联系我们 加入收藏
 
年期检索 高级检索
33
2025年8月3日 星期日
  2016, Vol. 24 Issue (10): 1500-1511    
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
高粱SbSKIP基因的克隆及其在烟草中的抗旱功能分析
刘洋1,姚新转2,吕立堂3,雷雨婷3,代婷婷3,赵德刚4
1.
2. 贵州大学农业生物工程重点实验室
3. 贵州大学 生命科学学院/农业生物工程研究院
4. 贵州大学
Cloning of SbSKIP Gene from Sorghum (Sorghum bicolor) and Analysis of Drought-resistant Function in Tobacco (Nicotiana tabacum)
全文: PDF (1569 KB)   HTML (1 KB) 
输出: BibTeX | EndNote (RIS)      
摘要 SKIP(ski-interacting protein)是一种RNA 代谢相关蛋白,在植物抵抗非生物胁迫伤害中起着很重要的调节作用,本研究克隆了高粱(Sorghum bicolor)SbSKIP基因,cDNA编码区全长1 485 bp,编码494个氨基酸;进化树分析表明,高粱SbSKIP基因编码的氨基酸序列与节节麦(Aegilops tauschii)相似性为52%;定量PCR分析表明,在模拟干旱条件下SbSKIP基因表达量上调,在干旱处理16 h后,表达量显著增加了2倍以上。构建植物表达载体pSH-SbSKIP,利用农杆菌(Agrobacterium tumefaciens)介导的叶盘法遗传转化烟草(Nicotiana tabacum),获得25个转基因植株。选取3个不同株系TP4、TP9和TP13,在不同浓度甘露醇模拟干旱条件下对种子发芽率和幼苗期进行抗旱性分析,结果发现,在300 mmol/L甘露醇处理10 d,种子的发芽率比野生型高60%以上,而且从苗期根的生长发现,野生型根的生长明显受到抑制。在自然条件下生长的烟草幼苗,用20% PEG 6000处理14 d,结果显示,转基因烟草正常生长,而野生型烟草大部分叶片黄化甚至出现萎蔫,其中转基因植株超氧化物歧化酶(super-oxide dismutase, SOD)的平均活性值比野生型高91.2%,H2O2和丙二醛(malondialdehyde, MDA)含量分别比野生型降低了42%和32.7%,可溶性糖(soluble sugar)含量达野生型的1.1倍。结果表明,SbSKIP基因表达提高了烟草植株的干旱耐受能力,本研究为进一步研究SbSKIP基因功能提供依据,同时该基因的克隆和功能分析可为创制转基因抗旱材料提供基础资料。
服务
把本文推荐给朋友
加入我的书架
加入引用管理器
E-mail Alert
RSS
作者相关文章
刘洋
姚新转
吕立堂
雷雨婷
代婷婷
赵德刚
关键词 SbSKIP基因同源序列比对qRT-PCR超氧化物歧化酶过氧化氢丙二醛可溶性糖    
Abstract:SKIP (ski-interacting protein) is a kind of metabolism related protein, playing an important role in regulating resistance to abiotic stress. In this study, the SbSKIP gene of Sorghum bicolor was cloned, whose cDNA encoding region included 1 485 bp and encoded 494 amino acids. Phylogenetic tree analysis showed that the SbSKIP gene of sorghum had the highest homology with Aegilops tauschii, and the second was Arabidopsis thaliana. According to phylogenetic tree analysis data, the amino acid sequence of SbSKIP had 52% similarity with Aegilops tauschii and 42% similarity with Populus trichocarpa (XP_002324443.2), Gossypium arboreum (KHG23995.1), Glycine max(ACY79501.1), Arabidopsis thaliana (NP_565151.1), respectively. Quantitative PCR analysis result showed that under the condition of simulated drought (16.5% PEG 6000), SbSKIP gene expression in leaves and roots did not changed significantly in 0 h, but it increased along with processing time. After 16 h of drought treatment, the expression significantly increased more than twice. And gene expression of sorghum leaf area was obviously higher than that of roots. We had constructed plant expression vector pSH-SbSKIP and transferred the vector to tabacco (Nicotiana tabacum) through leaf disc method transformation. And after resistance selection and PCR identification, we got a total of 25 strains of transgenic plants and harvested T0 generation seeds. TP4-4, TP4-9, TP4-13 had a high expression level of SbSKIP among transgenic plant. And the 3 different T0 plant strains TP4-4, TP4-9 and TP4-13 were used to analyze the seed germination and seedling drought tolerance at 0, 150 and 300 mmol/L mannitol simulated drought conditions, respectively. Results showed that the seed germination rate of transgenic plants were over 60% higher than that of wild type. As well as the root growth of wild type was obviously suppressed at the seedling stage under the 300 mmol/L mannitol treatment for 10 d. Under the natural condition with 20% PEG 6000, the seedlings which Long out of the 5~6 leaves were treated for 14 d. Results showed that the bottom of transgenic tobacco leaves margin slightly yellowed, and the bottom of wild type tobacco leaves mostly yellowed and even showed signs of wilting. And the average activity of super-oxide dismutase (SOD) of transgenic plants was 91.2% of that in the wild type, and H2O2, malondialdehyde (MDA) respectively than that of the wild type reduces the 42% and 32.7%, the content of soluble sugar was 1.1 times than that of wild type. Preliminary identification of SbSKIP gene expression to improve the drought tolerance of tobacco plants, and provides the basis for the further study SbSKIP gene function and the gene cloning and functional analysis and provide basic data for the creation of transgenic drought resistant materials.
Key wordsSbSKIP gene    Homologous sequence alignment    qRT-PCR    Super-oxide dismutase activity    Hydrogen peroxide content    Malondialdehyde content    Soluble sugar
收稿日期: 2016-05-15      出版日期: 2016-08-06
基金资助:国家自然科学基金
通讯作者: 赵德刚     E-mail: dgzhao@gzu.edu.cn
引用本文:   
刘洋 姚新转 吕立堂 雷雨婷 代婷婷 赵德刚. 高粱SbSKIP基因的克隆及其在烟草中的抗旱功能分析[J]. , 2016, 24(10): 1500-1511.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2016/V24/I10/1500
 
版权所有 © 2014 《农业生物技术学报》编辑部   京ICP备11035905号-3
地址:北京市海淀区圆明园西路2号中国农业大学生命科学楼1053室 邮编:100193
电话:010-62733684 传真:010-62731615 E-mail: nsjxb@cau.edu.cn