【摘要】：干旱和低溫脅迫是導(dǎo)致植物生長發(fā)育受阻和作物減產(chǎn)的重要環(huán)境因素。研究植物對干旱和低溫的響應(yīng)具有重要的理論意義和實踐價值。我們對小白菜的三個品系蘇州青(BcL.1)、黃心烏菜(BcL.2)和黑心烏菜(BcL.3)對干旱和低溫脅迫的耐受性進行了研究,主要研究結(jié)果如下：1.黑心烏菜比蘇州青和黃心烏菜更加耐寒,表現(xiàn)出較好的低溫耐受性。低溫脅迫后黑心烏菜的存活率明顯高于蘇州青和黃心烏菜(2-3倍),黑心烏菜的相對電導(dǎo)率明顯低于蘇州青和黃心烏菜,低溫脅迫下黑心烏菜相對于前兩者受到的傷害水平較低,植株組織的凍害較小。2.黑心烏菜相對于蘇州青和黃心烏菜對干旱脅迫更加耐受,黑心的存活率明顯高于蘇州青和黃心烏菜(2-3倍),單株離體實驗表明,黑心烏菜葉片失水率明顯小于蘇州青和黃心烏菜。3.為了研究黑心烏菜對低溫、干旱脅迫耐受的機制,對三種植株體內(nèi)可溶性糖含量進行檢測,其脅迫處理后可溶性糖含量均有上升但三者之間無顯著差異,表明黑心烏菜株系對低溫、干早的耐受性可能與可溶性糖積累關(guān)系不大。4.干旱處理后,植株體內(nèi)脯氨酸含量均有上升,黑心烏菜中脯氨酸含量顯著高于蘇州青和黃心烏菜。但在低溫脅迫下三種植株的脯氨酸含量無明差異。表明黑心烏菜對干旱脅迫的耐受性可能與脯氨酸積累有關(guān)。5.為了進一步驗證黑心烏菜抗旱和抗寒的機制,我們檢測了脅迫下脯氨酸代謝途徑上相關(guān)基因的表達,干旱處理后,黑心烏菜中脯氨酸合成關(guān)鍵基因P5CS表達水平明顯上升,脯氨酸降解途徑中PDH基因的表達與蘇州青和黃心烏菜無顯著差異,說明黑心烏菜中P5CS基因高度表達增加脯氨酸含量導(dǎo)致其對干旱脅迫的耐受。進一步檢測P5CS酶活,也支持上述結(jié)論。而低溫處理后,三種植株中P5CS和PDH在轉(zhuǎn)錄水平和P5CS酶活均無顯著差異。6.為了進一步研究黑心烏菜相對于蘇州青和黃心烏菜對低溫脅迫更加耐受的原因,我們檢測了在低溫脅迫下CBF/DREB1依賴的信號途徑中相關(guān)基因的表達水平。結(jié)果表明在低溫脅迫下,黑心烏菜中CBF/DREB1途徑中關(guān)鍵基因CBF1在其上游調(diào)控因子LOS4和CBF自身調(diào)控因子CBF2調(diào)控下高度表達,激活其下游COR15A基因表達,使黑心烏菜具有比蘇州青及黃心烏菜更強的抗寒性。7.以黑心烏菜的cDNA為模板擴增BrCBF1基因全長,將該基因連接到pART27載體上。獲得的重組載體轉(zhuǎn)化至農(nóng)桿菌菌株GV3101,通過浸花轉(zhuǎn)化法將BrCBF1重組載體轉(zhuǎn)化到擬南芥野生型植株中,利用轉(zhuǎn)基因篩選與遺傳鑒定。
[Abstract]:Drought and low temperature stress are the important environmental factors leading to plant growth and development hindrance and crop yield reduction. It is of great theoretical and practical value to study the response of plants to drought and low temperature. The tolerance of three lines of pakchoi, BcL.1, BcL.2 and BcL.3 to drought and low temperature stress were studied. The main results were as follows: 1. Acanthopsis nigra showed better cold tolerance and lower temperature tolerance than those from Suzhou and Huangxin. After low temperature stress, the survival rate of Acanthopsis nigra was significantly higher than that of Soochow and Huangxin (2-3 times), and the relative electrical conductivity was significantly lower than that of Soochow and Huangxin. Under low temperature stress, the injury level of Acanthopsis nigra was lower than that of the former two, and the freezing injury of plant tissue was less than that of the former. 2. 2. Acanthopsis nigra was more tolerant to drought stress than that of Soochow and Huangxin, and the survival rate of black heart was significantly higher than that of Soochow and Huangxin (2-3 times). The experiment in vitro showed that the survival rate of black heart was 2 ~ 3 times higher than that of Soochow and Huangxin. The rate of water loss in leaves of Acanthopsis nigra was significantly lower than that of Qingqing and Huangxin in Suzhou. In order to study the mechanism of tolerance to low temperature and drought stress, the content of soluble sugar in three plants was detected. The content of soluble sugar increased after stress treatment, but there was no significant difference among them. The results showed that the tolerance to low temperature and early drying of the black heart black cabbage strain may not be related to the accumulation of soluble sugar. 4. After drought treatment, the proline content in the plants increased, and the proline content in Acanthopsis nigra was significantly higher than that in Soochow and Huangxin. However, there was no obvious difference in proline content among the three plants under low temperature stress. The results suggested that the tolerance of Acanthopsis nigra to drought stress might be related to the accumulation of proline. 5. In order to further verify the mechanism of drought resistance and cold resistance, we detected the expression of proline metabolic pathway related genes under stress. After drought treatment, the P5CS expression level of proline biosynthesis key gene was significantly increased. There was no significant difference between the expression of PDH gene in proline degradation pathway and that in Soochow Qingxinwu and Huangxinqingcai, which indicated that the high expression of P5CS gene in Acanthopsis nigra led to its tolerance to drought stress due to the increase of proline content. Further detection of P5CS activity also supports the above conclusion. However, there was no significant difference in transcription level and P5CS enzyme activity between P5CS and PDH in the three plants after low temperature treatment. In order to further study the reason of the higher tolerance to low temperature stress, we detected the expression level of related genes in CBF/DREB1 dependent signaling pathway under low temperature stress. The results showed that CBF1, the key gene of CBF/DREB1 pathway, was highly expressed under the control of upstream regulatory factor LOS4 and CBF self-regulatory factor CBF2 under low temperature stress, and the downstream COR15A gene expression was activated. To make the black heart black cabbage has stronger cold resistance than Suzhou green and yellow black. 7. The full length of the BrCBF1 gene was amplified by using the cDNA of Acanthopsis nigra as a template, and the gene was ligated to the pART27 vector. The recombinant vector was transformed into Agrobacterium tumefaciens strain GV3101, to transform BrCBF1 recombinant vector into Arabidopsis wild-type plants by means of flower soaking. Transgenic screening and genetic identification were used.
【學(xué)位授予單位】：合肥工業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S634.3

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