Expression and screening of anther specific promoters

• Main Authors: Fang Li-Wen, 方麗雯
• Other Authors: Chen Liang-Jwu
• Format: Others
• Language: zh-TW
• Published: 1994
• Online Access: http://ndltd.ncl.edu.tw/handle/01998648599849639674

碩士 === 國立中興大學 === 分子生物研究所 === 82 === According to the sequence information for the promoter region of TA29, a pair of primers AS29A and AS28B that could specifically amplify this promoter region was synthesized and a DNA fragment of 366 bp containing the promoter region of TA29 was then amplified from tobacco genomic DNA by polymerase chain reaction ( PCR ). To test the tissue specific function of this PCR amplified promoter fragment, a chimeric gene construct pBIA129 that contains this 366 bps promoter region fused to GUS reporter gene was obtained. Through agrobacteria transformation system, five kanamycin resistant tobacco plants were screened and the existence of this chimeric gene for each kanamycin resistant tobacco plant was demonstrated by PCR and Southern analysis. Histochemical analysis demonstrated that the GUS enzyme activity was expressed only in the tapetal cells of the third stage of tobacco anther. The results indicated that the PCR amplified promoter fragment could program the temporal and spatial expression of GUS gene in transgenic tobaccos. A cDNA clone BA112 that derived from the mRNA of Brassic anther was previously demonstrated to be tapetal specific by Northern blot and in situ hybridization analysis. To search for this tapetal specific gene, a genomic library of Brassic napus was screened with BA112 cDNA probe. After screening for about 2.5×105 plaques, there were 36 clones showed positively hybridized to BA112 cDNA but only one clone gBA05 showed strongly hybridized to an oligonucleotide probe BA19, an oligonucleotide probe locates at the 5' end of BA112.