本文選題：不結(jié)球白菜 + 內(nèi)質(zhì)網(wǎng)應(yīng)激��； 參考：《南京農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：不結(jié)球白菜(Brassica rapa ssp.chinensis),又稱青菜,原產(chǎn)于中國(guó)。由于其口感好,營(yíng)養(yǎng)豐富,深受廣大人民喜愛(ài),特別是在南方,更是人們的家中常備蔬菜之一。隨著生活質(zhì)量的提升,人們對(duì)不結(jié)球白菜的需求也日益多樣化,如何在保證口感的同時(shí)提高產(chǎn)量以及提高其有益次級(jí)代謝產(chǎn)物,如花青素、維生素等,已經(jīng)成為人們重點(diǎn)關(guān)注的問(wèn)題。花青素作為植物的二級(jí)次生代謝產(chǎn)物,在植物生理及食物營(yíng)養(yǎng)方面的重要性日益凸顯。近年來(lái)關(guān)于它的報(bào)道越來(lái)越多,因此提高其含量及穩(wěn)定性等方面的研究日益重要。而在紫色不結(jié)球白菜中花青素含量極高,此外不結(jié)球白菜的產(chǎn)量極高,因此提高不結(jié)球白菜中花青素的含量具有很高的經(jīng)濟(jì)價(jià)值。本研究以南京農(nóng)業(yè)大學(xué)白菜系統(tǒng)生物學(xué)實(shí)驗(yàn)室提供的兩種不同品系的不結(jié)球白菜為試驗(yàn)材料,即NJZX1-3和NJZX1-0。其中NJZX1-3在成長(zhǎng)過(guò)程葉的正面是紫色,背面為綠色,受到脅迫后背面也會(huì)產(chǎn)生部分紫色;而NJZX1-0是NJZX1-3的綠色突變體,無(wú)論在何種條件下,其顏色變化不顯著;用DTT(二硫蘇糖醇)對(duì)兩種材料處理后,發(fā)現(xiàn)其在形態(tài)和生理方面會(huì)出現(xiàn)不同的響應(yīng)現(xiàn)象,于是重點(diǎn)分析了 DTT誘導(dǎo)的內(nèi)質(zhì)網(wǎng)應(yīng)激(ER應(yīng)激)并引起自噬與花青素之間的關(guān)系后,再通過(guò)BLAST獲得了與擬南芥中與內(nèi)質(zhì)網(wǎng)應(yīng)激響應(yīng)相關(guān)基因的同源基因BrcbZIP60,通過(guò)生物信息學(xué)和分子生物學(xué)方法初步斷定其功能。這些研究為進(jìn)一步研究花青素與自噬的關(guān)系奠定了基礎(chǔ)。具體結(jié)果如下:1.DTT處理后的形態(tài)觀察與生理指標(biāo)的檢測(cè)本研究以NJZX1-0和NJZX1-3兩種不結(jié)球白菜為材料,在5片葉齡時(shí)用DTT噴灑材料葉片,并在0h,2 h,12 h,24 h時(shí)取樣。隨后測(cè)定葉片花青素的含量、抗氧化酶SOD、POD和CAT活性,以及花青素合成相關(guān)的酶BrcCHS基因和相關(guān)的由ROS引起的核基因的表達(dá)情況,在處理2 h時(shí)拍照記錄。結(jié)果顯示:形態(tài)上可以看出,在NJZX1-3葉片背面顏色加深�？寡趸傅幕钚苑矫�,兩種材料出現(xiàn)不同的變化趨勢(shì),雖然都有上升趨勢(shì),但是NJZX1-0的POD,SOD,CAT等酶活性明顯高于NJZX1-3;在NJZX1-3中BrcCHS基因的表達(dá)量也顯著升高,而在NJZX1-0中沒(méi)有顯著變化;此外,核內(nèi)抗氧化基因BrcGPXH在兩種不同的品系中均呈現(xiàn)不斷升高后略有下降的表達(dá)模式,NJZX1-3中的變化幅度顯著低于NJZX1-0,這與之前酶活性的變化趨勢(shì)相同;而另一種核內(nèi)抗氧化基因BrcGST的表達(dá)量在兩種品系中呈現(xiàn)出與之前不同的變化,即在NJZX1-3中,該基因的表達(dá)量增加程度與NJZX1-0出現(xiàn)顯著性差別。綜上所述,在DTT處理的紫色不結(jié)球白菜中花青素比沒(méi)有處理的不結(jié)球白菜中提高2.5倍以上,最高產(chǎn)量達(dá)到了 2.7mg.L-1;NJZX1-3的變種NJZX1-0基本沒(méi)有變化,此外花青素在一定程度上起到了抗氧化的作用。2.DTT誘導(dǎo)的內(nèi)質(zhì)網(wǎng)應(yīng)激生物標(biāo)記基因以及自噬相關(guān)基因表達(dá)的測(cè)定利用擬南芥相關(guān)基因通過(guò)BLAST獲得不結(jié)球白菜內(nèi)質(zhì)網(wǎng)應(yīng)激標(biāo)志基因的同源基因BrcIRE1、BrcbZIP60、BrcERO1和BrcPDI,通過(guò)qRT-PCR測(cè)定以上基因在不同時(shí)間點(diǎn)的表達(dá)情況;以同樣的方法獲得并測(cè)定了不結(jié)球白菜中自噬相關(guān)基因BrcATG8C-1、BrcATG8C-2、BrcATG8C-3、BrcATG8D 和 BrcATI1 的表達(dá)情況。結(jié)果顯示,在DTT處理的不結(jié)球白菜中,內(nèi)質(zhì)網(wǎng)應(yīng)激標(biāo)志基因以及自噬相關(guān)基因都有表達(dá),其中四個(gè)內(nèi)質(zhì)網(wǎng)應(yīng)激標(biāo)志基因的表達(dá)模式是類似的,但是在NJZX1-3品系中,自噬相關(guān)的基因表達(dá)更為明顯,而在NJZX1-0中的表達(dá)情況顯著性并不高。這表明,通過(guò)引起內(nèi)質(zhì)網(wǎng)應(yīng)激導(dǎo)致細(xì)胞自噬的過(guò)程中,花青素的合成與自噬呈現(xiàn)出正相關(guān)的關(guān)系。3.不結(jié)球白菜BrcbZIP60基因的克隆及初步分析為了進(jìn)一步明確花青素的合成與內(nèi)質(zhì)網(wǎng)應(yīng)激引起的細(xì)胞自噬之間的關(guān)系,通過(guò)BLAST獲得不結(jié)球白菜的BrcbZIP60基因,并通過(guò)生物信息學(xué)與分子生物學(xué)方法對(duì)其初步分析,結(jié)果顯示:通過(guò)克隆技術(shù),成功獲得了BrcbZI 60的ORF序列,它包括450 bp堿基,編碼149個(gè)氨基酸,并將其構(gòu)建GFP標(biāo)簽的載體;通過(guò)軟件分析預(yù)測(cè),BrcbZIP60基因與bZIP60基因類似;再利用瞬轉(zhuǎn)技術(shù),將之前獲得的質(zhì)粒轉(zhuǎn)入擬南芥原生質(zhì)體內(nèi),衣霉素處理后,觀察該基因有大量表達(dá)。
[Abstract]:Brassica rapa ssp.chinensis, also known as green cabbage, is native to China. Because of its good taste and rich nutrition, it is very popular with the people. Especially in the south, it is one of the most common vegetables in the people's home. With the improvement of the quality of life, the demand for non heading Chinese cabbage is also increasingly diversified. Increasing production and improving its beneficial secondary metabolites, such as anthocyanin and vitamins, have become a major concern. As the two secondary metabolites of plants, anthocyanins are becoming more and more important in plant physiology and food nutrition. In recent years, more and more reports about it have been reported, thus increasing its content and stability. In the purple non heading Chinese cabbage, the content of anthocyanin is very high and the yield of the non heading Chinese cabbage is very high. Therefore, it is of high economic value to improve the anthocyanin content in the non heading Chinese cabbage. This study is based on two different strains of different strains of Chinese cabbage system biology Laboratory of Nanjing Agricultural University. The Chinese cabbage is a test material, NJZX1-3 and NJZX1-0., in which the front of NJZX1-3 in the growth process is purple, the back is green, and the back also produces some purple on the back, and NJZX1-0 is a green mutant of NJZX1-3. No matter under which conditions, the color change is not obvious; after the treatment of the two materials with DTT (two disulfidol), hair is made. There will be a different response in morphology and physiology. Then it focuses on the analysis of the DTT induced endoplasmic reticulum stress (ER stress) and the relationship between autophagy and anthocyanins. Then, the same gene BrcbZIP60, which is related to the stress response related to the endoplasmic reticulum in Arabidopsis, is obtained by BLAST, and through bioinformatics and molecular birth. The results of these studies lay a foundation for further study of the relationship between anthocyanin and autophagy. The results are as follows: the morphological observation and physiological indexes after 1.DTT treatment were based on two kinds of non heading Chinese cabbage, NJZX1-0 and NJZX1-3, with DTT spraying leaves at 5 leaf age, and in 0h, 2 h, 12 h, 2. Sampling at 4 h. Then the content of leaf anthocyanins, antioxidant enzyme SOD, POD and CAT activity, and the expression of BrcCHS gene related to anthocyanin synthesis and related nuclear gene expression caused by ROS were recorded. The photo was recorded at 2 h. The results showed that the color of the leaf on the back of the NJZX1-3 blade was deepened and the antioxidant enzyme activity was observed. On the other hand, the two kinds of materials have different trends, although all of them have an upward trend, but the activity of POD, SOD, CAT and other enzymes of NJZX1-0 is obviously higher than that of NJZX1-3; the expression of BrcCHS gene in NJZX1-3 is also significantly increased, but there is no significant change in NJZX1-0; moreover, the anti oxidative gene BrcGPXH in the nucleus is rising in two different strains. The change in NJZX1-3 was significantly lower than that of NJZX1-0, which was the same as that of previous enzyme activity, and the expression of BrcGST in the other nuclear antioxidant gene in the two strains was different from that before, that is, in NJZX1-3, the increase in the expression of the gene was significant with that of NJZX1-0. In summary, the anthocyanin in the purple non heading Chinese cabbage treated by DTT was more than 2.5 times higher than that in the non heading Chinese cabbage and the maximum yield reached 2.7mg.L-1; the variant NJZX1-0 of NJZX1-3 did not change basically. In addition, anthocyanins played an anti oxygen effect.2.DTT induced endoplasmic reticulum stress biologic mark. Determination of gene expression and autophagy related gene expression using Arabidopsis related genes to obtain homologous gene BrcIRE1, BrcbZIP60, BrcERO1 and BrcPDI of non heading Chinese cabbage endoplasmic reticulum stress marker gene by BLAST. The expression of the above gene at different time points was measured by qRT-PCR; the non heading White was obtained and measured by the same method. The expression of autophagy related genes, BrcATG8C-1, BrcATG8C-2, BrcATG8C-3, BrcATG8D and BrcATI1, showed that the endoplasmic reticulum stress marker gene and autophagy related genes were expressed in the non heading Chinese cabbage treated by DTT, and the expression pattern of four endoplasmic reticulum stress marker genes was similar, but in the NJZX1-3 strain, The expression of autophagy related genes is more obvious, but the expression in NJZX1-0 is not significant. This indicates that the synthesis of anthocyanins and autophagy in the process of endoplasmic reticulum stress induced autophagy is positively related to autophagy. The cloning and preliminary analysis of the BrcbZIP60 gene of.3. non heading Chinese cabbage is to further clarify anthocyanins. The relationship between synthesis and autophagy induced by endoplasmic reticulum stress, the BrcbZIP60 gene of non heading Chinese cabbage was obtained by BLAST, and its preliminary analysis by bioinformatics and molecular biological methods showed that the ORF sequence of BrcbZI 60 was successfully obtained by cloning technology, which includes 450 BP bases and 149 amino acids. It is used to construct the carrier of the GFP label; the BrcbZIP60 gene is similar to that of the bZIP60 gene by software analysis. Then the plasmids obtained before are transferred into the protoplasm of Arabidopsis thaliana by using the transient technique. After the treatment of ycomycin, the gene has a large number of expression.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S634.3

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