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2025年8月15日 星期五
  2016, Vol. 24 Issue (6): 837-846    
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
花生β-1,3-葡聚糖酶基因启动子功能区的缺失分析
宋汝涛1,王合春1,于明洋1,孙明明2,郭悦2,隋炯明2,王晶珊3,乔利仙4
1. 青岛农业大学生命科学学院
2. 青岛农业大学 生命科学学院
3.
4. 青岛农业大学
Deletion Analysis on The Peanut (Arachis hypogaea) Promoter of β-1,3-glucanase Function Region
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摘要 花生β-1,3-葡聚糖酶基因启动子(Arachis hypogaea promoter of β-1,3-glucanase, Ah-Glu-Pro)属于诱导型的启动子。为分析该启动子序列中对外源信号分子响应的重要顺式调控元件,利用交错式热不对称PCR(thermal asymmetric interlaced polymerase chain reaction, TAIL-PCR)方法扩增得到长度为970 bp的Ah-Glu-Pro序列。PLACE 和 PlantCARE在线预测结果表明,该启动子序列中含有对病原菌及水杨酸(salicylic acid, SA)响应的顺式调控元件,如GRWAAW、GT1-motif、W-box、RAV1AAT、INRNTPSADB、AMMORESIVDCRNIA1和BIHD1OS。根据预测结果,在5'端设计5个正向引物Glu-F、Glu-P4、Glu-P3、Glu-P2和Glu-P1,3'端设计一个反向引物Glu-R,5个引物对扩增得到该启动子序列Ah-Glu-P以及5'端4个缺失片段Ah-Glu-P4~Ah-Glu-P1,长度分别为931、767、650、376和217 bp。将这5个片段分别克隆到pCAMBIA1301-xylA载体中,构建以木糖异构酶基因(xylose isomerase gene, xylA)作为安全筛选标记、以β-葡萄糖苷酸酶基因(β-glucuronidase gene, GUS)作为报告基因的相应5个植物表达载体pCAMBIA1301-xylA-Glu-P~pCAMBIA1301-xylA-Glu-P1。将这5个表达载体分别转化洋葱(Allium cepa)表皮细胞,进行GUS蛋白组织化学染色及GUS酶活性检测,结果表明,经SA诱导后,转入Ah-Glu-P~Ah-Glu-P3 3个载体的洋葱表皮细胞中的GUS酶活性分别提高1.45、2.16和1.27倍,转入Ah-Glu-P2~Ah-Glu-P1载体的洋葱表皮细胞在SA诱导前后GUS酶活性无明显差别。结合软件预测结果推测,在启动子Ah-Glu-P、Ah-Glu-P4和Ah-Glu-P3内部存在的RAV1AAT、MYBCOREATCYCB1和INRNTPSADB为对SA响应的正调控元件;在Ah-Glu-P~Ah-Glu-P4之间存在的GT1-motif和AMMORESIVDCRNIA1为对SA响应的负调控元件。对这些重要顺式调控元件功能的进一步确认将为通过调控实现花生内源β-1,3-葡聚糖酶基因的高效表达、提高花生(Arachis hypogaea)的抗病性、以及在花生遗传转化过程中特异诱导表达启动子的有效利用提供理论依据。
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宋汝涛
王合春
于明洋
孙明明
郭悦
隋炯明
王晶珊
乔利仙
关键词 花生β-1,3-葡聚糖酶基因诱导型启动子顺式作用元件    
Abstract:The plant β-1,3-glucanase belongs to one of the important pathogenesis-related proteins (PR-proteins), which could be accumulated when induced by exogenous signal molecule. The Arachis hypogaea promoter of β-1,3-glucanase (Ah-Glu-Pro) has been proved to be an inducible promoter, and there might contain some cis-regulatory elements which could respond to exogenous signal molecule. In order to clarify the promoter's function and further analyze and confirm the key cis-regulatory elements within the promoter, thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was conducted with peanut genomic DNA as template, and a 970 bp fragment was obtained successfully, named by Ah-Glu-Pro. Online function prediction by PLACE and PlantCARE showed that Ah-Glu-Pro contained some typical cis-elements, such as TATA box and CAAT box, some cis-acting elements which could respond to pathogens and salicylic acid (SA), such as GRWAAW, GT1-motif, W-box, RAV1AAT, INRNTPSADB, AMMORESIVDCRNIA1 and BIHD1OS. Five forward primers Glu-F, Glu-P4, Glu-P3, Glu-P2 and Glu-P1 at the 5' end with the XbaⅠrestriction enzyme cutting site and one reverse primer Glu-R at the 3' end with the BglⅡrestriction enzyme cutting site were designed and synthesized according to predictive results. PCR was conducted with Glu-F/ Glu-R, Glu-P4/Glu-R, Glu-P3/Glu-R, Glu-P2/Glu-R and Glu-P1/Glu-R, and Ah-Glu-P and 4 deleted fragments Ah-Glu-P4~Ah-Glu-P1 with the length of 931, 767, 650, 376 and 217 bp were obtained successfully, respectively. The 5 fragments were then transferred to plasmid pCAMBIA1301-xylA, replacing the Cauliflower mosaic virus 35S promoter (CaMV35S), and 5 corresponding plant expression vectors pCAMBIA1301-xylA-Glu-P~ pCAMBIA1301-xylA-Glu-P1 with xylose isomerase gene (xylA) as safe maker and with β-glucuronidase gene (GUS) as reporter gene were constructed successfully. These plant expression vectors were then transformed to Agrobacterium tumefaciens EHA105 using freezing and thawing method, and further transformed to onion (Allium cepa) epidermal cells by Agrobacterium mediated transformation. GUS histochemical staining and GUS enzyme activity in these transgenic onions were detected. GUS staining on the onion epidermal cells transformed by Ah-Glu-Pro showed that the cells appeared blue when were induced by 5.0 mmol/L SA, while appeared light blue without induction by SA. Those onion epidermal cells transformed by pCAMBIA1301-xylA-Glu-P4~pCAMBIA1301-xylA-Glu-P1 appeared blue when induced by 5.0 mmol/L SA. Those onion epidermal cells transformed by Ah-Glu-P~Ah-Glu-P4 appeared much deep blue than those cells transformed by Ah-Glu-P3~Ah-Glu-P1. GUS enzyme activity in onions transformed by Ah-Glu-P~Ah-Glu-P3 were increased of 1.45, 2.16 and 1.27 times, respectively, after SA induction than that without SA induction. GUS enzyme activity in onions transformed by Ah-Glu-P2~Ah-Glu-P1 had no significant difference between with and without SA induction. Consideration of the predictive cis-regulatory elements showed that RAV1AAT, MYBCOREATCYCB1 and INRNTPSADB within Ah-Glu-P, Ah-Glu-P4 and Ah-Glu-P3 might be positive cis-elements, and GT1-motif and AMMORESIVDCRNIA1 between Ah-Glu-P and Ah-Glu-P4 might be negative cis-elements. The further function confirmation on these cis-regulatory elements will provide theoretical basis for regulating the peanut endogenous β-1,3-glucanase gene efficient expression, improving the disease resistance of peanut, and effective utilization of inducible promoter in the process of peanut genetic transformation.
Key wordsPeanut    β-1, 3-glucanase gene    Inducible promoter    Cis-elements
收稿日期: 2015-12-03      出版日期: 2016-05-06
基金资助:花生耐盐突变体M34耐盐相关基因的克隆及功能分析;花生小GTP结合蛋白基因Ahrab7提高耐盐性的分子机理研究;花生耐盐种质创新利用及相关基因挖掘
通讯作者: 乔利仙     E-mail: lxqiao73@163.com
引用本文:   
宋汝涛 王合春 于明洋 孙明明 郭悦 隋炯明 王晶珊 乔利仙. 花生β-1,3-葡聚糖酶基因启动子功能区的缺失分析 [J]. , 2016, 24(6): 837-846.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2016/V24/I6/837
 
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