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.
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