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园艺学报 ›› 2019, Vol. 46 ›› Issue (1): 25-36.doi: 10.16420/j.issn.0513-353x.2017-0786

• 研究论文 • 上一篇    下一篇

梨糖转运相关基因PbTMT4启动子克隆及功能分析

程寅胜1,2,*,陈健秋1,*,陈 丹1,吕佳红1,张 俊1,张绍铃1,伍 涛2,**,张虎平1,**   

  1. (1南京农业大学园艺学院,南京 210095;2湖北省农业科学院果树茶叶研究所,武汉 430064)
  • 出版日期:2019-01-25 发布日期:2019-01-25

Cloning and Functional Analysis of the Promoter of PbTMT4 Gene Related Sugar Transport in Pear

CHENG Yinsheng1,2,*,CHEN Jianqiu1,*,CHEN Dan1,Lü Jiahong1,ZHANG Jun1,ZHANG Shaoling1,WU Tao2,**,and ZHANG Huping1,**   

  1. (1College of Horticulture,Nanjing Agricultural University,Nanjing 210095,China;2Institute of Fruit Tree Tea,Hubei Academy of Agricultural Sciences,Wuhan 430064,China)
  • Online:2019-01-25 Published:2019-01-25

摘要:

以‘砀山酥梨’(Pyrus bretschneideri‘Dangshan Suli’)为材料,利用梨基因组数据库,通过PCR获得了糖转运相关基因PbTMT4(Pbr032130.1)2 211 bp的CDS序列及其编码起始位点上游长度为1 220 bp的启动子序列。使用农杆菌介导法将PbTMT4导入拟南芥,与野生型对照植株相比,转基因拟南芥植株的生长速度更快,抽薹、开花时间更早,叶片糖积累量更高。生物信息学分析表明,该启动子中含有多个与逆境应答、激素信号和光信号相关的顺式作用元件。为进一步分析PbTMT4启动子功能,构建了该启动子与GUS基因融合的植物表达载体并转化拟南芥。对T3代转基因拟南芥各组织进行GUS活性染色和半定量分析,发现GUS基因在根、茎、叶、花和果荚中均有表达,NaCl、干旱、GA3、MeJA以及光照处理均能一定程度上提高转基因拟南芥中GUS基因的转录水平。逆境处理发现,PbTMT4转基因株系较野生型植株受到的伤害小。研究结果初步表明,PbTMT4可促进转基因拟南芥发育并提高糖积累量,可能在抗非生物胁迫中起重要的调控作用。

关键词: 梨, 启动子, 糖, 液泡膜单糖转运蛋白(TMT), GUS基因

Abstract:

The 2 211 bp CDS sequence of PbTMT4 gene(Pbr032130.1)and the promoter sequence of 1 220 bp upstream of the transcription initiation site were obtained using Pyrus bretschneideri‘Dangshan Suli’as the material based on the pear genome database. When the PbTMT4 gene was introduced into Arabidopsis by Agrobacterium-mediated method,the growth rate of transgenic Arabidopsis was faster,and the flowering time was earlier than the wild type. At the same time,the amount of sugar accumulation in transgenic Arabidopsis was significantly higher than that in wild type. Bioinformatics analysis indicated that the promoter contains multiple cis-acting elements associated with stress response,hormone signals and light signals. To further analyze the promoter function of PbTMT4 gene,a promoter-reporter vector was constructed and transformed into Arabidopsis. Analysis of T3 transgenic Arabidopsis tissues with histochemical staining and semi-quantitative analysis showed that GUS gene was expressed in roots,stems,leaves,flowers and fruit pod. And sodium chloride(NaCl),drought,gibberellin(GA3),methyl jasmonate(MeJA)and light intensity can induce the transcription level of GUS gene in Arabidopsis to some extent. Stress treatment revealed that the PbTMT4 gene overexpression transgenic line had less damage than the wild type plants. The results of the study preliminarily suggest that PbTMT4 gene can promote the development of transgenic plants and increase sugar accumulation,and may play an important regulatory role in the abiotic stress.

Key words: Pyrus bretschneideri, promoter, sugar, tonoplast monosaccharide transporter, GUS gene

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