【作者】 高翔；

【导师】 叶兴国；

【作者基本信息】 中国农业科学院 ， 作物遗传育种， 2010， 博士

【摘要】 果聚糖(Fructan)广泛存在于温带、亚温带草本及禾本植物中,能提高植物对干旱、盐碱和低温等非生物胁迫的抗性。异源六倍体小麦是重要的粮食作物,果聚糖合成和降解是小麦碳水化合物的主要代谢途径之一。研究小麦中果聚糖合成酶(Fructan biosynthesis enzymes, FBEs)基因结构特性、表达特性、遗传特性和功能,解析果聚糖合成相关基因与抗逆性之间的关系等,对认识小麦的进化过程、明晰果聚糖合成酶基因参与抗旱的机理及通过转基因方法提高小麦等作物的抗逆性具有重要意义。本研究以二倍体乌拉尔图小麦(AA)、西尔斯山羊草(SS)、粗山羊草(DD),四倍体硬粒小麦(AABB),六倍体普通小麦(AABBDD)等小麦族植物为材料,利用Touch-down PCR技术分离出果聚糖合成酶基因蔗糖:蔗糖-1-果糖基转移酶( Sucrose: sucrose 1-fructosyltransferase, 1-SST)、果聚糖:果聚糖-1-果糖基转移酶(Fructan: fructan1-fructosyltransferase, 1-FFT)和蔗糖:果聚糖-6-果糖基转移酶(Sucrose: fructan 6-fructosyltransferase, 6-SFT),进行了基因结构和序列分析;以普通小麦品种扬麦6号为材料,采用I-PCR及TAIL-PCR技术,分别克隆了三个果聚糖合成酶基因的启动子序列,利用生物信息学技术对克隆的启动子功能元件进行了预测,通过功能元件缺失的方式构建功能缺失融合载体,运用基因枪轰击小麦幼胚,通过GUS基因瞬时表达进行启动子的功能研究;利用Southern杂交分析了三个果聚糖合成酶基因在不同倍性小麦族植物的拷贝数。利用绿色荧光蛋白(GFP)和6-SFT基因构建融合表达载体,轰击洋葱表皮细胞,对6-SFT进行了亚细胞定位分析;以6个普通小麦品种为材料,采用Real-time技术分析了在不同干旱胁迫时期果聚糖合成酶基因的表达模式。利用农杆菌介导法将克隆的小麦6-SFT基因转入了烟草,对转基因烟草植株进行了抗旱、抗盐和抗低温鉴定。主要结果如下:1.小麦族植物中1-SST基因序列比较保守。gDNA长度为3326 bp,cDNA大小为1989 bp,由4个外显子和3个内含子构成,4个外显子大小依次为406 bp、9 bp、872 bp、702 bp,3个内含子大小分别为161 bp、146 bp、1030 bp。小麦族植物中的1-SST基因都含有一个只有9个核酸序列(ATCCCAACG)组成的小外显子,是目前植物中发现的最小的外显子。在氨基酸水平上,AABB同AA和SS的序列相似性分别为95.62%和96.83%。AABBDD同AA、SS和DD的相似性分别为97.89%、99.09%和99.24%;2.小麦族植物中的1-FFT基因大小在AA、SS、AABB、AABBDD基因组上都为2652 bp, cDNA大小均为1947 bp,由4个外显子和3个内含子构成,4个外显子大小分别为367 bp、9 bp、869 bp和702 bp,3个内含子大小分别为156 bp、377 bp和172 bp。在DD基因组上gDNA序列长度是2603 bp,cDNA大小为1935 bp,4个外显子大小分别为355 bp、9 bp、869 bp和699 bp,3个内含子大小分别为160 bp、393 bp和118 bp。1-FFT基因也有一个只含有9个碱基(ATCCCAACG)的外显子序列。在氨基酸水平上,AABB同AA和SS的序列相似性分别为97.84%和99.38%。AABBDD同AA、SS和DD的相似性分别为99.07%、99.07%和88.63%。3.小麦族植物6-SFT基因在进化上比较活跃。其gDNA在AA、SS、DD、AABB和AABBDD基因组中分别为3122 bp、3302 bp、3100 bp、3138 bp和3146 bp,cDNA大小同为1851 bp。与1-SST和1-FFT基因相同,6-SFT也含有4个外显子和3个内含子。在第一个外显子上出现了插入/缺失突变,它们的gDNA长度主要差异在第三个内含子上。在氨基酸水平上,AABB同AA和SS的序列相似性分别为97.08%和98.54%。AABBDD同AA、SS和DD在DNA序列上的相似性分别为97.24%、98.70%和96.92%。4.明确了果聚糖合成酶基因在不同倍性小麦族植物中的拷贝数。1-SST基因在AA基因组和DD组中存在3个拷贝,在SS基因组中至少有1个拷贝,在AABB基因组中有5～6个拷贝,在AABBDD基因组中至少含有7～8个拷贝;1-FFT基因在SS和DD基因组中存在3个拷贝,在AA基因组中至少存在2个拷贝,在四倍体AABB基因组中有6个拷贝,在普通小麦AABBDD基因组中至少有7个拷贝出现;6-SFT基因在AA基因组中有2～3个拷贝,在SS基因组中至少有1个拷贝,在DD基因组中存在3个拷贝,在AABB中有5～6个拷贝,在AABBDD中至少存在7～8个拷贝。5.成功从小麦基因组中克隆了3个果聚糖合成酶基因启动子区段,命名为PSFT、PSST及PFFT,长度分别为1064 bp、1927 bp及2142 bp。功能元件预测结果表明,3个启动子区段皆有完整的TATA盒及CAAT元件,同时也具有逆境胁迫响应的元件,如MYB、LTR、ABRE及GARE,同时也存在控制生长昼夜节律性变化的Circadian元件及胚乳特异表达的Skn-1元件(PFFT除外)。对1-SST及1-FFT启动子进行了功能元件缺失研究表明它们受胁迫诱导。6.明确了果聚糖合成酶基因在不同干旱胁迫阶段的表达特性。1-SST和1-FFT基因在干旱胁迫7天时表达量达到最高,6-SFT在干旱胁迫26天时表达量达到最高。进一步分析表明,不同小麦材料果聚糖合成酶基因具有有不同的表达特性,1-SST基因在扬麦6号中表达量最大,其次是扬麦12,而在整个干旱胁迫处理过程中中国春的表达量基本保持在同一水平,表达量较低,这也许是中国春不耐旱的原因之一。1-FFT的表达模式和1-SST相似,只是在胁迫处理到第7天时,旱选10号的表达量最高。6-SFT对干旱胁迫的响应要慢于1-SST和1-FFT,也是旱选10号的表达量最高。7.利用GFP的瞬时表达进行6-SFT基因亚细胞定位分析,构建了融合表达载体16318-hGFP::6-SFT,借助基因枪转化技术转化洋葱表皮细胞,获得在洋葱表皮细胞中融合表达的GFP及6-SFT,共聚焦显微镜观测表明6-SFT基因定位在洋葱表皮细胞质膜上。8.利用烟草这种模式植物进行6-SFT抗逆功能分析,获得了转小麦6-SFT基因的烟草。通过对转基因烟草株系的旱胁迫、盐胁迫和低温胁迫试验,相对于未转基因的野生型对照来说,转基因烟草表现出较强的抗旱、抗盐和抗低温能力。更多还原

【Abstract】 Fructans, being widespread in temperate and sub-temperate herbaceous and grass plants, which biosynthetic and degraded processes characterize one of major metabolic pathways of wheat carbohydrate, can enhance the resistances of plants to drought, salinity, low temperature and other abiotic stresses. As an important allohexaploid plant and a staple crop, wheat has received great attention in the study on FBEs gene structure features, expression characteristics, genetic features, the mechanism of wheat fructan accumulation, the analysis of the relationship between fructan synthesis-related genes and the resistances, which shows great significance on the enhancement of the resistance of wheat and other crops of important agricultural traits. In this study, three diploid species (Triticum urartu Thum: AA; Aegilops speltoides Gren: SS; Aegilops tauschii Coss: DD), one tetraploid accession (Triticum turgidum: AABB) and one hexaploid variety (Triticum aestivum: AABBDD) were chosen as plant materials for FBEs isolation. Touch-down PCR technique were employed to clone genes coding for fructan biosynthetases: sucrose: sucrose-1-fructosyltransferase, 1-SST; fructan:fructan-1-fructosyltransferase, 1-FFT and sucrose: fructan-6-fructosyltransferase, 6-SFT. Sequence alignment and structural analysis were also performed among them and those of different origins. Using I-PCR and TAIL-PCR technology, partial length of promoters for FBEs were identified from common wheat cv. Yangmai 6. Functional elements were predicted through online bioinformatics. Functional elements were confirmed through the construction of fusion vector with truncated promoter fragments and GUS and then transformation into the wheat immature embryos using biolistics. Prediction on the gene copies in common wheat was carried out using Southern blotting analysis. Six common wheat cultivars were chosen for expression profile assay by real-time technique under drought stress at different time intervals. Agrobacterium-mediated transformation was adopted to characterize the function of the cloned wheat 6-SFT in tobacco (Nicotiana tobacum) under drought, salt and low temperature stresses. Main results are covered as the following:1. High conserved 1-SST in Triticeae plants. The full length of gDNA and cDNA were 3326 bp and 1989 bp, respectively, containing four exons and three introns in composition with 406 bp, 9 bp, 872 bp, 702 bp in exon length and 161 bp, 146 bp, 1030 bp in intron length. At the amino acid level, AABB-originated 1-SST shared the similarity of 95.62% and 96.83% with the AA- and SS-originated. In AABBDD genome, it had 97.89%, 99.09% and 99.24% identity with that in AA, SS, and DD genomes at the amino acid level, respectively.2. The gDNA and cDNA lengths of 1-FFT were the same from AA, SS, AABB, AABBDD genomes with 2652 bp and 1947 bp in length, respectively, composed of four exons and three introns. The sizes of four exons were 367 bp, 9 bp, 869 bp, 702 bp and the sizes of the 3 introns, 156 bp, 377 bp, 172 bp, respectively. In the DD genome, gDNA length was 2603 bp and cDNA length, 1935 bp consisting of 4 exons: 355 bp, 9 bp, 869 bp and 699 bp, and 3 introns: 160 bp, 393 bp and 118 bp. At the amino acid level, 97.84% and 99.38% similarities were found between AABB vs AA and AABB vs SS. Compared to AABBDD genome-originated, the similarity was 99.07%, 99.07%, 88.63% in AA, SS and DD genome, respectively. 3. 6-SFTs from Triticeae plants were more active during evolution. Different gDNA lengths were found to be 3122 bp, 3302 bp, 3100 bp, 3138 bp and 3146 bp in AA, SS, DD, AABB, and AABBDD genome, respectively. And the cDNA sizes were 1851 bp. 6-SFT also contains four exons and three introns. High efficiency of insertion/deletion mutation in the first exon accounted for the full-length difference in Triticeae plants. At the amino acid level, 97.08% and 98.54% identity was found between AABB vs AA, AABB vs SS, respectively. 97.24%, 98.70% and 96.92% similarity was shared by AABBDD-originated 6-SFT with AA-, SS-, and DD-originated at the amino acid level, respectively.4. Multi-copies of FBEs in Triticeae were found. For 1-SST, there were three copies in the AA group and the DD group, while in the SS group, at least one copy was found. In the AABB and AABBDD genomes, there were 5 to 6 copies and more than 7 to 8 copies, respectively. In the SS and DD groups, there existed three copies of 1-FFT, while in the AA genome, at least two copies existed. Then in tetraploid wheat, AABB, there were six copies and in the common wheat, AABBDD, at least seven copies were present; 6-SFT in AA group, there are 2 or 3 copies; in the SS group, at least one copy existed; in the DD group, there were three copies and at least 5 to 6 and 7 to 8 copies were present in AABB and AABBDD genomes, respectively.5. Successful isolation of promoter for FBEs from wheat cv. Yangmai 6, designated as PSFT, PSST and PFFT with 1064 bp, 1927 bp and 2142 bp in length, respectively. Functional elements prediction online showed the presence of TATA box and CAAT element, stress response elements, such as MYB, LTR, ABRE and GARE and Circadian element responsible for circadian rhythm in plants and Skn-1 components controlling endosperm-specific expression (except PFFT) in all the three promoters. Functional truncated assay on PSST and PFFT demonstrated that they were abiotic stress-responsive.6. Expression pattern was characterized on FBEs under different time-interval drought stresses. The highest expression levels of 1-SST and 1-FFT reached at seven days, while that of 6-SFT reached after 26-day treatment. Further analysis showed different expression characteristics in different wheat varieties. As for 1-SST expression, Yangmai 6 was high, followed by Yangmai 12, while during the whole process of drought stress in common wheat cv. Chinese Spring, the expression remained at the same level with relatively lower expression, which may answer for the low drought-tolerance in part. 1-FFT and 1-SST shared the same expression pattern with the highest expression in Hanxuan 10 at 7-day drought stress. 6-SFT responded to drought stress slower than 1-SST and 1-FFT with the same highest expression level presenting in Hanxuan 10.7. Biolistics-mediated transformation of onion epidermal cell with fusion vector 16318-hGFP::6-SFT. Transient expression of GFP showed 6-SFT may be located at cell membrane using confocal microscope.8. Obtaining transgenic tobacco lines with wheat 6-SFT. When treated with no watering, 300 mM NaCl and -20℃for 30 min, transgenic tobaccos showed strong tolerance to drought, salt and low temperature stresses while wild types display wilting, growth retardment and turn white.更多还原