| As a member of Citcurbiteae, bitter melon is not only a vegetable crop but also an important medical herb in China and East Asia. These years, phytochemists have isolated a number of potential medical components from this plant, such as the ribosome inactivating protein (RIP) , MAP30 (Momordica anti-Hiv protein), which suppresses HIV (human immunodeficiency virus) activity, momordica charantia lectin (MCL), momordica charantia inhibitor (MCI) and momordicoside A and B, both of which can inhibit tumor growth. However, few materials related to tissue culture and the in vitro regeneration system has not been established yet. These were harmful to the conservation and propagation of rare varieties. Here we reported the establishment of in vitro regeneration system of bitter melon and developed a new way for improving the varieties of bitter melon and laid the base for establishment of genetic transformation system of bitter melon. At the same time, we cloned a flower specific MADS-box gene from bitter melon. The results as following:1 Explants of bitter melon were easily induced to grow callus. The frequency of callus formation was about 90.0%. The kind and proportion of phytohormones had not noticeably influenced on callus formation of different vegetative organs. The callus of young root, hypocotyls and cotyledon grew faster than those of epicotyl and young leaf. The kind of organs and phytohormones did not make remarkable difference on the time when the callus were produced. In general, explants began to swell in four days after being inoculated on media.2. Explants of bitter melon could produce three types of callus: green, yellow green and fragile yellow callus. In the process of callus differentiating adventitious bud, the kind, proportion and quantity of phytohormone and the type of callus were very important. Theadventitious buds had been induced successfully on medium (MS+6-BA 4.0 mgl"l+KT 2.0 mgl-1) by yellow green callus. The frequency was about 66.7%. Yellow callus had notIXdifferentiated adventitious buds even under the most suitable conditions.3. The medium (MS + ZT 5.0 mgl"1 +KT 0.5 mgl'1) was suitable for the proliferation of bud; the coefficient of proliferation was about 5-6.4. The media (1/2 MS + ZT 0.02 mgl"1 or 1/2 MS) were suitable for rooting in vitro of shoot, the shoot on them could produce new young root 6-7 in three weeks.5. After plantation test, the survival rate of tube plantlets was about 70%; their characteristics were the same as those from seed by field test.6. The flower specific gene of bitter melon, named BAG (GeneBank accession number AY 178837), was cloned from flower bud. A full-length cDNA of lOOlbp contained a complete open reading frame of 228 amino acids, 5' and 3' nontranslated regions and a long ploy (A) tail. The predicted amino acids sequence of BAG showed 95% sequence identity to MADS-box protein CUM10 [Cucwnis sativus], 93% to CAG1 [Cucumis sativjts], 84% to MADS box protein GHMADS-2 [Gossipier hirsutum], 83% to MADS-box protein 5 [Vitas vinifera], 76% to MADS-box protein [Mains x domestica], 72% to MADS-box protein AGL11 [Arabidopsis thaliana], respectively. A comparison between the putative translation product of BAG and the other MADS-box related proteins demonstrated that the highest conservation domain is found within the putative MADS-box DNA-binding domain. A second domain, the K-box involved in dimerization, which is conserved among the MADS-box proteins, is also found. Genomic Southern blot analysis suggested the existence of at least two or three copies of the gene. Northern blot and reverse transcriptase polymerase chain reaction indicated that the BAG cDNA is carpels and stamens specific in bitter melon.7. One pairs of specific nested PCR primers were designed according to the pubilished sequence of MADS-box gene "BAG" in bitter melon. The promoter of Mads-box gene (BAGP) with a length of 664 bp was cloned by DNA walking technology. The sequence of BAGP was analyzed by software DNATools 5.1 and CallMat, which... |
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