本文選題：轉(zhuǎn)錄組測序 + 富含亮氨酸的類受體蛋白激酶　； 參考：《山東大學(xué)》2017年博士論文

【摘要】：南極地區(qū)由陸地和海洋組成,是地球上的極寒、極冰、極風(fēng)和極干的區(qū)域。南極獨特的地理構(gòu)成和極端的氣候使得南極沿海地區(qū)隱花植物(苔蘚和地衣)的種類和特征有別于世界其他地區(qū)的植物資源,具有獨特的抗低溫、耐高鹽、耐干旱、抗輻射等適應(yīng)能力。苔蘚是南極大陸主要的植物類群,其逆境適應(yīng)機(jī)制仍不清楚。富含亮氨酸的類受體蛋白激酶(Leucine-rich repeat receptor-like kinases,LRR-RLK)作為最初感知外界環(huán)境變化和傳導(dǎo)信號類受體蛋白激酶最大的亞家族,可能在苔蘚適應(yīng)極地嚴(yán)酷環(huán)境過程中發(fā)揮著關(guān)鍵性作用。本研究中,我們利用Illumina公司的高通量測序技術(shù)分析了南極絲瓜蘚(Pohlia nutans)在高鹽脅迫下的轉(zhuǎn)錄組,從基因組水平上篩選了LRR-RLK 亞家族基因并分析了其對鹽脅迫的響應(yīng)特征;篩選出3個對鹽脅迫正響應(yīng)的LRR-RLKs(PnLRR-RLK19、PnLRR-RLK27和PnLRR-RLK44)進(jìn)行了深入研究,探討了其在模式植物擬南芥和/或小立碗蘚中的功能,并探討了其可能的分子機(jī)制;對于揭示南極苔蘚適應(yīng)極端環(huán)境的作用機(jī)制,促進(jìn)極地獨特基因資源的開發(fā)和利用具有一定的理論意義和應(yīng)用價值。1植物PnLRR-RLK基因的轉(zhuǎn)錄組分析利用高通量測序技術(shù)分析了南極絲瓜蘚(Pohlia nuans)在正常條件和高鹽脅迫下(200 mM NaCl,1 h)的基因轉(zhuǎn)錄組特征。轉(zhuǎn)錄組測序后最終共得到72922個單一基因序列,平均長度為704 bp;單一基因序列大于500 bp的共有23747個。以轉(zhuǎn)錄組數(shù)據(jù)庫為基礎(chǔ),在南極絲瓜蘚篩選出56個候選的富含亮氨酸類受體蛋白激酶的蛋白序列,命名為PnLRR-RLK1-56,根據(jù)胞內(nèi)激酶結(jié)構(gòu)域?qū)RR-RLK分為11個亞家族(LRR Ⅰ-ⅩⅢ)。鹽脅迫下有4個PnLRR-RLKs表達(dá)水平顯著上調(diào),有10個PnLRR-RLKs的表達(dá)水平顯著下調(diào),進(jìn)一步研究了在鹽脅迫條件下3個正響應(yīng)PnLRR-RLKs(19/27/44)的生物信息學(xué)分析,發(fā)現(xiàn)3個基因在高鹽、低溫、干旱、紫外輻射和脫落酸(ABA)處理下顯著上調(diào)表達(dá)。蛋白序列比對分析表明,PnLRR-RLK19/27/44具有保守的蛋白激酶結(jié)構(gòu)域,與其他物種PnLRR-RLKs的同源性為 41%-60%;PnLRR-RLK19 與 PnLRR-RLK27 和 PnLRR-RLK44 的同源性分別為43.7%、16.9%和19.6%。系統(tǒng)進(jìn)化樹分析發(fā)現(xiàn),南極苔蘚基因PnLRR-RLK19 與 PnLRR-RLK27 屬于 LRRⅡ型的 RLK 亞家族,PnLRR-RLK44屬于LRR Ⅺ型的RLK亞家族。我們進(jìn)一步對3個受多種脅迫誘導(dǎo)的PnLRR-RLKs基因進(jìn)行深入的生物學(xué)功能研究。1.1 PnLRR-RLK27提高了植株對鹽脅迫和氧化脅迫的抗性將GFP和PnLRR-RLK27:GFP的瞬時表達(dá)載體分別轉(zhuǎn)化擬南芥和小立碗蘚原生質(zhì)體,暗培養(yǎng)一段時間后,共聚焦顯微鏡觀察熒光分布發(fā)現(xiàn),對照組GFP綠色熒光均勻分布在細(xì)胞膜、細(xì)胞質(zhì)和細(xì)胞核中;PnLRR-RLK27:GFP綠色熒光可能在細(xì)胞膜上分布。進(jìn)一步利用與H+-ATPase-RFP載體(細(xì)胞膜定位的marker基因,發(fā)紅色熒光)共轉(zhuǎn)或細(xì)胞膜特異性染料FM4-64(發(fā)紅色熒光)分析PnLRR-RLK27的亞細(xì)胞定位,結(jié)果發(fā)現(xiàn)PnLRR-RLK27:GFP綠色熒光與H+-ATPase-RFP在細(xì)胞膜上紅色熒光或FM4-64的紅色熒光完全重疊,呈黃色;表明,PnLRR-RLK27定位在細(xì)胞膜。在小立碗蘚中的功能及機(jī)制研究:通過同源重組構(gòu)建小立碗蘚過表達(dá)載體pTFH15.3:PnLRR-RLK27,通過遺傳轉(zhuǎn)化使PnLRR-RLK27在小立碗蘚中組成型表達(dá)。取單個長度為3 mm野生型和過表達(dá)PnLRR-RLK27小立碗蘚植株的莖尖放入含0 mM和125 mM NaCl的BCD中,培養(yǎng)7周。發(fā)現(xiàn)正常情況下,PnLRR-RLK27不影響小立碗蘚的生長(直徑均為13 mm左右);而鹽處理下(125 mM BCD),過表達(dá)PnLRR-RLK27顯著提高了植株對鹽脅迫的抗性,野生型植株配子體直徑為4.7mm,過表達(dá)PnLRR-RLK27植株配子體為10.0mm左右。在含10μM和15μM ABA的BCD培養(yǎng)基上,過表達(dá)PnLRR-RLK27植株配子體直徑比野生型大1.6倍和1.8倍。與野生型植株相比,鹽脅迫下轉(zhuǎn)基因小立碗蘚的鹽脅迫響應(yīng)基因(PpENA2、PpSHP1和PpSHP2)、和脅迫誘導(dǎo)基因(PpDBF、PpCOR47和PpCORTMC-AP3)和ABA信號途徑負(fù)調(diào)控基因(PpABI3a和PpABI3b)的表達(dá)量明顯提高。表明,過表達(dá)PnLRR-RLK27顯著增強了小立碗蘚在配子體生長階段對鹽脅迫抗性和降低了對ABA的敏感性。在擬南芥中的功能及機(jī)制研究:構(gòu)建了PnLRR RLK27的pROK2載體,通過農(nóng)桿菌侵染擬南芥花序,獲得了過表達(dá)PnLRR-RLK27轉(zhuǎn)基因擬南芥純系植株(以下簡稱轉(zhuǎn)基因植株)。鹽脅迫下,轉(zhuǎn)基因植株種子萌發(fā)率比野生型植株高1.5倍,轉(zhuǎn)基因植株主根是野生型植株1.3倍左右。ABA處理下,轉(zhuǎn)基因植株的萌發(fā)率是野生型植株的2倍,主根比野生型植株長10.0倍。在H202處理下,轉(zhuǎn)基因植株主根比野生型長1.4倍,側(cè)根數(shù)多1.6倍。表明,過表達(dá)PnLRR-RLK27提高了擬南芥對鹽脅迫和氧化脅迫的抗性,降低了對ABA的敏感性。與野生型擬南芥相比,鹽處理下過表達(dá)PnLRR-RLK27降低了植株H202和MDA的含量,增加了脯氨酸的含量,提高了抗氧化酶超氧化物岐化酶(superoxide dismutase,SOD)、過氧化物酶(peroxidase,POD)和過氧化氫酶(catalase,CAT)的活性,上調(diào)了抗氧化酶基因At4PX1、AtAPX2、AtCAT2和AtZAT10的表達(dá)水平。鹽處理下,鹽脅迫響應(yīng)基因(AtHKT和AtSOS3)和ABA信號途徑基因(AtABF3、AtMYB2、AtRD22、AtDREB2A、AtRD29A、AtRD29B、AtKIN1 和 AtCOR47)的表達(dá)量在轉(zhuǎn)基因植株中比野生型明顯提高。以上結(jié)果表明,PnLRR-RLK27基因的功能保守,在兩種模式植物中功能類似,均能顯著提高植物對鹽脅迫抗性,其作用機(jī)制可能與提高植物對ROS的清除能力以及脅迫相關(guān)激素ABA信號途徑相關(guān)。1.2 PnLRR-RLK19提高了植株對鹽脅迫和干旱脅迫的抗性亞細(xì)胞定位分析發(fā)現(xiàn),PnLRR-RLK19在細(xì)胞膜分布。PnLRR-RLK19在擬南芥中過表達(dá),對植物的正常生長沒有影響。鹽處理后,轉(zhuǎn)基因植株種子萌發(fā)率比野生型植株高25.0%,轉(zhuǎn)基因植株的主根比野生型植株長2.0倍。在0.5 μMABA處理下,轉(zhuǎn)基因植物種子萌發(fā)率比野生型植株高11.5%以上;在7.5 μM ABA處理后,轉(zhuǎn)基因植株的主根比野生型植株長1.5倍。H202和甘露醇處理后,過表達(dá)植株側(cè)根數(shù)比野生型植株分別多1.5倍和2.0-4.0倍。盆栽實驗顯示,鹽處理后,轉(zhuǎn)基因植株葉片黃化比野生型擬南芥少;干旱處理后,轉(zhuǎn)基因植株的失水率較低。鹽脅迫處理后,轉(zhuǎn)基因植株ABA信號途徑基因(AtAREB1、AtAREB2、AtABF3、AtMYB2、AtDREB2A、AtRD22和AtRD29A)和鹽脅迫響應(yīng)基因(AtP5CS1和AtSOS3)均顯著上調(diào)表達(dá)。干旱處理后,轉(zhuǎn)基因植株ABA信號途徑基因(AtABF3、AtMYB2、AtDREB2A、AtRD22、AtRD29A、AtRD29和AtKIN1)顯示明顯上調(diào)的表達(dá)模式。以上結(jié)果表明,PnLRR-RLK19不僅能夠提高了植物對鹽脅迫的抗性,也增強了植物對干旱脅迫及氧化脅迫的抗性;其機(jī)制與鹽脅迫信號途徑和ABA信號途徑正相關(guān)。1.3 PnLRR-RLK44提高了植株對鹽脅迫和脫落酸的抗性PnLRR-RLK44定位于細(xì)胞膜。構(gòu)建了PnLRR-RLK4 的過表達(dá)載體,利用農(nóng)桿菌侵染擬南芥,篩選和繁種,最終獲得過量表達(dá)的轉(zhuǎn)基因純合植株。在100 mMNaCl培養(yǎng)基上,過表達(dá)植株的萌發(fā)率比野生型植株高17.6%,轉(zhuǎn)基因植株主根比野生型長28.1%。在0.5 μMABA處理下,轉(zhuǎn)基因植株的種子萌發(fā)率比野生型植株高20.0%,主根比野生型長28.9%。進(jìn)而,利用Real-time qPCR技術(shù)檢測了鹽脅迫信號途徑和ABA途徑中相關(guān)的脅迫響應(yīng)基因的表達(dá)情況,發(fā)現(xiàn)過表達(dá)植株鹽脅迫離子通道蛋白基因(AtHKT1和AtSOS3)、鹽脅迫響應(yīng)基因(AtP5CS1和AtADH1)和和ABA不敏感基因ABI1都呈顯著的上調(diào)表達(dá)。同時,ABA合成途徑關(guān)鍵基因(AtNCED3、AtABA1 和AtAAO3)和ABA早期響應(yīng)基因(AtMYB2、AtRD22、AtRD29A和AtDREB2A)均下調(diào)表達(dá)。這些結(jié)果表明,PnLRR-RLK44通過參與鹽脅迫信號途徑和與ABA信號途徑的負(fù)調(diào)控因子作用來提高植物的耐鹽能力。綜上所述,PnLRR-RLK27提高了植物對鹽脅迫抗性和降低了對ABA的敏感性,其作用機(jī)制可能與提高植物對ROS的清除能力以及鹽脅迫/ABA信號途徑相關(guān)。PnLRR-RLK19不僅提高了植物對鹽脅迫抗性和ABA不敏感性,也增強了植物對干旱脅迫及氧化脅迫的抗性,鹽脅迫、滲透脅迫和ABA信號途徑相關(guān)基因表達(dá)上調(diào)。PnLRR-RLK44通過參與鹽脅迫信號途徑和與ABA信號途徑的負(fù)調(diào)控因子作用來提高植物的耐鹽能力,ABA合成和響應(yīng)途徑下調(diào),ABA負(fù)調(diào)控途徑上調(diào)。PnLRR-RLK09/27/44同屬一個基因亞家族,其基因功能有類似之處,但也有一定的分化,在提高植物的抗逆機(jī)制中有自己獨特之處。2 一個泛素連接酶在南極苔蘚對非生物脅迫應(yīng)答中的作用E3泛素連接酶(E3 ubiquitin ligase,E3)可以特異地識別靶蛋白,在植物生長發(fā)育和對非生物脅迫響應(yīng)的過程中發(fā)揮著重要的作用。但是,泛素連接酶在低等植物中作用的研究目前還沒有報道。本文從南極苔蘚中克隆了一個泛素連接酶基因,命名為PnE3。PnE3基因的cDNA含有1個2097 bp開放性閱讀框,編碼698個氨基酸,分子量為76.2 kDa,等電點為6.79。氨基酸序列分析發(fā)現(xiàn),PnE3包含1個Ubox結(jié)構(gòu)域(氨基酸288-351)、4個ARM重復(fù)結(jié)構(gòu)域(氨基酸418-625)。低溫、高鹽、干旱和ABA處理均能誘導(dǎo)該基因表達(dá)上調(diào),表明PnE3基因參與南極苔蘚對非生物脅迫響應(yīng)。將PnE3:GFP的瞬時表達(dá)載體轉(zhuǎn)化擬南芥,發(fā)現(xiàn),PnE3:GFP綠色熒光主要在細(xì)胞質(zhì)中分布,表明,PnE3定位在細(xì)胞質(zhì)。在小立碗蘚中的功能及機(jī)制研究:125 mM NaCl處理下,野生型植株配子體直徑比轉(zhuǎn)基因植株大1.4倍;5 μM ABA處理下,野生型植株配子體直徑比轉(zhuǎn)基因植株大1.6倍。ABA信號途徑基因PpAB13A、PpABI3B和PpABI3C在轉(zhuǎn)基因小立碗蘚中均下調(diào)表達(dá)。在擬南芥中的功能及機(jī)制研究:鹽處理下,野生型植株的子葉張開率比過表達(dá)系高40%,野生型植株的主根根長比過表達(dá)系長2.4倍。ABA處理下,野生型植株的子葉張開率比過表達(dá)系高25.6%,野生型植株的主根比過表達(dá)系長3.7倍。鹽脅迫下,脅迫響應(yīng)基因AtABI3、AtABFA、AtDREB2A和AtRD29A在轉(zhuǎn)基因擬南芥的表達(dá)水平顯著下調(diào)。綜上所述,PnE3基因功能保守,在兩種模式植物中均能提高植物對鹽和ABA的敏感性,其作用機(jī)制主要與泛素化修飾植物ABA途徑響應(yīng)基因相關(guān)。
[Abstract]:The Antarctic region is composed of land and sea. It is the extreme cold, polar ice, extreme wind and extreme dry areas on the earth. The unique geographical composition and extreme climate of the Antarctic make the species and characteristics of the Antarctic Coastal Areas (moss and lichen) different from the plant resources in other parts of the world, and have unique resistance to low temperature, salt tolerance, drought resistance and resistance. Bryophyte is a major plant group in the Antarctic continent, and its adaptation mechanism is still unclear. Leucine-rich repeat receptor-like kinases (LRR-RLK), which is rich in leucine, is the first subfamily to perceive the changes in the external environment and the largest protein kinase of conduction signals, and may be suitable for mosses. In this study, we should play a key role in the extreme environmental process. In this study, we used Illumina company's high throughput sequencing technology to analyze the transcriptional group of Pohlia nutans under high salt stress. We screened the LRR-RLK subfamily gene from the genome level and analyzed its response to salt stress; 3 of them were screened out. LRR-RLKs (PnLRR-RLK19, PnLRR-RLK27 and PnLRR-RLK44) of positive response to salt stress were studied in depth, and their functions in the model plant Arabidopsis and / or small erect moss were discussed, and their possible molecular mechanisms were discussed. The mechanism of revealing the adaptation of Antarctic moss to the extreme environment and the development of the unique genetic resources of polar regions were also discussed. The transcriptome analysis of the PnLRR-RLK gene of.1 plants with a certain theoretical and applied value was used to analyze the gene transcriptome characteristics of the Antarctic silk moss moss (Pohlia nuans) under normal conditions and high salt stress (200 mM NaCl, 1 h). After the transcriptional group was sequenced, a total of 72922 single gene sequences were obtained and the average length was long. A total of 704 BP; a total of 23747 single gene sequences greater than 500 BP. Based on the transcriptional database, 56 candidate protein kinases rich in leucine receptor protein kinase were screened in the Antarctic gourd moss, named PnLRR-RLK1-56, and LRR-RLK was divided into 11 subfamilies (LRR I - III) based on the intracellular kinase domain. 4 under Salt Stress The expression level of PnLRR-RLKs was significantly up-regulated and the expression level of 10 PnLRR-RLKs decreased significantly. Further study of bioinformatics analysis of 3 positive responses to PnLRR-RLKs (19/27/44) under salt stress showed that 3 genes were significantly up-regulated in high salt, low temperature, drought, ultraviolet radiation and decuquic acid (ABA) treatment. The analysis shows that PnLRR-RLK19/27/44 has a conservative protein kinase domain, and the homology of PnLRR-RLKs with other species is 41%-60%. The homology of PnLRR-RLK19 with PnLRR-RLK27 and PnLRR-RLK44 is 43.7%. 16.9% and 19.6%. phylogenetic tree analysis found that the Antarctic moss gene PnLRR-RLK19 and PnLRR-RLK27 belong to RLK subtype of LRR II. Family, PnLRR-RLK44 belongs to the LRR RLK subfamily. We further study the biological function of 3 PnLRR-RLKs genes induced by multiple stresses..1.1 PnLRR-RLK27 enhanced the plant resistance to salt stress and oxidative stress. The transient expression vectors of GFP and PnLRR-RLK27:GFP were transformed into Arabidopsis and small erect bowl moss, respectively. After a period of dark culture, the fluorescence distribution of the GFP was observed by confocal microscopy. The green fluorescence of the control group was distributed evenly in the cell membrane, cytoplasm and nucleus, and the green fluorescence of PnLRR-RLK27:GFP might be distributed on the cell membrane. Further use of the H+-ATPase-RFP vector (the marker gene located in the cell membrane, the red fluorescence) was further used. Or cell membrane specific dyestuff FM4-64 (red fluorescence) analysis of the subcellular localization of PnLRR-RLK27, the results showed that the PnLRR-RLK27:GFP green fluorescence overlapped the red fluorescence of the cell membrane or the red fluorescence of FM4-64 in the cell membrane. It showed that PnLRR-RLK27 was located in the cell membrane. The function and mechanism in the small erect moss A small bowl of moss overexpression vector pTFH15.3:PnLRR-RLK27 was constructed by homologous recombination, and PnLRR-RLK27 was formed in the small bowl of mosses by genetic transformation. A single length of 3 mm wild type and over expressed PnLRR-RLK27 small erect moss plants were placed in BCD containing 0 mM and 125 mM NaCl, and cultured for 7 weeks. There was no influence on the growth of small erect moss (about 13 mm in diameter), while under salt treatment (125 mM BCD), overexpression of PnLRR-RLK27 significantly increased the resistance to salt stress. The gametophyte diameter of the wild type plant was 4.7MM, and the overexpressed PnLRR-RLK27 plant gametophyte was about 10.0mm. On the BCD medium containing 10 mu M and 15 u M ABA, the overexpressed PnLRR-RLK27 The plant gametophyte diameter was 1.6 times larger and 1.8 times larger than that of the wild type. Compared with the wild type, the salt stress response genes (PpENA2, PpSHP1 and PpSHP2), and the negative regulatory genes (PpABI3a and PpABI3b) of the stress induced genes (PpDBF, PpCOR47 and PpCORTMC-AP3) and ABA signal pathways were significantly increased under salt stress. The expression of PnLRR-RLK27 significantly enhanced the resistance to salt stress and the sensitivity to ABA in the gametophyte growth stage. The function and mechanism of the Arabidopsis in Arabidopsis thaliana: the pROK2 vector of PnLRR RLK27 was constructed and the Arabidopsis flower was infected by Agrobacterium tumefaciens, and the pure lines of the over expressed PnLRR-RLK27 transgenic Arabidopsis were obtained. Under salt stress, the seed germination rate of the transgenic plant was 1.5 times higher than that of the wild type plant. The main root of the transgenic plant was about 1.3 times.ABA of the wild type plant. The germination rate of the transgenic plants was 2 times that of the wild type, and the main root was 10 times longer than that of the wild type. Under the H202 treatment, the main root of the transgenic plant was 1. longer than the wild type. 4 times and 1.6 times the lateral root number, indicating that overexpression of PnLRR-RLK27 increased the resistance to salt stress and oxidative stress in Arabidopsis and reduced sensitivity to ABA. Compared with wild Arabidopsis, overexpression of PnLRR-RLK27 under salt treatment reduced the content of H202 and MDA, increased the content of prolysine and increased the antioxidant enzyme superoxide dismutase. (superoxide dismutase, SOD), the activity of peroxidase (peroxidase, POD) and catalase (catalase, CAT), up regulation of the expression level of the antioxidant enzyme gene At4PX1, AtAPX2, AtCAT2 and AtZAT10. The expression of 1 and AtCOR47 was significantly higher in the transgenic plants than in the wild type. The above results showed that the function of the PnLRR-RLK27 gene was conservative, and the function was similar in the two pattern plants, which could significantly improve the plant resistance to salt stress. The mechanism of its action may be to improve the scavenging ability of plant to ROS and the signal of stress related hormone ABA signal. .1.2 PnLRR-RLK19 enhanced the subcellular location analysis of plant resistance to salt stress and drought stress. It was found that the distribution of PnLRR-RLK19 in the cell membrane was overexpressed in Arabidopsis thaliana, and did not affect the normal growth of plants. After salt treatment, the germination rate of transgenic plants was 25% higher than that of wild type plants, and the main transgenic plants were dominant. The root of the plant was 2 times longer than that of the wild type. Under the treatment of 0.5 MABA, the seed germination rate of transgenic plants was more than 11.5% higher than that of the wild type plants. After 7.5 u M ABA treatment, the main roots of the transgenic plants were 1.5 times.H202 and mannitol treatment, and the number of overexpressed plants was 1.5 times and 2.0-4.0 times more than that of the wild type plants. After salt treatment, the leaf yellow of transgenic plants was less than that of wild type Arabidopsis. After drought treatment, the water loss rate of transgenic plants was low. After salt stress treatment, the ABA signal pathway genes (AtAREB1, AtAREB2, AtABF3, AtMYB2, AtDREB2A, AtRD22 and AtRD29A) and salt stress response genes (AtP5CS1 and AtSOS3) were significantly up-regulated after salt stress treatment. After drought treatment, the ABA signaling pathway genes of transgenic plants (AtABF3, AtMYB2, AtDREB2A, AtRD22, AtRD29A, AtRD29 and AtKIN1) showed obvious up-regulated expression patterns. The results showed that PnLRR-RLK19 not only enhanced the resistance of plants to salt stress, but also enhanced plant resistance to drought stress and oxidative stress; its mechanism and salt were also enhanced. The stress signal pathway and the positive correlation of the ABA signal pathway.1.3 PnLRR-RLK44 enhanced the plant's resistance to salt stress and abscisic acid in the cell membrane. The overexpression vector of PnLRR-RLK4 was constructed, the Arabidopsis thaliana was infected with Agrobacterium tumefaciens, and the transgenic homozygous plants were overexpressed. On the 100 mMNaCl medium. The germination rate of overexpressed plants was 17.6% higher than that of wild type plants. The seed germination rate of transgenic plants was 20% higher than that of wild type, and the main root of transgenic plants was 20% higher than that of wild type, and the main root of the transgenic plant was 20% higher than that of wild type, and the main root of the transgenic plant was 20% higher than that of the wild type. The signal pathway of salt stress and the related threat in the ABA pathway were detected by Real-time qPCR technology. In response to the expression of response genes, Salt Stressed ion channel protein genes (AtHKT1 and AtSOS3) were overexpressed. Salt stress response genes (AtP5CS1 and AtADH1) and ABA Insensitive Gene ABI1 were all significantly up-regulated. At the same time, the key genes of ABA synthesis pathway (AtNCED3, AtABA1 and AtAAO3) and ABA early response genes were found. Both RD29A and AtDREB2A were downregulated. These results suggest that PnLRR-RLK44 improves salt tolerance by participating in salt stress signaling pathways and with negative regulatory factors of ABA signaling pathways. In summary, PnLRR-RLK27 increases plant resistance to salt stress and reduces sensitivity to ABA. The mechanism may be associated with increasing plant resistance. The scavenging ability of ROS and the salt stress of /ABA signal pathway related.PnLRR-RLK19 not only improve plant resistance to salt stress and ABA insensitivity, but also enhance plant resistance to drought stress and oxidative stress, salt stress, osmotic stress, and ABA signal pathway related gene expression up regulation of.PnLRR-RLK44 through participation in salt stress signal pathway and A The negative regulatory factor of the BA signal pathway improves the salt tolerance of plants, the ABA synthesis and response pathway is down, and the negative regulation of ABA is a subfamily of.PnLRR-RLK09/27/44. The function of the gene is similar, but it also has some differentiation, which has its own uniqueness in improving the resistance mechanism of plants,.2 a ubiquitin. The role of enzyme in the response of Antarctic moss to abiotic stresses E3 ubiquitin ligase (E3 ubiquitin ligase, E3) can identify target proteins in a special way and play an important role in plant growth and response to abiotic stress. However, the study of the role of ubiquitin ligase in lower plants has not yet been reported. A ubiquitin ligase gene was cloned from the moss of Antarctica. The cDNA, named PnE3.PnE3, contains 1 2097 BP open reading frames, encoding 698 amino acids, and the molecular weight is 76.2 kDa. The isoelectric point is found to be 6.79. amino acid sequence analysis. PnE3 contains 1 Ubox domains (288-351 of aminoacid) and 4 ARM repeat domains (amino acid 418-625). Low temperature, high salt, drought and ABA can all induce the up-regulated gene expression, indicating that PnE3 gene participates in the response of Antarctic moss to abiotic stress. The transient expression vector of PnE3:GFP is converted to Arabidopsis thaliana. It is found that the green fluorescence of PnE3:GFP is mainly distributed in the cytoplasm, indicating that PnE3 is located in the cytoplasm. The function and mechanism in the small erect moss Under the treatment of 125 mM NaCl, the gametophyte diameter of wild type plants was 1.4 times larger than that of transgenic plants. Under 5 u M ABA treatment, the gametophyte diameter of wild type plants was 1.6 times larger than that of transgenic plants,.ABA signal pathway gene PpAB13A, PpABI3B and PpABI3C were down down in the transgenic small erect moss. The cotyledon opening rate of wild type plants was 40% higher than that of overexpression line, and the root length of wild type plant was 2.4 times longer than that of overexpression line. The cotyledon opening rate of wild type plants was 25.6% higher than that of overexpression line, and the main root of wild type plants was 3.7 times longer than that of overexpression line. Under salt stress, the stress response gene AtABI3, AtABFA, AtDREB2A and AtRD29A were under salt stress. The expression level of transgenic Arabidopsis was significantly downregulated. In conclusion, PnE3 gene is conservative in function, and can enhance plant pairs in two kinds of plants.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：Q943.2

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