Constructing The Subtractive CDNA Library About Gibberellin And Ethylene Treated In Abscission Zone Of Gossypium Hirsutum

Cotton is one of the most important economic crops in the world and cotton fiber is the major natural fiber source for textile industry. Cotton is also an important strategic material. With the rapid development of the textile industry, demand of cotton is increasing annually. At present, cotton varieties have improved to a large extent regarding yield and quality, but the phenomenon of abortion and abscission of young fruit is still very common. To estimate the role of hormone in abortion and abscission of young fruits, isolation and analyses of genes expressed preferentially in cotton abscission zone will be helpful in further understanding of this mechanism at genomic level and ultimately can do well to improve cotton fiber yield. In this research, upland cotton genetic standard line TM-1 was used to construct the SSH library. The main results obtained are as follows:1. Two hormones gibberellin (GA₃) and ethephon (CEPA) were designed and used in three different concentrations i.e. (GA₃: 10ppm, 50ppm, 100ppm; CEPA:10ppm, 250ppm, 1000ppm). This application affected the abscission zone of flowering cotton fruits. Abscission rates were investigated after seven days. In 2006, the abscission rates of cotton fruits, which were treated with the concentration of 10ppm and 50ppm gibberellin solutions, had significant difference with the control while the abscission rates of cotton fruits which were treated with 100ppm gibberellin solutions had no significant difference with the control. In 2007, all gibberellin solutions of 10ppm, 50ppm and 100ppm concentrations had significant difference with the control. As the ethephon treatment, in 2006, 250ppm and 1000ppm had significant difference but 10ppm had no significant difference with the control. In 2007, all three concentrations of ethephon had significant difference with the control. The treatments of each gibberellin solution had significant difference in each year, which showed that climate had great effect on the performance of gibberellin.2. The application of 50ppm gibberellin had distinct effect on protected cotton fruits while 250ppm ethephon had sigificantly effect on accelerating cotton fruits abscission. Two cDNA libraries were constructed from upland cotton genetic standard line TM-1 by suppression subtractive hybridization (SSH). The cDNA from abscission zones, which were treated with 50ppm gibberellin solution and 250ppm ethephon solution, were used as testers of the two SSH libraries while cDNA from abscission zones which were treated with water were used as the same driver. Through differential screening, 80 clones in both libraries were sequenced. 28 unique sequences were found in two SSH libraries by sequence analysis. In EST, the known functional sequences were analyzed involved in translation, signal transduction, metabolism, transcription, energy metabolism, protein synthesis, stress defense and about 29% ribosomal genes, which were related to translation. This showed that there was a great deal of proteins synthesis after hormonal treatment in abscission zone cell. 14% genes were related to signal transduction and metabolism while 11 % were related to transcription.3. For the space-time expression mode of genes, which specifically expressed in abscission zone cell, 10 genes from these libraries were analyzed in roots, stems, leaves and abscission zones by RT-PCR. For more accurate results, abscission zones were treated with 50ppm gibberellin solutions and analyzed after 0.5h, 2h, 4h, 8h and 12h by RT-PCR. Chalcone synthase gene (CHS) was expressed in every tissue and every phase, but its expression was stronger in abscission zone cell, 2 hours after the application of gibberellin solutions. Cytosolic cyclophilin gene (ROC3) was expressed stronger in roots and stems than leaves and abscission zone cells. It did not express in abscission zone cells after 2 hours of gibberellin solution treatment. Translationally controlled tumor protein (TCTP) expressed stronger in roots and stems, but did not express in leaves. It expressed weaker in abscission zone cell after 0.5 hours and became the most stronger after 12 hours of gibberellin solution treatment.