Molecular Cloning and Promoter Activity Analysis of Auxin Influx Carrier Genes from Momordica charantia

• Main Authors: Min-yi Chung, 鐘敏禕
• Other Authors: Yi-Yin Do
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/31197172834107514784

碩士 === 國立臺灣大學 === 園藝學研究所 === 93 === To obtain auxin influx carrier gene McLAX1 from bitter gourd（Momordica charantia）, pMAIC11 cDNA was used as the probe for screening the genomic library. Based on restriction map and Southern analysis, λMAIC1 was delegated to be subcloned and sequenced. The McLAX1 gene in λMAIC1 is consisted of 4,346 base pairs with eight exons and seven introns. The splice junctions between introns and extrons of McLAX1 are according to the GT/AG rule except the first intron. The start codon locates in the first exon. McLAX1 encodes a polypeptide containing 469 amino acids with a calculated molecular mass of 52.70 kDa and a predicted isoelectric point of 8.28. The identity of nucleotide sequences and amino acid sequences between λMAIC1 and pMAIC11 cDNA is 99.3% and 99.1%, respectively, therefore the gene in λMAIC1 was supposed to be an allele of McLAX1. The homology of the amino acid sequences of McLAX1 with AUX1 and AUX1-like proteins is 61%∼82%. According to the result of analysis for upstream promoter sequence of McLAX1, several predicted responsive elements related to auxin, abscisic acid, Methyl jasmonate, salicylic acid, ethylene, light, low-temperature, and wounding were found. To analyze the promoter activity of McLAX, the plasmid 20P12 containing McLAX2::GUS had been constructed. The expression plasmid was transformed into petal discs of Phalaenopsis, young leaf, mature leaf, male flower, and female flower of bitter gourd via particle bombardment. The result of transient expression analysis indicated that the promoter activity of McLAX2 in female flower of bitter gourd is higher than CaMV 35S promoter. The stable expression analysis in Arabidopsis and tobacco was performed by Agrobacterium-mediated transformation. Southern analysis had verified the transfomants. The results of GUS histochemical staining indicated that the promoter activity of McLAX2 exists in the leaf, inflorescence, stamen, pistil and pod of Arabidopsis, and in the leaf and root of tobacco.