甘蔗SoSnRK2.1和SoACLA-1基因的功能鉴定与分析

发布时间：2018-05-13 12:20

本文选题：甘蔗 + SnRK2.1　；参考：《广西大学》2016年博士论文

【摘要】：干旱是影响甘蔗生长、发育、产量和质量的一种重要因素。ATP-柠檬酸裂解酶(ACL)和非酵解型蛋白激酶(SnRK2)是植物正常生长和发育关键调控酶。ACL催化作用柠檬酸在细胞质中转变成乙酰辅酶A,SnRK2在响应多种非生物胁迫方面发挥着重要作用。在ABA信号转导途径中,这两个基因参与植物非生物胁迫下的发育。我们研究了SoACLA-1与SoSnRK21在原核生物和真核生物中的调控机制。主要研究结果如下：1.根据SoSnRK2.1和SoACLA.1基因序列,我们克隆了这两个基因。使用特异性的引物扩增SoSnRK2.1和SoACLA-1基因的全长并且根据原核表达载体pET30a(+)和植物表达载体pRI101-ON,pRI101-AN和pUBTC设计限制性酶切位点的。基于序列分析表明SoACLA-1和SoSnRK2.1基因的完整开放阅读框长度分别为1272 bp和1002 bp。氨基酸序列分析表明,SoACLA-1和SoSnRK2.1与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均在原核生物获得表达。将没有内含子的SoSnRK2.1和SoACLA-1基因插入到pET30a(+)载体,其中该载体包含T7启动子进行原核表达。原核表达结果显示重组的pET-SoACLA-1和SoSnRK2.1分别为46 kDa和38 kD a.此外pET-SoACLA-1和SoSnRK2.1重组蛋白以包涵体形式存在。SoACLA-1和SoSnRK2.1重组蛋白通过Ni2+NTA柱亲和层析纯化和透析浓缩。此外,SoACLA-1和soSnRK2.1进行响应干旱(PEG)处理,鉴定它们的干旱耐受性3.利用模式植物烟草鉴定SoSnRK2.1的功能。将受CaMV 35S启动子控制的SoSnRK2.1-GFP-pBI121转入烟草中。植株转化率达到75%。结果表明,转基因烟草植株比非转基因烟草有较低水平的离子泄漏(IL),低浓度丙二醛(MDA)和H202,含量。而且,转基因烟草植株体内超氧化物歧化酶(SOD),过氧化物酶(POD)和过氧化氢酶(CAT)的三种抗氧化酶活性较高,叶绿素含量和叶片相对含水量(RWC)也比非转基因烟草高。SoSnRK2.1能够通过有性繁殖稳定传播到下一代。研究结果表明,转SoSnRK2.1基因的烟草的生长和形态学显示该基因的过量表达提高了烟草的耐旱性,说明SoSnRK2.1是甘蔗响应非生物胁迫并且是调控生长和发育的关键基因。4.通过转基因烟草鉴定甘蔗SoACLA-1基因的耐旱功能。将受CaMV 35S启动子控制的SoACLA-1-pRI101-AN表达载体转入农杆菌,再转入野生型烟草植株。采用PCR方法检测转基因烟草的转化率达到56%。观察转基因烟草的形态和多种生理特性和生化指标,结果与转SoSnRK2.1基因的相似,过量表达甘蔗SoACLA-1基因可以增强烟草植株的耐旱性。结果显示,SoACLA-1基因与植物的耐旱性相关。5.成功获得转SoSnRK2.1基因甘蔗。将SoSnRK2.1连接到pRI101-ON载体,利用农杆菌介导转入ROC22和YL6胚性愈伤组织。通过PCR检测目标片段和NPTII标记基因,ROC22和YL6转基因植株率分别为26.5%和16.7%。初步证明,转基因甘蔗植株对干旱胁迫有可能增强。6.成功获得转SoACLA-1基因甘蔗。甘蔗植株表达载体pUBTC与SoACLA-1构建成功并且使用用Ubi启动子控制,ROC22愈伤组织作为受体,使用农杆菌介导的遗传转化体系,转化率达到16%。转基因植株与对照植株进行表型的观察和对比。结果表明在干旱胁迫下,转基因植株的耐旱性明显比对照植株提高。
[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.

【学位授予单位】：广西大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S566.1

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