Sps Cloning Of Sucrose Phosphate Synthase And Cotton Genetic Transformation Research

Cotton is an important economical crop in China and the world. Cotton fiber is an excellent textile raw material because it is a natural fiber with low cost, high production capacity and predominant thermal properties. With the development of the textile industry, higher quality fiber is needed for the new textile technology. In recent years, researchers have been obtained many cotton varieties by cross breeding. But the crossing breeding always develops slowly and need long periods because of lack good quality cotton cultivar. As a result, it is important to improve cotton fiber quality and the resistance by genetic engineering methodsSucrose phosphate synthase can catalyze the UDPG and fructose 6-phosphate generated 6-phosphate sucrose and UDP. It is a key enzyme for sugar metabolism. It also can improve plants endurance to kinds of stresses treatment. Meanwhile the SPS can affect the development of secondary cell wall and cellulose synthesis. In recent years, researchers also found it has close relation with the flower development. In this research, we cloned AtSPS5.1 by RT-PCR and the GhSPS1 by gene-walking and over-lap PCR. Then we constructed the AtSPS5.1-pART and RNAi-GhSPS1-pART vector. We transferred two vectors into cotton, respectively, and gained the transgenic callus by Agrobacterium-mediated system. All these results offer a basic on further study the function of sucrose phosphate synthase.The main results are as follows:1. In this research, we coloned the AtSPS5.1 from Arabidopsis thaliana by RT-PCR. The full sequence of the gene is 3251 bp, including 3132 bp coding region which encoding 1043 amino acids. Comparison with other homologue genes, the sequence has higher similarity, even more than 60%. AtSPS5.1 also has conserved motifs of SPS family. We constructed the over-expression vector of AtSPS5.1 using middle vector, pKannibal and expression vector, pART27.2. We cloned DNA sequence of GhSPS1 from cotton genomic by three times gene-walking and cDNA sequence by over-lap PCR. The full sequence of the gene is 4545 bp, including 12 introns and 13 extrons. The 3108 bp ORF of GhSPS1 coded 1035 amino acids. Comparison the nucleotide and deduced amino acid sequences of GhSPS1 with other homology genes, the results showed GhSPS1 belong to SPSA family. We constructed the RNAi expression vector of GhSPS1 using specific 200 bp segment according to alignment result.3. Up to data, four genes, GhSPS1, GhSUS3, GhINV and GhCESA4 may have close relation with fiber development. We analyzed the genes expression pattern by real-time quantitative PCR. The results showed that GhSPS1 and GhSUS3 expressed in all tissues. However the expression of GhSPS1 and GhSUS3 was highest in the 0 DAF ovules,3 DAF and 18 DAF fibers. GhINV transcripts were higher in 3-15 DAF fibers than in other tissues. GhCESA4 transcripts had the highest expression in 18 DAF fibers.4. The expression patterns of GhSPS1 under abiotic stresses treatment were analyzed by semi-quantitative RT-PCR. It can be concluded that the mRNA levels of GhSPS1 increased when the cotton seedlings were treated by ABA and lower temperature, while decreased under drought treatment. With higher temperature treatment, the expression of GhSPS1 decreased firstly, and then followed with light increase.5. Hypocotyls and cotyledons were cultured on different medium and induced callus forming. It showed that the callus initiating rates of hypocotyls were higher than that of cotyledons. And the callus amounts and state were also better in the former. The initiating rates and callus were different when the hypocotyls were cultured on different medium. The callus had the significantly advantage in MSB+0.1 mg/L 2,4-D+0.1 mg/L KT+3% glucose+2% phytagel.6. During the research, we also pay more attention on preventing the browning phenomenon in tissue culture of Gossypium hirsutum. The results showed that PVP could prolong the culture time of callus, but it could not restrain browning completely. Cotton callus browning were inhibited by adding glucose into medium instead of sucrose as carbon source and cut off the sterilization time to 15 min.7. In this research, we optimized the Agrobacterium-mediated transformation system about cotton and investigated some factors affecting transformation efficiency. The transformation efficiency could get to the highest when the O.D.600 of Agrobacterium got to 0.4, sterile seedlings grew up to 9 days, transformation time was 5 min and co-culture time was 48 h.8. Agrobacterium tumefaciens strain LBA4404 was used for cotton transformation to generate SPS+ and SPSi resistant callus of CRRI 24 and CRRI 10. The transgenic callus was identified by PCR and RT-PCR. Over-expressing AtSPS5.1 callus showed much better growth state than that of transfer SPSi callus.