Cloning and functional analysis of the chitinase gene promoter in peanut.
Chitinase is an important pathogenesis-related protein in plants, and it can accumulate when induced by salicylic acid (SA) or other elicitors. Here, we found that chitinase mRNA levels were 4.5-times greater when peanut seedlings were sprayed with 1.5 mM SA, as compared to water. The upstream promoter sequence of the chitinase gene was cloned by TAIL-PCR and the potential cis-regulatory elements in this promoter were predicted by the cis-element databases PLACE and plantCARE. Elements in the promoter related to SA induction and disease resistance response included AS-1, GT1-motif, GRWAAW, TGTCA, W-box, and WB-box. The full-length promoter (P) and a series of 5'-deleted promoters (P1-P5) were cloned and then substituted for the 35S promoter of pCAMBIA1301-xylA, which carries the xylose isomerase gene as the selectable marker and GUS as the reporter gene. Six plant expression vectors (pCAMBIA1301-xylA-P-pCAMBIA1301-xylA-P5) were obtained. The six expression vectors were then transferred into onion epidermal cells and peanut plants by Agrobacterium-mediated transformation. Both the full-length and deleted promoters resulted in GUS staining of the onion epidermis cells when induced by SA. In onion epidermis cells, GUS enzyme activity was greater after SA induction. In transgenic peanut plants, GUS mRNA levels were greater after SA induction. Consideration of the cis-regulatory elements predicted by PLACE and plantCARE suggested that AS-1, GRWAAW, and W-box are positive regulatory elements in P2 and P3 and that GT1-motif and TGTCA are negative regulatory elements between P and P2.
Key Lab of Plant Biotechnology in Universities of Shandong Province, Qingdao Key Lab of Germplasm Innovation and Application of Major Crops, College of Life Science, Qingdao Agricultural University, Qingdao, Shandong, China.
X L
XL
Chen
Key Lab of Plant Biotechnology in Universities of Shandong Province, Qingdao Key Lab of Germplasm Innovation and Application of Major Crops, College of Life Science, Qingdao Agricultural University, Qingdao, Shandong, China.
R T
RT
Song
Key Lab of Plant Biotechnology in Universities of Shandong Province, Qingdao Key Lab of Germplasm Innovation and Application of Major Crops, College of Life Science, Qingdao Agricultural University, Qingdao, Shandong, China.
M Y
MY
Yu
Key Lab of Plant Biotechnology in Universities of Shandong Province, Qingdao Key Lab of Germplasm Innovation and Application of Major Crops, College of Life Science, Qingdao Agricultural University, Qingdao, Shandong, China.
J M
JM
Sui
Key Lab of Plant Biotechnology in Universities of Shandong Province, Qingdao Key Lab of Germplasm Innovation and Application of Major Crops, College of Life Science, Qingdao Agricultural University, Qingdao, Shandong, China.
J S
JS
Wang
Key Lab of Plant Biotechnology in Universities of Shandong Province, Qingdao Key Lab of Germplasm Innovation and Application of Major Crops, College of Life Science, Qingdao Agricultural University, Qingdao, Shandong, China.
L X
LX
Qiao
D010367:Arachis / Q000201:enzymology* / Q000235:genetics*; D002688:Chitinases / Q000235:genetics*; D018506:Gene Expression Regulation, Plant / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*
D010940:Plant Proteins; D002688:Chitinases