì)干旱和鹽脅迫的耐受性。(6)在本生煙葉片中瞬時(shí)表達(dá)Zm LTPL63,氣相色譜檢測(cè)表明葉片中不飽和脂肪酸如亞油酸、亞麻酸較對(duì)照明顯上升。說明Zm LTPL63參與脂肪酸代謝的調(diào)節(jié)。(7)表達(dá)Zm LTPL63影響植株的生長(zhǎng)發(fā)育進(jìn)程。轉(zhuǎn)基因擬南芥在生長(zhǎng)發(fā)育后期表現(xiàn)為抽苔和開花延遲。q RT-PCR結(jié)果發(fā)現(xiàn),參與開花正向調(diào)控的關(guān)鍵基因的表達(dá)量明顯降低,推測(cè)Zm LTPL63可能通過抑制關(guān)鍵成花基因的表達(dá)導(dǎo)致開花延遲。
[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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2;S513

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 李倩;王景一;毛新國(guó);李昂;高麗鋒;劉惠民;景蕊蓮;;小麥脂質(zhì)轉(zhuǎn)運(yùn)蛋白基因TaLTP克隆及功能分析[J];作物學(xué)報(bào);2015年05期

2 張藝能;周玉萍;陳瓊?cè)A;黃小玲;田長(zhǎng)恩;;擬南芥開花時(shí)間調(diào)控的分子基礎(chǔ)[J];植物學(xué)報(bào);2014年04期

3 劉梅;生華;化文平;儲(chǔ)君;王U喼，

本文編號(hào)：2201570