本文選題：吡咯啉--羧酸還原酶　切入點(diǎn)：剛毛檉柳　出處：《林業(yè)科學(xué)》2017年07期

【摘要】：【目的】脯氨酸在植物對(duì)鹽和干旱脅迫應(yīng)答中起重要的滲透調(diào)節(jié)作用,增加植物體內(nèi)脯氨酸含量可以提高植物抗逆性。吡咯啉-5-羧酸還原酶(P5CR)是合成脯氨酸的重要還原酶,本研究擬從抗旱耐鹽植物剛毛檉柳中克隆獲得1個(gè)ThP5CR基因,研究該基因的抗逆功能,為該基因用于林木基因工程育種奠定理論基礎(chǔ)�！痉椒ā恳�"Pyrroline 5 Carboxylate Reductase"作為關(guān)鍵詞,對(duì)實(shí)驗(yàn)室前期構(gòu)建的剛毛檉柳轉(zhuǎn)錄組序列進(jìn)行比對(duì)查找獲得ThP5CR基因c DNA序列,并通過RT-PCR和測(cè)序驗(yàn)證克隆獲得的ThP5CR基因序列。利用生物信息學(xué)工具進(jìn)行序列分析。利用Real time RT-PCR分析不同逆境處理下ThP5CR基因在剛毛檉柳根和葉中的表達(dá)情況。為了進(jìn)一步分析ThP5CR基因的抗逆功能,構(gòu)建了植物過表達(dá)載體pROKⅡ-ThP5CR,并進(jìn)行剛毛檉柳瞬時(shí)轉(zhuǎn)化,同時(shí)以pROKⅡ瞬時(shí)侵染剛毛檉柳作為對(duì)照。分析比較Na Cl和甘露醇脅迫后2種瞬時(shí)轉(zhuǎn)化剛毛檉柳的脯氨酸、MDA、H2O2含量和二氨基聯(lián)苯胺(DAB)、氯化硝基四氮唑藍(lán)(NBT)及伊文思藍(lán)(Evans blue)染色情況,以綜合評(píng)定ThP5CR基因的抗逆功能�！窘Y(jié)果】ThP5CR基因編碼273個(gè)氨基酸,編碼蛋白分子量為28.29 k Da,理論等電點(diǎn)為9.22。含有典型的NADP結(jié)構(gòu)域和P5CR二聚體結(jié)構(gòu)域。2種脅迫(Na Cl和PEG6000)和3種激素(ABA、GA3和JA)處理后,剛毛檉柳ThP5CR基因的表達(dá)都發(fā)生了不同程度的改變,且每種處理后至少有1個(gè)時(shí)間點(diǎn)發(fā)生了明顯改變。此外,Na Cl脅迫、PEG6000脅迫和ABA激素處理對(duì)ThP5CR基因的表達(dá)產(chǎn)生的影響更為顯著。對(duì)2種瞬時(shí)表達(dá)剛毛檉柳植株中ThP5CR基因的表達(dá)分析顯示成功獲得瞬時(shí)過表達(dá)轉(zhuǎn)基因株系(標(biāo)記為OX)。進(jìn)一步的生理指標(biāo)和生理染色結(jié)果顯示,非脅迫條件下,過表達(dá)植株脯氨酸含量高于對(duì)照植株(標(biāo)記為Con);150 mmol·L-1Na Cl和200 mmol·L-1甘露醇脅迫后,2種轉(zhuǎn)基因株系脯氨酸含量差異則更顯著;NBT、DAB染色和H2O2含量測(cè)定結(jié)果顯示,OX植株的O-·2和H2O2含量明顯低于對(duì)照;而Evans blue染色結(jié)果和MDA含量測(cè)定表明,與對(duì)照相比,過表達(dá)ThP5CR植株著色更淺、MDA含量更低�！窘Y(jié)論】ThP5CR基因能對(duì)Na Cl、PEG脅迫和ABA等激素處理做出應(yīng)答,可能參與了剛毛檉柳抗旱耐鹽脅迫應(yīng)答。在150 mmol·L-1Na Cl和200 mmol·L-1甘露醇脅迫下瞬時(shí)過表達(dá)ThP5CR植株可通過增加脯氨酸含量增強(qiáng)細(xì)胞內(nèi)清除活性氧能力,使O-·2和H2O2的積累減少,從而減少細(xì)胞受損或細(xì)胞死亡,增強(qiáng)植物的抗逆性。本研究初步證實(shí)ThP5CR基因是一個(gè)逆境脅迫應(yīng)答候選基因。
[Abstract]:[objective] Proline plays an important role in the osmotic regulation of plant responses to salt and drought stress. Increasing the content of proline in plants can improve the resistance of plants to stress, and pyrrolidin-5-carboxylate reductase (P5CRs) is an important reductase for the synthesis of proline. In this study, a ThP5CR gene was cloned from Tamarix chinensis, a salt-tolerant plant, and its stress-resistant function was studied. [methods] "Pyrroline 5 Carboxylate Reductase" was used as the key word. The c DNA sequence of ThP5CR gene was obtained by comparing the transcriptional sequences of Tamarix chinensis constructed in laboratory. The sequence of the cloned ThP5CR gene was verified by RT-PCR and sequencing. The sequence analysis was carried out by bioinformatics tools. The expression of ThP5CR gene in the roots and leaves of Tamarix chinensis under different stress conditions was analyzed by Real time RT-PCR. To further analyze the stress resistance function of ThP5CR gene, The plant overexpression vector pROK 鈪，

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