抗逆基因LcLycE、LcF3H及衰老相關(guān)基因SAG172的功能研究
發(fā)布時(shí)間：2019-03-25 21:02
【摘要】：日益增多的人口導(dǎo)致糧食需求量增多,而植物的生長(zhǎng)發(fā)育與生長(zhǎng)環(huán)境密切相關(guān),逆境脅迫及早衰嚴(yán)重影響植物正常的生長(zhǎng)發(fā)育,降低植物的存活率,嚴(yán)重制約了農(nóng)作物的產(chǎn)量。植物生長(zhǎng)過(guò)程中產(chǎn)生豐富的次生代謝產(chǎn)物,如酚類(lèi)和萜類(lèi)等化合物,是植物自身進(jìn)化的一種應(yīng)對(duì)脅迫因素的防御機(jī)制。類(lèi)胡蘿卜素和黃酮類(lèi)化合物有助于逆境脅迫條件下活性氧的消除,對(duì)植物抗逆有重要的調(diào)節(jié)作用。本研究從枸杞中分離了類(lèi)胡蘿卜素代謝途徑中的LYCE基因和黃酮類(lèi)化合物代謝途徑中上游基因F3H,并命名為L(zhǎng)cLycE和LcF3H。顏色互補(bǔ)實(shí)驗(yàn)證明LcLycE能夠催化phytoene生成δ-carotene。qPCR結(jié)果顯示LcLycE基因表達(dá)水平受冷脅迫誘導(dǎo),表明LcLycE基因可能參與調(diào)控耐寒脅迫。將LcLycE基因在擬南芥中過(guò)表達(dá),發(fā)現(xiàn)轉(zhuǎn)基因植物中Lutein含量升高,光氧化損傷減弱,植物耐冷性提高。薄層層析實(shí)驗(yàn)證實(shí)LcF3H能夠催化NAR生成DHK。qPCR結(jié)果顯示LcF3H基因表達(dá)受干旱脅迫誘導(dǎo),表明LcF3H可能參與調(diào)控耐旱脅迫。過(guò)表達(dá)LcF3H基因促進(jìn)了轉(zhuǎn)基因煙草的flavan-3-ols水平,并且促進(jìn)了轉(zhuǎn)基因植物的抗氧化系統(tǒng),有效清除干早脅迫產(chǎn)生的ROS,最終提高了植物的耐早性。葉片的衰老是葉片發(fā)育過(guò)程的最后一個(gè)階段,伴隨營(yíng)養(yǎng)物質(zhì)從源組織向庫(kù)組織的轉(zhuǎn)移,葉片的早衰制約果實(shí)的成熟和產(chǎn)量。因此,植物衰老相關(guān)機(jī)理的研究對(duì)調(diào)節(jié)植物生長(zhǎng)發(fā)育和提高產(chǎn)量具有重要的理論和實(shí)踐意義。本研究在擬南芥中發(fā)現(xiàn)了一個(gè)衰老相關(guān)LRR-RLK基因SAG172。sag172突變體表現(xiàn)葉片晚衰,葉片失水減慢;而誘導(dǎo)過(guò)表達(dá)SAG172基因的植物葉片提前衰老,葉片失水加快,ABA誘導(dǎo)的氣孔敏感性降低,說(shuō)明SAG172通過(guò)調(diào)節(jié)氣孔運(yùn)動(dòng),加速葉片失水,進(jìn)而促進(jìn)葉片衰老。qPCR與GUS報(bào)告基因檢測(cè)結(jié)果顯示SAG172的表達(dá)受衰老和ABA誘導(dǎo),并且誘導(dǎo)表達(dá)趨勢(shì)與AtNAP一致,推測(cè)AtNAP與S4G172存在調(diào)控關(guān)系。酵母單雜及體內(nèi)GUS酶活實(shí)驗(yàn)證實(shí)SAG172受AtNAP轉(zhuǎn)錄因子直接調(diào)控。當(dāng)用激素處理離體葉片時(shí)發(fā)現(xiàn),sag172突變體葉片對(duì)ABA敏感,而對(duì)JA、ETH、SA表現(xiàn)不敏感。qPCR結(jié)果顯示,sag172突變體中ABA誘導(dǎo)的標(biāo)記基因表達(dá)量有所抑制,說(shuō)明SAG172基因通過(guò)ABA信號(hào)通路調(diào)控葉片衰老。綜上,本研究對(duì)LcLycE、LcF3H和SAG172基因生理功能的分析為植物抗逆、抗衰老、提高產(chǎn)量的相關(guān)分子育種研究提供了良好的科學(xué)依據(jù)。
[Abstract]:The growing population leads to the increase of food demand, and the growth and development of plants are closely related to the growth environment. Stress and early decline seriously affect the normal growth and development of plants, reduce the survival rate of plants, and seriously restrict the yield of crops. The production of abundant secondary metabolites such as phenols and terpenoids during plant growth is a defense mechanism of plant self-evolution against stress factors. Carotenoids and flavonoids contribute to the elimination of reactive oxygen species (Ros) under stress and play an important role in regulating stress resistance of plants. In this study, the LYCE gene in carotenoid metabolism pathway and the upstream gene F3H in flavonoids metabolism pathway were isolated from Lycium barbarum L. and named as LcLycE and LcF3H.. Color complementation experiments showed that LcLycE could catalyze the production of 未-carotene.qPCR from phytoene. The results showed that the expression level of LcLycE gene was induced by cold stress, suggesting that LcLycE gene might be involved in regulation of cold stress. The over-expression of LcLycE gene in Arabidopsis showed that the content of Lutein in transgenic plants increased, the photooxidative damage weakened, and the cold tolerance of transgenic plants increased. Thin layer chromatography (TLC) showed that LcF3H could catalyze the production of DHK.qPCR from NAR. The results showed that the expression of LcF3H gene was induced by drought stress, suggesting that LcF3H might be involved in the regulation of drought tolerance. Over-expression of LcF3H gene promoted the level of flavan-3-ols in transgenic tobacco and promoted the antioxidant system of transgenic plants, which effectively eliminated the ROS, produced by dry-early stress and finally improved the early tolerance of transgenic plants. Leaf senescence is the last stage of leaf development. Along with the transfer of nutrients from the source tissue to the sink tissue, the premature senescence of the leaves restricts the ripening and yield of the fruit. Therefore, the study of plant senescence-related mechanism has important theoretical and practical significance for regulating plant growth and development and increasing yield. In this study, a SAG172.sag172 mutant of senescence-related LRR-RLK gene was found in Arabidopsis thaliana, which showed late leaf senescence and decreased leaf water loss. However, the over-expression of SAG172 gene induced leaf senescence, accelerated leaf water loss and decreased stomatal sensitivity induced by ABA, indicating that SAG172 could accelerate leaf water loss by regulating stomatal movement. The results of qPCR and GUS reporter gene analysis showed that the expression of SAG172 was induced by senescence and ABA, and the induced expression trend was consistent with that of AtNAP. It was suggested that there was a regulatory relationship between AtNAP and S4G172. Single hybrid and in vivo GUS activity assay confirmed that SAG172 was directly regulated by AtNAP transcription factors. When the leaves were treated with hormone, it was found that the leaves of sag172 mutants were sensitive to ABA, but insensitive to JA,ETH,SA. The results of qPCR showed that the expression of marker genes induced by ABA in sag172 mutants was inhibited. These results suggest that SAG172 gene regulates leaf senescence through ABA signaling pathway. In conclusion, the analysis of physiological functions of LcLycE,LcF3H and SAG172 genes provides a good scientific basis for molecular breeding of stress resistance, senescence resistance and yield improvement in plants.
【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q943.2
本文編號(hào)：2447315
[Abstract]:The growing population leads to the increase of food demand, and the growth and development of plants are closely related to the growth environment. Stress and early decline seriously affect the normal growth and development of plants, reduce the survival rate of plants, and seriously restrict the yield of crops. The production of abundant secondary metabolites such as phenols and terpenoids during plant growth is a defense mechanism of plant self-evolution against stress factors. Carotenoids and flavonoids contribute to the elimination of reactive oxygen species (Ros) under stress and play an important role in regulating stress resistance of plants. In this study, the LYCE gene in carotenoid metabolism pathway and the upstream gene F3H in flavonoids metabolism pathway were isolated from Lycium barbarum L. and named as LcLycE and LcF3H.. Color complementation experiments showed that LcLycE could catalyze the production of 未-carotene.qPCR from phytoene. The results showed that the expression level of LcLycE gene was induced by cold stress, suggesting that LcLycE gene might be involved in regulation of cold stress. The over-expression of LcLycE gene in Arabidopsis showed that the content of Lutein in transgenic plants increased, the photooxidative damage weakened, and the cold tolerance of transgenic plants increased. Thin layer chromatography (TLC) showed that LcF3H could catalyze the production of DHK.qPCR from NAR. The results showed that the expression of LcF3H gene was induced by drought stress, suggesting that LcF3H might be involved in the regulation of drought tolerance. Over-expression of LcF3H gene promoted the level of flavan-3-ols in transgenic tobacco and promoted the antioxidant system of transgenic plants, which effectively eliminated the ROS, produced by dry-early stress and finally improved the early tolerance of transgenic plants. Leaf senescence is the last stage of leaf development. Along with the transfer of nutrients from the source tissue to the sink tissue, the premature senescence of the leaves restricts the ripening and yield of the fruit. Therefore, the study of plant senescence-related mechanism has important theoretical and practical significance for regulating plant growth and development and increasing yield. In this study, a SAG172.sag172 mutant of senescence-related LRR-RLK gene was found in Arabidopsis thaliana, which showed late leaf senescence and decreased leaf water loss. However, the over-expression of SAG172 gene induced leaf senescence, accelerated leaf water loss and decreased stomatal sensitivity induced by ABA, indicating that SAG172 could accelerate leaf water loss by regulating stomatal movement. The results of qPCR and GUS reporter gene analysis showed that the expression of SAG172 was induced by senescence and ABA, and the induced expression trend was consistent with that of AtNAP. It was suggested that there was a regulatory relationship between AtNAP and S4G172. Single hybrid and in vivo GUS activity assay confirmed that SAG172 was directly regulated by AtNAP transcription factors. When the leaves were treated with hormone, it was found that the leaves of sag172 mutants were sensitive to ABA, but insensitive to JA,ETH,SA. The results of qPCR showed that the expression of marker genes induced by ABA in sag172 mutants was inhibited. These results suggest that SAG172 gene regulates leaf senescence through ABA signaling pathway. In conclusion, the analysis of physiological functions of LcLycE,LcF3H and SAG172 genes provides a good scientific basis for molecular breeding of stress resistance, senescence resistance and yield improvement in plants.
【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q943.2
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