發(fā)布時(shí)間：2018-05-13 12:20

  本文選題：甘蔗 + SnRK2.1��； 參考：《廣西大學(xué)》2016年博士論文

【摘要】：干旱是影響甘蔗生長、發(fā)育、產(chǎn)量和質(zhì)量的一種重要因素。ATP-檸檬酸裂解酶(ACL)和非酵解型蛋白激酶(SnRK2)是植物正常生長和發(fā)育關(guān)鍵調(diào)控酶。ACL催化作用檸檬酸在細(xì)胞質(zhì)中轉(zhuǎn)變成乙酰輔酶A,SnRK2在響應(yīng)多種非生物脅迫方面發(fā)揮著重要作用。在ABA信號(hào)轉(zhuǎn)導(dǎo)途徑中,這兩個(gè)基因參與植物非生物脅迫下的發(fā)育。我們研究了SoACLA-1與SoSnRK21在原核生物和真核生物中的調(diào)控機(jī)制。主要研究結(jié)果如下：1.根據(jù)SoSnRK2.1和SoACLA.1基因序列,我們克隆了這兩個(gè)基因。使用特異性的引物擴(kuò)增SoSnRK2.1和SoACLA-1基因的全長并且根據(jù)原核表達(dá)載體pET30a(+)和植物表達(dá)載體pRI101-ON,pRI101-AN和pUBTC設(shè)計(jì)限制性酶切位點(diǎn)的�；�于序列分析表明SoACLA-1和SoSnRK2.1基因的完整開放閱讀框長度分別為1272 bp和1002 bp。氨基酸序列分析表明,SoACLA-1和SoSnRK2.1與玉米和栽培稻中的同源基因有很高的同源性。2.SoSnRK2.1和SoACLA-1均在原核生物獲得表達(dá)。將沒有內(nèi)含子的SoSnRK2.1和SoACLA-1基因插入到pET30a(+)載體,其中該載體包含T7啟動(dòng)子進(jìn)行原核表達(dá)。原核表達(dá)結(jié)果顯示重組的pET-SoACLA-1和SoSnRK2.1分別為46 kDa和38 kD a.此外pET-SoACLA-1和SoSnRK2.1重組蛋白以包涵體形式存在。SoACLA-1和SoSnRK2.1重組蛋白通過Ni2+NTA柱親和層析純化和透析濃縮。此外,SoACLA-1和soSnRK2.1進(jìn)行響應(yīng)干旱(PEG)處理,鑒定它們的干旱耐受性3.利用模式植物煙草鑒定SoSnRK2.1的功能。將受CaMV 35S啟動(dòng)子控制的SoSnRK2.1-GFP-pBI121轉(zhuǎn)入煙草中。植株轉(zhuǎn)化率達(dá)到75%。結(jié)果表明,轉(zhuǎn)基因煙草植株比非轉(zhuǎn)基因煙草有較低水平的離子泄漏(IL),低濃度丙二醛(MDA)和H202,含量。而且,轉(zhuǎn)基因煙草植株體內(nèi)超氧化物歧化酶(SOD),過氧化物酶(POD)和過氧化氫酶(CAT)的三種抗氧化酶活性較高,葉綠素含量和葉片相對(duì)含水量(RWC)也比非轉(zhuǎn)基因煙草高。SoSnRK2.1能夠通過有性繁殖穩(wěn)定傳播到下一代。研究結(jié)果表明,轉(zhuǎn)SoSnRK2.1基因的煙草的生長和形態(tài)學(xué)顯示該基因的過量表達(dá)提高了煙草的耐旱性,說明SoSnRK2.1是甘蔗響應(yīng)非生物脅迫并且是調(diào)控生長和發(fā)育的關(guān)鍵基因。4.通過轉(zhuǎn)基因煙草鑒定甘蔗SoACLA-1基因的耐旱功能。將受CaMV 35S啟動(dòng)子控制的SoACLA-1-pRI101-AN表達(dá)載體轉(zhuǎn)入農(nóng)桿菌,再轉(zhuǎn)入野生型煙草植株。采用PCR方法檢測轉(zhuǎn)基因煙草的轉(zhuǎn)化率達(dá)到56%。觀察轉(zhuǎn)基因煙草的形態(tài)和多種生理特性和生化指標(biāo),結(jié)果與轉(zhuǎn)SoSnRK2.1基因的相似,過量表達(dá)甘蔗SoACLA-1基因可以增強(qiáng)煙草植株的耐旱性。結(jié)果顯示,SoACLA-1基因與植物的耐旱性相關(guān)。5.成功獲得轉(zhuǎn)SoSnRK2.1基因甘蔗。將SoSnRK2.1連接到pRI101-ON載體,利用農(nóng)桿菌介導(dǎo)轉(zhuǎn)入ROC22和YL6胚性愈傷組織。通過PCR檢測目標(biāo)片段和NPTII標(biāo)記基因,ROC22和YL6轉(zhuǎn)基因植株率分別為26.5%和16.7%。初步證明,轉(zhuǎn)基因甘蔗植株對(duì)干旱脅迫有可能增強(qiáng)。6.成功獲得轉(zhuǎn)SoACLA-1基因甘蔗。甘蔗植株表達(dá)載體pUBTC與SoACLA-1構(gòu)建成功并且使用用Ubi啟動(dòng)子控制,ROC22愈傷組織作為受體,使用農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化體系,轉(zhuǎn)化率達(dá)到16%。轉(zhuǎn)基因植株與對(duì)照植株進(jìn)行表型的觀察和對(duì)比。結(jié)果表明在干旱脅迫下,轉(zhuǎn)基因植株的耐旱性明顯比對(duì)照植株提高。
[Abstract]:Drought is an important factor affecting the growth, development, yield and quality of sugarcane,.ATP- citrate lyase (ACL) and non glycolytic protein kinase (SnRK2) are the key regulatory enzymes of plant growth and development..ACL catalyzes the transformation of citric acid to acetyl coenzyme A in the cytoplasm, and SnRK2 plays an important role in response to a variety of abiotic stresses. In the ABA signal transduction pathway, these two genes are involved in the development of plant abiotic stress. We have studied the regulation mechanism of SoACLA-1 and SoSnRK21 in prokaryotes and eukaryotes. The main results are as follows: 1. according to the sequence of SoSnRK2.1 and SoACLA.1, we augmentation these two genes. Using specific primers to expand the gene. The length of the SoSnRK2.1 and SoACLA-1 genes was increased and the restriction sites were designed based on the prokaryotic expression vector pET30a (+) and the plant expression vector pRI101-ON, pRI101-AN and pUBTC. Based on the sequence analysis, the complete open reading frame length of the SoACLA-1 and SoSnRK2.1 genes was 1272 BP and 1002 bp. amino acid sequences, respectively. The homologous genes in the SoSnRK2.1 and the maize and cultivated rice are highly homologous.2.SoSnRK2.1 and SoACLA-1 are expressed in the prokaryotes. The SoSnRK2.1 and SoACLA-1 genes without introns are inserted into the pET30a (+) carrier, and the carrier contains the T7 promoter for the prokaryotic expression. The prokaryotic expression results show the recombinant pET-SoACLA-1 and So SnRK2.1 was 46 kDa and 38 kD A. respectively, in addition to pET-SoACLA-1 and SoSnRK2.1 recombinant proteins in inclusion body,.SoACLA-1 and SoSnRK2.1 recombinant proteins were purified and concentrated by Ni2+NTA column affinity chromatography. Furthermore, SoACLA-1 and soSnRK2.1 responded to drought (PEG) treatment, and identified their drought tolerance 3. using pattern plant tobacco identification. The function of SoSnRK2.1 was to be transferred into tobacco by the SoSnRK2.1-GFP-pBI121 controlled by the CaMV 35S promoter. The conversion of the plant to 75%. showed that the transgenic tobacco plants had a lower level of ion leakage (IL), low concentration of malondialdehyde (MDA) and H202, and the content of the transgenic tobacco plants. SOD), three kinds of antioxidant enzyme activities of peroxidase (POD) and catalase (CAT) are higher, chlorophyll content and leaf relative water content (RWC) are also higher than non transgenic tobacco, which can be propagated steadily through sexual reproduction to the next generation. The results showed that the growth and morphology of tobacco transferred from SoSnRK2.1 gene showed the gene. Excessive expression improves the drought resistance of tobacco, indicating that SoSnRK2.1 is a key gene for sugarcane response to abiotic stress and is the key gene for regulating growth and development..4. can identify the drought resistant function of sugarcane SoACLA-1 gene through transgenic tobacco. The SoACLA-1-pRI101-AN expression vector controlled by CaMV 35S promoter is transferred into Agrobacterium and then transferred into wild type tobacco. PCR method was used to detect the transformation rate of transgenic tobacco to 56%. to observe the morphology, physiological characteristics and biochemical indexes of transgenic tobacco. The result was similar to that of the transgenic SoSnRK2.1 gene. The overexpression of the sugarcane SoACLA-1 gene could enhance the drought resistance of the tobacco plants. The results showed that the SoACLA-1 gene was related to the drought tolerance of the plant.5.. SoSnRK2.1 gene sugarcane was obtained. SoSnRK2.1 was connected to pRI101-ON vector and transferred into ROC22 and YL6 embryogenic callus by Agrobacterium tumefaciens. By PCR detection of target fragment and NPTII marker gene, the rate of ROC22 and YL6 transgenic plants were 26.5% and 16.7%. respectively. SoACLA-1 gene sugarcane was obtained. The plant expression vector, pUBTC and SoACLA-1, was successfully constructed and used with Ubi promoter, ROC22 callus as a receptor, using Agrobacterium mediated genetic transformation system, and the transformation rate was observed and compared between the 16%. transgenic plants and the control plants. The results showed that the drought stress was under drought stress. The drought tolerance of transgenic plants was significantly higher than that of control plants.

【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S566.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 郭盈添;范琨;白果;石杰霞;董開茂;關(guān)雪蓮;鄭健;;金露梅幼苗對(duì)高溫脅迫的生理生化響應(yīng)[J];西北植物學(xué)報(bào);2014年09期

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本文編號(hào)：1883124