Cloning And Function Analysis Of GhSAMDC2/3/4 Gene In Cotton(Gossypium Hirsutum L)

Cotton is the world’s important economic crops,and it also one of pioneer crops against saline. Most planted cotton blong to upland cotton, accounting for more than 90% of the world’s total cotton output. Xinjian is a big province of cotton, maintaining the first in yield and total output first in the country for 20 years in a row. Cotton industry has become the important pillar of xinjiang economy and recorded in the main source of income for the farmers. However, due to various adversities such as global warming, water resource shortage, caused serious soil salinization, and it has heavily effect on cotton normal growth and development, and it also make cotton-food fight increasingly prominent. Numerous studies show that the response to abiotic stress is mainly dependent on stress perception, signal transduction related genes, stress response-related genes and metabolic pathways to active the expression of a serious of molecular networks. The traditional cotton breeding target pay more attention to yield form factors, fiber quality characteristics, and the disease and insect resistance, but few research focus on drought, salinity, heavy metal, extreme temperature adversity resistance.Many studies have shown that the increase of polyamine content can significantly improve resistance in plants. SAMDC is one of three key enzymes of polyamine synthesis, and it plays an important role in adversity resistance. SAMDC gene has been cloned in a variety of plants, and the study on a certain gene function. In this study we cloned SAMDC homology cloning genes from upland cotton, using the QRT-PCR technology analysis the homologous gene expression characteristics under different conditions, then select adversity advantage genes converse into model plant Arabidopsis thaliana and upland cotton, and related physiological studies to alter the expression levels of SAMDC gene, so as to regulate polyamines content, improve its resistant on abiotic stress, and provide a new way for plant stress resistance breeding.1. On the basis of the known Gh SAMDC1 gene, using electronic cloning combined with QT-PCR technique for upland cotton(Gossypium hirsutum L) S-adenosine methionine decarboxylase(S- adenosylmethionine decarboxylase, SAMDC) gene family three homologous genes, named Gh SAMDC2, Gh SAMDC3 and Gh SAMDC4 respectively. Sequence analysis showed that the gene m ORF is 1068 bp, 1110 bp and 1032 bp respectively, and encoding 355, 369, and 343 amino acids. Cluster analysis showed that Gh SAMDC2/3 and the cacao tree(Theobroma cacao) SAMDC gathered for a class, and Gh SAMDC2 and Gh SAMDC3 protein’s closest relative;Gh SAMDC4 and arabidopsis thaliana At SAMDC4 together for a class.Real-time fluorescent quantitative PCR analysis showed that Gh SAMDC2 expressed in stem is relatively high, and with the fiber development, expressing the increasing volume in the late fiber development express quantity highest; Gh SAMDC2/3/4 under different stress showed different patterns of expression, Gh SAMDC2 affected by low temperature and drought stress induced the most intense, Gh SAMDC3 response to salt stress significantly, Gh SAMDC4 induced by ABA.2. Transform p GWB17-Gh SAMDC2/3/4 overexpressing vector into the model plant arabidopsis thaliana respectively, kana filter to obtain pure fit and verified by PCR and QRT- PCR, all obtained positivetransgenic plants. Choose the high amount of gene expression to Na Cl stress strain.The results show that with 200 m M Na Cl treatment, seed germination rate of the third days is significantly higher than the wild type, which is 10.89 times that of the wild type; seed germination rate of the 7 days in arabidopsis seed germination rate is 1.34 times that of WT, indicated Gh SAMDC3 gene increase significantly the arabidopsis seed germination under salt stress. With the treatment of 200 m M Na Cl, wild type with and turn Gh SAMDC3 gene in arabidopsis seedlings, the results show that Gh SAMDC3 genes expressed in 48 h a significant rise in quantity; Using high pressure liquid chromatography(HPLC) analysis polyamine content in wild type and transgenic arabidopsis under high salinity, we found that the transgenic arabidopsis central Spd content increased significantly, and the transgenic plants of polyamine metabolism is also active.3. Constructed p GWB17-Gh SAMDC2/3/4 plant expression vector respectively, using the method of agrobacterium infect cotton hypocotyl transformation YZ-1 and xinluzao 33, successive transfer culture and Kana screening for resistant plants. Extraction resistance of cotton genomic DNA for PCR verification, in addition to p GWB17-Gh SAMDC4 for embryonic callus, the others all had positive transgenic cotton plants.In addition to observe turn p GWB17-Gh SAMDC3 and turn light of embryonic callus growth situation, found that genetically modified for embryonic callus growth was significantly higher than that of no load growth.