Isolation of the endosperm-specific LPAAT gene promoter from coconut (Cocos nucifera L.) and its functional analysis in transgenic rice plants.
As one of the key tropical crops, coconut (Cocos nucifera L.) is a member of the monocotyledonous family Aracaceae (Palmaceae). In this study, we amplified the upstream region of an endosperm-specific expression gene, Lysophosphatidyl acyltransferase (LPAAT), from the coconut genomic DNA by chromosome walking. In this sequence, we found several types of promoter-related elements including TATA-box, CAAT-box and Skn1-motif. In order to further examine its function, three different 5'-deletion fragments were inserted into pBI101.3, a plant expression vector harboring the LPAAT upstream sequence, leading to pBI101.3-L1, pBI101.3-L2 and pBI101.3-L3, respectively. We obtained transgenic plants of rice by Agrobacterium-mediated callus transformation and plant regeneration and detected the expression of gus gene by histochemical staining and fluorometric determination. We found that gus gene driven by the three deletion fragments was specifically expressed in the endosperm of rice seeds, but not in the empty vector of pBI101.3 and other tissues. The highest expression level of GUS was at 15 DAF in pBI101.3-L3 and pBI101.3-L2 transgenic lines, while the same level was detected at 10 DAF in pBI101.3-L1. The expression driven by the whole fragment was up to 1.76- and 2.8-fold higher than those driven by the -817 bp and -453 bp upstream fragments, and 10.7-fold higher than that driven by the vector without the promoter. Taken together, our results strongly suggest that these promoter fragments from coconut have a significant potential in genetically improving endosperm in main crops.
Xu
Li
L
Key Laboratory of Tropical Crops Germplasm Resources Utilization of Ministry of Agriculture, Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences, 571737, Danzhou, Hainan, China.
Ye
Rongjian
R
Zheng
Yusheng
Y
Wang
Zhekui
Z
Zhou
Peng
P
Lin
Yongjun
Y
Li
Dongdong
D
D000217:Acyltransferases / Q000235:genetics; D016960:Agrobacterium tumefaciens / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003059:Cocos / Q000201:enzymology / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics; D056625:Endosperm / Q000235:genetics*; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D012275:Oryza / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic
D018744:DNA, Plant; D000217:Acyltransferases