| The development of Bt cottons has greatly decreased the yield loss of cotton production caused by cotton boll worm and the utility of chemical insecticides for boll worm-protection have been decreasing these years. However, the anti-insect activity of the Bt cottons were found to be higher at the early developmental stages of cotton and decreases thereafter. A possible solution to this problem may be the utility of new promoters which can drive the insecticidal gene expressing in the developing cotton bolls instead of the constitutive CaMV35S promoter. In the present work, we here report the cloning of several genes/gene fragments and their expression patterns with respect to the reproductive cotton organ.To obtain RNA of high-quality of cotton, we have first compared the RNA extraction efficiency of phenol-SDS method and the proteinase K-hot borate method. The results showed both methods gave a similar yield and integrity of RNA, but the latter method gave a better removal of impurities such as proteins. RNA solution thus obtained using the hot borate method behaves to be colorless, suggesting that the oxidation of cotton phenols was inhibited perfectly.A cDNA library with a tittering of 7+106 pfu/mL was constructed basing on the cotton boll shell (10 days post anthesis, dpa) RNA obtained above. The recombinant ratio was found to be ca. 90% by blue-white counting.The abundance level of gene expressing in 10-dpa cotton boll shell were analyzed using PCR method. Two cDNA cloned were obtained, one encodes a fragment of Rubisco activase 2, while the other one remains unclear. Further Northern blot analysis revealed that the former gene/fragment has relatively strong expression in the green tissues such as boll shell, stem and the expanding leaf, while no expression or only weak expression has been detected in the fiber, flower and root of cotton. The latter gene/fragment was found to be expressed strongly in the leaf, boll shell, fiber and root of cotton, while no expression signal or only weak signal could be detected in the flower and stem of cotton. The expression gave the strongest signal in the leaf, with next the boll, root and the fiber.In the present work, 17 cDNA fragments were cloned from the cotton boll shell(10dpa) and flower RNA by RT-PCR technology using degenerate primers designed according to the conservative domain of MADS-box protein family. To classify the genes represented by the cDNA frgments obtained, the phylogenetic tree was constructed using phylip 3.5. The results revealed that 5 of the 17 fragments belong to the B function gene family, 11 of the 17 fragments belong to C function gene subfamily, and the other one exclusive according to the classic ABC model of the flower development. Obviously, with the cloning of the cDNA fragments, useful information may be obtained by further study on comparison with the known features of MADS-box genes among the same subfamily, and such information may be useful to the cloning of the full-length cDNAs and their promoters. Moreover, we have tried to clone the full length cDNA of some fragments mentioned above using 5'RACE technology, and two cDNA clones were obtained. One of them encodes a lipid transfer precursor protein (Itpp), while the other one showed high homology to may ESTs of a 7-10 dpa wood cotton fiber cDNA library. Northern blot analysis showed that Itpp was preferentially expressed in the cotton fiber (10 dpa), and only very weak expression could be detected in the cotton boll and flower. This suggests that the gene may have close relative to the fiber development of cotton.
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