| Dendrobium (Dedrobium. Pink BEAUTY 'Queen'×Dedrobium. Pink GEM 'ELEGANCE') were treated under low temperature of 10±1℃/23±1℃for 12h/12h (night/day). The effect of chilling to flower buds inducement and differentiation was studied; The content of carbohydrate, phytohormones and activities of EST, POD, SOD were measured; An MADS-box gene that may related to flowering was cloned, and some analyses about this gene were done. The results were as follows:(1) The Dendrobium finished vernalization in about 40 days. The induction rate of buds was 98%, and all of them were floral bud, the rate of single node with 2-3 buds was high, attaining 80%, each one contained more than 14 flowers. The induction rate of buds for 10 days, 20 days and 30days were lower, and most of them were leaf buds.(2) The content of carbohydrate was variety in low temperature process. In the former 20 days, the dissoluble sugar content increased notably, then decreased. and 30 days later, it increased again, attained the peak of 179.03 mg/g on 40th day. In the former 20 days, the starch content decreased gradually, 30 days later, it increased notably, attained the peak 632.96mg/g on the 40th day. The CK almost kept constant. The variety trend of starch and dissoluble sugar was just opposite, which showed that the increasement of sugar may result from the decomposition of the starch.(3) The Z variety presented as a pair of curves. It got two peaks on 20th day and 35th day; IAA increased gradually, got the peak(about 60 p mol/g FW) on 20th day, and maintained to 35th day, then it declined; GA3 showed an increaseing trend, got the highest on 30th day, it declined gradually after 45th day; ABA increased rapidly at the beginning, got the peak (51 pmol/g FW) on 10th day, declined later, and kept the lower level. The IAA/Z increased rapidly between 5th day and 35th day. Declined quickly after 35th day. All of these showed that phytohormone had some relationship with flowering.(4) There were some changes in EST, SOD, POD activities during the process. Some special EST isozyme bands disappeared or appeared. On 30th day, a band, Rf=0.609, appeared. There were some obvious changes in SOD. A band, Rf=0.40, disappeared on 30th day. The above showed that isozyme had some relationship with vernalization.(5) Many studies showed that most of genes related to floral differentiation and development belonged to MADS box gene family. According to the special 3' end sequence of plant cDNA, design a peculiar primer (primer2: 5'-GACTC GAGTC GATCG ATTTT TTTTT TTTTT TTT-3'); Basing on reported conservative sequence of MADS, design a degenerate primer (primer1:5'-GATCA AG(A/C)G(G/C) ATCGA GAA-3'), a MADS box cDNA fragment of 895 bps was cloned, named D-MADS. It contains a ORF, coding for 246 amino acids. Multiple alignment about this sequence showed that D-MADS had 97% similarity with DthyrFL1 cloned from Dendrobium thyrsiflorum. Semi-quantitative RT-PCR analysis of D-MADS showed that it was expressed only in flower bud, and its expression increased with the development of flower bud. All of these showed that D-MADS had some relationship with flower bud differentiation of Dendrobium.
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