Molecular Cloning, Expression Analysis And Function Verification Of The Gene ProDH In Upland Cotton(Gossypium Hirsutum L.)

Proline is a kind of small molecule osmotic substance and the most common compatible soluble amino acids. Pro will accumulate with high quantity in plants when they were subjected to osmotic stress. Pro accumulation is a well-known response to osmotic stress in plants. This amino acid is a compatible solute and has been proposed to stabilize subcellular structures and to scavenge free radicals. In addition, Pro plays an important role in the growth and development of plants. A high content can be detected in many plant tissues and organs, especially in reproductive tissues such as pollen and pistils. In plants, Pro accumulates through an increase in its synthesis concomitantly with inhibition of its catabolism. Proline dehydrogenase is a flavoprotein that catalyzes the first and rate-limiting step of two reactions converting Pro into Glu at the mitochondria. This enzyme is located at the matrix side of the inner mitochondrial membrane. Many researches suggest that ProDH is sensitive to environmental stresses. Dehydration, extreme temperatures, and other abiotic injuries repress ProDH and stimulate Pro synthesis, thus leading to net Pro accumulation. Once the stress is relieved, ProDH becomes activated to consume the stored Pro.In order to research the function of ProDH gene in gossypium hirsutum L, we cloned a ProDH gene from sumian22. We did some experiments to verify the function of the gene. Firstly, we put sumian22with3-4pieces of main leaves under salt stress for24h, then recovered for24h; at the meanwhile, we treated another group of materials with L-Proline(0.1M) for24h.Then we did QPCR with all the deals to watch the expressions of the gene. Secondly, we watched the expression site of the gene through subcellular localization. Thirdly, an over-expression vector was constructed to transfer the gene into tobacco, by which we verified the function of the gene. At last, we constructed an RNAi to transfer cotton. The results of our experiments are as follows:1. We designed the degenerate primers according homologous sequence and clone the middle fragment of the ProDH gene through RT-PCR, then5’and3’fragments are cloned by rapid amplification of cDNA ends (RACE). The full cDNA sequence is1633bp and ORF is1545bp which encodes a57.07KDa protein with515amino acids, and the isoelectric point of it is8.010009765625. Further comparison shows that certain similarly exists between the gene we cloned and the ProDH genes of Vitas viniferaproline, Medicago truncatula et al. We deducted the gene is the ProDH gene of Gossypium hirsutum L. Cluster analysis also suggests that ProDH has closed phylogenetic relationship with Populus trichocarpa, Vitis vinifera, and Cucumis sativus and so on. Secondly structure analysis suggestes that ProDH has the structure of Alpha helix, Beta turn, Random coil, Extended strand.2. Real-time quantitative PCR analysis showed that the expression was up at the first hour when treated with200mM NaCl, then began to down from the third hour. At the twenty fourth hour, it is so low that can not be detected. However, it up-regulated very rapidly and drastically at the first hour of recovery with40folds of the untreated ones. Then the gene began to down-regulate to a normal level at the24th hour of this progress. When we treated them with0.1M L-Proline, the expression of the gene began to down-regulate till the6th hour, then began to up-regulate, and reached the top at the12th hour with only1.67fold of the untreated. With a consistent treatment for24th, there is no distinct different expression of the gene with the untreated.3. Transient expression of the ProDH protein in onion epidermal cells showed that ProDH was localized in mitochondria.4. We constructed an over-expression vector of the gene and transfered it into tobacco to verify the function of the gene.At this part, we extracted the DNA and RNA of the18transgenic tobaccos, from which we kewn that we have succeed in getting17transgenic tobacco.The transgenic efficiency is up to94.4%.We treated the transgenic ones and wild type tobaccos with drought stress for7days finding the ability of drought resistance is lower than the WT.5. We constructed a RNAi of the gene in order to transfer it into Gossypium hirsutum L and get ProDH silence cotton in the future. Through a series of work, we are sure that the vector is successful constructing.lt is.That is for us to get salt and drought cotton varieties in the future.In this study, we got a ProDH gene from Gossypium hirsutum L. The gene codes for the crucial enzyme in the progress of Proline metabolish.This study laid molecular basis and theoretical instruction for deeper study of the gene and breeding of cotton.