花生β-1,3-葡聚糖酶基因启动子功能区的缺失分析  

Deletion Analysis on The Peanut(Arachis hypogaea) Promoter of β-1,3-glucanase Function Region

作　　者：宋汝涛 王合春[1] 于明洋[1] 孙明明[1] 郭悦[1] 隋炯明[1] 王晶珊[1] 乔利仙[1] 

机构地区：[1]青岛农业大学生命科学学院/山东省高校植物生物技术重点实验室,青岛266109

出　　处：《农业生物技术学报》2016年第6期837-846,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31471542和No.31301356);山东省科技发展计划(No.2014GNC110002)

摘　　要：花生β-1,3-葡聚糖酶基因启动子(Arachis hypogaea promoter ofβ-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Plant CARE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p CAMBIA1301-xyl A载体中,构建以木糖异构酶基因(xylose isomerase gene,xyl A)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p CAMBIA1301-xyl A-Glu-P^p CAMBIA1301-xyl A-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-P33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AhGlu-P3内部存在的RAV1AAT、MYBCOREATCYCB1和INRNTPSADB为对SA响应的正调控元件;在AhGlu-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�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

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 

分 类 号：Q786[生物学—分子生物学]