Cloning and analysis of the cDNA clones encoding the auxin efflux carriers in bitter gourd

• Main Authors: Yi-Li Li, 李宜麗
• Other Authors: Yi-Yin Do
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/55587895560126228795

碩士 === 國立臺灣大學 === 園藝學研究所 === 88 === To understand the molecular mechanism of response to auxin in plants, three cDNAs encoding auxin efflux carrier were isolated from bitter gourd ‘Moon Shine’. PIN1 and PIN2 gene fragments synthesized by polymerase chain reaction using Arabidopsis genomic DNA as template were used for screening the cDNA library constructed by poly(A)+ RNA from fruit of bitter gourd. The cDNA pMAEC28 (MCm-AEC1) sequence consists of 2,924 base pairs with an intron and encodes an open reading frame of 607 amino acids with a calculated molecular mass of 66 kDa and a predicted pI of 9.26. The cDNA pMAEC43 (MCm-AEC2) sequence consists of 2,270 base pairs encoding a polypeptide of 608 amino acids with a calculated molecular mass of 66 kDa and a predicted pI of 9.4. The cDNA pMAEC93 (MCm-AEC3) sequence consists of 2,511 base pairs and encodes an open reading frame of 635 amino acids with a calculated molecular mass of 68 kDa and a predicted pI of 8.59. All of the amino acid sequences of three encoded proteins show homology to Arabidopsis auxin efflux carrier PIN genes between 60 to 70% and possess 8 or 10 putative transmembrane domains interrupted by a central hydrophilic loop. According to the results of Southern analysis using specific probes from cDNA pMAEC43 and pMAEC93, these genes belong to single or low-copy gene family. Three or more related auxin efflux carrier genes exist in genome of bitter gourd. On the other hand, Northern blot analysis indicates that both MCm-AEC2 and MCm-AEC3 were highly expressed in flowers and early development stage of fruit. Accumulation of MCm-AEC2 or MCm-AEC3 mRNA was induced after bitter gourd fruit treated with 10-5 M auxin indoleacetic acid for 30 minutes or 0.1 mL/L exogenous ethylene for 24 hours. Gene expression was repressed by higher concentration of ethylene as 10mL/L. Total RNA isolated from fruit sections soaked in other kinds of auxin or plant hormones was performed in Northern hybridization.