【作者】 曹新有；

【导师】 陈耀锋；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2009， 博士

【摘要】 Ⅰ、大豆抗逆相关转录因子GmDREB5互作蛋白GmTPR1的功能分析转录因子在高等植物抗逆反应及基因表达调控中起着重要的作用。DREB(dehydration resistance element binding protein)是一类重要的逆境响应转录因子,DREB基因的过表达能显著提高植物的抗逆性。DREB转录因子的活性受到很多互作蛋白的调控,目前对于DREB转录因子互作蛋白的研究还很少。因此,对其互作蛋白的深入研究,将有助于阐明DREB转录因子的调控机制。本课题组的前期工作已经从大豆中筛选到可以显著提高转基因植物抗逆性的DREB基因(GmDREB5),并利用GmDREB5蛋白为诱饵,采用酵母双杂交的方法筛选到一个编码互作蛋白的基因GmTPR1。该基因目前研究较少,生物学功能未知。本研究对GmDREB5互作蛋白GmTPR1的特性及生物学功能进行了较为系统研究。具体研究结果如下:1、GmTPR1基因的克隆及序列比对:从大豆中克隆转录因子GmDREB5互作蛋白编码基因GmTPR1。氨基酸序列分析显示:GmTPR1只含有一个TPR基序(tetratricopeptide repeat motif),将GmTPR1与拟南芥中含有相同保守域的蛋白进行氨基酸同源性比较,结果显示与其同源性最高的只有14%,推测其可能为一个新的TPR蛋白家族成员。2、GmTPR1蛋白与GmDREB5蛋白互作鉴定:分别将GmTPR1、GmDREB5构建到含His标签的pEASY-E1和含GST标签的pGEX 4T-1载体上,形成融合表达载体,进行Pull-down、western blot技术分析。结果表明:GmTPR1蛋白与GmDREB5蛋白在体外具有强烈的相互作用;再分别将GmTPR1、GmDREB5构建到pUC-SPYNE和pUC-SPYCE双分子荧光互补载体上,基因枪法轰击洋葱表皮,荧光共聚焦显微镜下观察。结果表明:GmTPR1蛋白与GmDREB5蛋白在体内也具有强烈的相互作用。3、GmTPR1蛋白与GmDREB5蛋白亚细胞定位:采用含有绿色荧光蛋白基因(GFP)的163hGFP载体与GmTPR1、GmDREB5基因分别构建融合表达载体,转化洋葱表皮细胞进行亚细胞定位分析。结果表明:GmTPR1蛋白定位于细胞核内,GmDREB5蛋白定位于细胞核内及细胞膜上,说明GmTPR1蛋白与GmDREB5蛋白相互作用可能发生于细胞核内。4、GmTPR1基因的表达特征分析:RT-PCR和Real-time PCR结果表明:GmTPR1基因受干旱、低温、高盐、高温、脱落酸(ABA)、乙烯(ETH)、水杨酸(SA)等多种胁迫或激素处理诱导表达。脱落酸(ABA)、水杨酸(SA)胁迫1 h时,GmTPR1基因表达量达到最高;高盐处理6 h时,GmTPR1基因表达量达到最高;干旱处理时,GmTPR1基因表达量逐渐增高,24 h达到峰值;低温胁迫,表达量12 h达到最高,24 h有所降低。同时GmTPR1也对乙烯(ETH)、高温等激素或胁迫处理有所应答。这些结果表明:GmTPR1基因受多种胁迫处理的诱导,可能参与多种信号转导途径。5、在酵母中,GmTPR1可以提高转录因子GmDREB5转录激活活性:将GmTPR1插入到pGADT7载体上,GmDREB5插入到YepGAP载体上,分别形成pGADTPR1和YepD5融合表达载体,将其共同转化到含报告基因HIS3和LacZ的野生型DRE酵母报道子,结果发现其能在附加20 m mol/L 3-AT的四缺营养缺陷性平板(SD/His-/Ura-/Trp-/Leu-)上生长,而单独转化YepD5,酵母报道子则不能生长,结果说明,在酵母中,GmTPR1对GmDREB5转录因子的转录激活活性具有一定的促进作用。6、GmTPR1与GmDREB5蛋白互作位点分析:将全长GmTPR1基因及分段缺失基因插入pGADT7载体上,构建含不同GmTPR1基因片段(1-896,1-675,675-896,1-573,573-896)的载体,与酵母双杂交诱饵载体pGBKT7-GmDREB5共同转化酵母AH109,结果发现,当只有缺失GmTPR1的C端(573-896)时,酵母转化子不能在四缺营养缺陷性平板(SD/His-/Ade-/Trp-/Leu-)上生长,而其他片段均能正常生长,表明GmTPR1的C端对GmTPR1与GmDREB5相互作用可能起关键作用。7、GmTPR1基因的功能分析:将GmTPR1基因转化烟草和拟南芥进行功能鉴定。在干旱和高盐胁迫条件下,转基因烟草地上和地下部分的长度明显长于对照;转基因拟南芥的萌发率明显高于对照,根长明显长于对照;在盐胁迫条件下,转基因烟草的叶绿素含量明显高于对照,证明GmTPR1基因的过表达能够显著提高转基因烟草及拟南芥对干旱和高盐胁迫的抗性。综上所述:GmTPR1蛋白与GmDREB5蛋白在细胞核内具有强烈的相互作用,其GmTPR1的C端对GmTPR1与GmDREB5相互作用可能起关键作用。在酵母中,GmTPR1可以提高转录因子GmDREB5转录激活活性,且受各种胁迫诱导表达,其过量表达能明显提高转基因烟草及拟南芥对干旱和高盐胁迫的抗性。Ⅱ、利用核蛋白筛选系统克隆抗逆基因核蛋白(Nuclear proteins)在植物发育、逆境胁迫响应等生理过程中发挥重要的调控作用。转录水平的基因调控大部分发生在细胞核内,研究植物核蛋白的组成及动态变化是研究植物基因调控网络的基础。目前研究核蛋白的方法操作复杂,而且成本较高。因此,建立简便快速、低成本的研究方法来研究核蛋白的动态组成对于剖析植物细胞发育、环境胁迫响应等过程中的基因表达调控机制具有重要的理论意义。本研究对本课题组前期建立的核蛋白筛选(NTT)系统进行了鉴定,在此基础上,采用NTT系统对小白麦干旱处理5小时cDNA文库进行了筛选,并对候选克隆TaABP1进行了进一步的分析。具体结果如下:1、NTT筛选系统的鉴定及应用:为了验证NTT系统的可靠性,将具有核定位功能的转录因子基因GmAREB插入NTT筛选载体pLEXAD中,转化酵母EGY48,在SD/His-/Leu-营养缺陷性平板能够正常生长,证明NTT筛选系统是可靠的;同时,构建了小白麦干旱处理5小时cDNA文库,插入筛选载体pLEXAD中进行筛选,共得到大约200个候选克隆,测序后分析发现,在所有克隆中,参与胁迫类蛋白占29%;信号传导类蛋白占23%;转运类蛋白占8%;能量及基础代谢类蛋白占23%;未知蛋白占17%。59.4%的蛋白可以预测具有核定位信号。2、候选克隆TaABP1的克隆及功能分析:在获得的克隆中,有一个bZIP(basic leucine zipper,碱性亮氨酸拉链)转录因子基因,命名为TaABP1,同源性分析表明:TaABP1与TaABI5、TmABI5等都具有较高的同源性;转录激活分析表明TaABP1具有转录激活活性;亚细胞定位分析表明TaABP1定位于细胞核中;TaABP1基因表达特性分析表明,TaABP1基因的表达受ABA、高盐、低温和干旱的诱导,可能参与这些逆境信号传导;在不同逆境胁迫处理下,TaABP1基因在小白麦的根、茎、叶中的表达量也存在差异:叶>茎>根;将TaABP1转化了烟草进行功能鉴定,结果表明,干旱胁迫条件下,转基因烟草表现明显的抗旱性。3、小麦成熟胚愈伤组织诱导及分化研究:为了利用小麦成熟胚进行遗传转化,本研究尝试优化小麦组织培养条件。以小麦成熟胚为外植体进行离体培养,研究了不同预处理、不同激素浓度、组合等因素对愈伤组织诱导及分化的影响。结果表明:低温预处理对愈伤组织诱导及分化有利;在不同预处理条件下,2,4-D浓度对出愈率及再生苗率的影响与基因型有关,附加KT能缓解高浓度2,4-D对再生苗率的抑制作用。更多还原

【Abstract】 Ⅰ. Functional analysis of interactional protein GmTPR1 with stress-related transcription factor GmDREB5Transcription factors play important role in the regulation of plant growth, development, and response to environment. The DREB (dehydration resistance element binding protein) is a family of stress-related transcription factors. DREB family can enhance plant tolerance to abiotic stresses. The activity of DREB is regulated by some specific interactive proteins. Therefore, isolation of genes coding DREB interactive proteins will be contributed for the clarifying of the regulative mechanisms of DREB transcription factors on translational level. In previous research, overexpression of GmDREB5 was proved to enhance stress tolerances of transgenic plants. In order to study regulative mechism of GmDREB5, GmTPR1 gene coding GmDREB5 interactive protein had been isolated by screening cDNA library of soybean treated with drought using yeast-two-hybrid systemt. So far, few members of TPR gene family were studied and functions of most members are unknown. In this study, characteristic and function of GmTPR1 were analyzed. Some main results have been achieved as follows:1. Isolation and phylogenetic analysis of GmTPR1 gene A gene coding GmDREB5 interactive protein, GmTPR1, was isolated from soybean cDNA library. The GmTPR1 protein has one TPR (tetratricopeptide repeat) motif. The homologic analysis of the TPR motif with the similar Arabidopsis indicated that the highest identity of with other TPR proteins was 14% only, suggesting that GmTPR1 is a novel member of TPR protein family.2. Identification of the interaction between GmTPR1 and GmDREB5 The fragment encoding GmTPR1 was inserted into pEASY-E1 (with 6×His in N’end) and the fragment encoding GmDREB5 was inserted to pGEX 4T-1 (with GST tag), respectively to form fusion expression vector,which was analyzed by pull-down and western blot .and results suggested GmTPR1 interacts with GmDREB5 obviously in vitro; full-length sequences of GmTPR1 and GmDREB5 were cloned into the pUCSPYNE and pUCSPYCE BIFC (Bimolecular fluorescence complementation) vectors, respectively,for transient expression analysis in onion (Allium cepa) epidermal cells using the particle bombardment,and fluorescence was observed with a laser scanning confocal microscope. Result indicated that GmTPR1 interacts with GmDREB5 in vivo strongly also.3. Subcellular localization assay of the GmTPR1 and GmDREB5 GmTPR1 and GmDREB5 under the control of the cauliflower mosaic virus (CaMV) 35S promoter were inserted to 163hGFP vector before the 5’end of the coding region of green fluorescence protein (GFP) gene. The fused of plasmids 163hGFP- GmTPR1 and 163hGFP-GmDREB5 were transformated into onion epidermal cells for transient activity analysis. The results indicated that GmTPR1 is located in nuclei and GmDREB5 in the nuclei and membrane, suggesting this two proteins might interact each other in the nuclei of plant.4. Expression pattern analysis of GmTPR1 RT-PCR and Real-time PCR results showed that the expression of GmTPR1 gene was induced by drought, low temperature, high-salt high temperature, ABA, ETH, SA. Under ABA and SA treatment for 1 h , maximal expression pattern of GmTPR1 was found. Under high-salt condition for 6 h, GmTPR1 expression level reached the maximal.Under drought treatment, GmTPR1 mRNA began to accumulate at 1 h and reached maximum at 24 h after treatment. Under low-temperature treatment, GmTPR1 mRNA began to accumulate at 1 h and reached its maximum at 12 h. Interestingly, the GmTPR1 gene was also induced by exogenous ETH and high-temperature. thereby expression of GmTPR1 gene was thought to be induced by multitudinal treatment, and it might be involved in different signal transduction pathway.5. GmTPR1 can enhance transcriptional activation activity of GmDREB5 in yeast cells YepD5 and pGADTPR1 recombine plasmid were co-transformed into yeast strains carrying the wild type DRE element, which was site before HIS3 and LacZ reporter genes in chromosome. The results showed that co-transformed yeast grew well on SD/-Ade/-His/-Leu/-Trp medium plus 20 m mol/L 3-AT, but yeast cells transforamed without pGADT7-GmTPR1 didn’t grow at all on the same medium, suggesting GmTPR1 might enhance transcriptional activation activity of GmDREB5.6. Interactive site analysis between GmTPR1 and GmDREB5 proteins The full-length and different length of GmTPR1 fragments were inserted into the pGADT7 vector using site-sepecific recombinant enzyme, and co-transformed into the yeast AH109 with pGBKT7-GmDREB5. The results indicated that only the yeast transformated with the vector containing C-terminal sequencing of GmTPR1 could grow on SD/His- /Ade- /Trp- /Leu- medium, suggesting that C-terminal sequencing of GmTPR1 might play a crucial role in the interaction between GmTPR1 and GmDREB5.7. Functional analysis of GmTPR1 gene GmTPR1 gene was transformed into tobacco and Arabidopsis thaliana by Agrobacterium method approach for fuctional analysis.The results indicatied that the over-expression of GmTPR1 gene significantly improved drought and high-salt stress resistance in the two kinds of transgenic plants, and the size of parts above and under ground form the transgenic plants were longer than the corresponding parts form wild-type after stresses treatment. The Chlorophyll contents in transgenic lines were higher than in wild-type.Above results suggested GmTPR1 and GmDREB5 might interact in the nuclei of plant; C-terminal sequence of GmTPR1 might play a crucial role in the interaction between GmTPR1 and GmDREB5; GmTPR1 can enhance transcriptional activation activity of GmDREB5 in yeast cells; Expression of GmTPR1 gene was induced by multitudinal treatments and the over-expression of GmTPR1 gene significantly improved drought and high-salt stress resistance of transgenic tobacco and Arabidopsis thaliana.Ⅱ. Isolation of stress responsive genes using nuclear transportation trapNuclear proteins play vital roles in the growth and development of plants. The research on the component and the dynamic change of plant nuclear protein is essential to study on resistance-related genes of the regulation network. Current research methods are complex to operate and high cost. Therefore, it is important to build some simple and low cost techniques. In previous research, a high-efficiency nuclear transportation trap (NTT) system was constructed. In this study, the screening of wheat (Variety xiaobaimai treated under drought condition for 5 h ) cDNA library was completed using NTT system,and one (named TaABP1) of clones belonged to bZIP genes family was further studied.The major results are following:1. Identification and application of nuclear transportation trap (NTT) system In order to identify the reliability of NTT system, the one coding GmAREB with NLS (Nuclear location signal) motif was inserted into the pLEXAD vector and transformed into the yeast EGY48. The results showed that the yeast transformed with recombinant vector could survious on SD/His-/Leu- medium, suggesting that nuclear transportation trap (NTT) system is worked. We constructed wheat cDNA library and screened up 200 yeast clone using NTT system. The sequence results showed that, 29% genes among them is related to stresses response; 23% genes is related with energy and primary metabolism; The ratio of signal transduction related genes was 23%; The ratio of transport genes was 8%; And unknown protein was 17%.However, in all proteins, the most ones belonged to the type of nuclear proteins, which was accounted for 59.4%.2. Isolation and functional analysis of TaABP1 gene A novel basic leucine zipper transcription factor was isolated from wheat cDNA library using NTT system, and was named as TaABP1. Phylogenetic analysis showed that TaABP1 had higher homology with TaABI5 and TmABI5. The transcriptional activation activity analysis showed that TaABP1 did have the activation activity in yeast. The subcellular localization assay indicated that TaABP1 was localized in nucleus. Analysis of gene expression pattern indicated that the expression of TaABP1 was intensively induced by ABA, high salt, low temperature and drought. Under multitudinal treatment condition, tissue-specific expression pattern analysis indicated that TaABP1 expressed in the root, stem and leaf of wheat, and the expression quantity was decreased successively in the tissues of leaf, stem, and root. The functional analysis results indicated that TaABP1 gene improved the resistance to drought stress of transgenic tobacco significantly.3. Study on induction and differentiation of the callus from mature embroys of wheat Mature embryos of wheat as explants were cultured on the medium with different concentrations and combinations of 2,4-D and KT after different pretreatments to studyt their effects on callus induction and differentiation . It was shown that low temperature pretreatment(at 4℃) increased the calluses induction rates, Empleying this technique, regeneration rate of some genotypes was more than 30%.the results also indicated that 2,4-D concentration was related to genotypes and the addition of KT could abate the inhibition of high concentrations 2,4-D.更多还原