Isolation And Functional Analysis Of Abiotic Stress Related Aquaporin Genes From Glycine Soja

Low temperature, drought and salt stresses are the major environmental factors that place limits on plant productivity all over the world. Enhancing the crops'resistance to drought,salt and cold stress is very important for increasing the crop production and solving the famine problem.With the rapid development of molecular biology and plant bioengineering, a better way is found to solve the problem by molecular breeding using gene transfomation. However, the complexity of molecular mechanism of plant response to stresses and the intellectual property rights of stress-related genes restrict the execution of molecular breeding. It's important to discover novel gene sources which play important roles in plant response to stresses and can be used for molecular breeding at liberty. Many kinds of stresses affect the expression levels of aquaporins, while studies demonstrating the relationship between aquaporins and plant stress tolerance remains limited. Isolating aquaporin genes and analyzing their effects to stress tolerance can provide information for the research on aquaporin functions or the mechanism of plant response to stresses, and can also enlarge the gene resource for bioengineering and molecular breeding to improve plant tolerance to stress.Glycine soja contains abundant abiotic stress correlative genes. In this study, 3 groups of G.soja were stressed by cold, sality or drought, samples from these groups and unstressed plants were hybridized with the genechip of G.max seperately. Expression profiles of ESTs in our constructed EST library of sality-stressed G.soja were inferred with these data, and 2 ESTs were selected which response to abiotic stresses early from Glycine soja and belong to the aquaporin(AQP), which is now called the major intrinsic protein (MIP) family, and the full-length genes corresponding to these ESTs were obtained by in silico cloning and SMART-RACE technique. Amino acid sequences of their protein products were analyzed with kinds of bio-informatics methods, so that characters, structures and functions of the proteins were predicted. Two novel aquaporin genes including one plasma membrane intrinsic protein (PIP) and one tonoplast intrinsic protein (TIP) were isolated. Expression pattern of genes, subcellular location of gene products and stress tolerance of Arabidopsis overexpressing the two genes were analyzed and effects of the two genes were concluded. This research will provide some information for researches on molecular mechanism of stress tolerance of plant and enlarge the gene for plant stress-tolerance genetic engineering. Results were summarized as follows:1. Selection of ESTs early responding to abiotic stresses of Glycine soja 3 groups of G.soja seedlings were stressed by cold, sality and drought, and samples from these groups as well as unstressed seedlings were subjected to Affymetrix Co. to hybridize with the genechip of G.max seperately. EST library of Glycine soja under salty stress was scanned and all the 2003 ESTs were aligned with more than 60 000 probe-sets of the genechip. Results of the chip were verified with real-time fluorescent quantitive RT-PCR and the matching is about 78%. Expression profiles of ESTs under these stresses were infered, and real-time fluorescent PCR was used to verify results of the chip. 241 unique ESTs were recognized to be up-regulated early(in one hour) by abiotic stresses , 5 of which are aquaporin ESTs.2. Isolation of aquaporin genes of G. soja early responding to abiotic stressThe 5 MIP ESTs were analyzed with Blast N and Blast X. 1 EST were determined as the EST of the TIP (tonoplast intrinsic protein) subfamily, and the other 4 ESTs belong to the PIP (plasma membrane intrinsic protein) subfamily. 2 full-length MIP genes, both of which are up-regulated by cold, sality and drought stresses, were obtained using in silico cloning and a modified 5'-RACE technique (talent applying), one encodes a tonoplast intrinsic protein and was named as GsTIP, the other encodes a plasma membrane intrinsic protein and was named as GsPIP2. The amino acid sequence of the former gene has a similarity of 84% with Medicago truncatula, and the one of the latter has a similarity of 90% with Mimosa pudica.Analysis of physical features including conserved domains, secondary structures, subcellular positions of the two genes were carried out with softwares, such as Clustal X, DNAman, Antheprot and so on, as well as databases and tools published in the internet. Both the 2 proteins contains the feature transmembrane structures and hydrohobicity of MIP family, and have enough similarity with MIPs of other plants, which shows these 2 proteins belongs to the TIP and PIP subfamily relatively. Both proteins have potential phospho-sites and myristyl-sites, which suggests these proteins might be regulated or activated by post-translation modifications.3. Subcellular analysis of aquaporins of G. sojaTwo transient expression vector were constructed to express the two genes with GFP merged at the C-terminal of gene products. Vectors were transformated into tobacco leaves to get fused proteins express in the epidermis cells. After transient expression, epidermis cells were observed with laser-confocal microscope with blue laser stimulating the green fluorescence from GFP. Subcellular location of the two gene products were analyzed according to the laser-confocal images. The results shows that GsTIP locates at tonoplast and GsPIP2 locates at plasma membrane, which consists with the predicted results. The result of GsPIP2 also shows GsPIP2 forms conglomeration, which suggests GsPIP2 might form homotetramer or hereotetramer.4. Expression pattern analysis of aquaporins of G. sojaAccording to the result of genechip, the expression level of the two aquaporin genes were supposed to be stimulated by cold, salt and drought stress. More detailed experiments were carried out to analyze expression pattern of the two genes in G. soja under different stress conditions. Semi-quantitive RT-PCR was used to analyze the expression levels of the two genes in roots and leaves of G. soja stressed with cold, salt and drought for 0h, 0.5h, 1h, 3h and 6h. The result of Semi-quantitive RT-PCR comfirmed the result of genechip that both genes are induced by all the three stress conditions in the leaves, and the expression level changes in roots are contrary to the changes in leaves. In roots, expression level of GsTIP is suppressed by salt and drought and doesn't change under cold stress. Expression of GsPIP2 in roots is suppressed by all the three stress conditions. Expression pattern under salt stress condition is similar to that under drought stress and is different from that under cold stress for both genes in both tissues. There are also similarities between the expression pattern of GsTIP and GsPIP2: both are stimulated by all the three stress conditions in leaves, and are suppressed by salt and drought condition, and the expression levels of both genes raises to a higher level 0.5h after cold stressed.5. Effects of G. soja aquaporins to stress toleranceTwo overexpressing vectors were constructed for the overexpression of the two genes in Arabidopsis. Genes are constructed downstream the CaMV35S promoter to constantly express in transgenic Arabidopsis. Overexpressing vectors were transformed into Arabidopsis with the floral dip method, and the lines overexpressing each aquaporin gene were determined with Kanamycin tolerance screening, PCR, RT-PCR and semi-quantitive RT-PCR and the lines overexpressing the relative aquaporin genes were preserved and analyzed. Germination and growth behaviours including survival rate, root length and fresh weight under cold, salt and stimulated drought stress conditions were investigated, and dehydration/ rehydration speed, water potential in shoots and transpiration speed under different conditions were measured. The results show that both overexpressing aquaporins decrease plant stress tolerance to salt and drought stress conditions, but by different ways. Arabidopsis plants overexpressing GsTIP have higher speed of dehydration and transpiration than wild type plants, suggesting GsTIP affects stress tolerance by enhancing the transpiration. Whereas plants overexpressing GsPIP2 has a greater decline in water potential of shoots than wild type plants, suggesting GsPIP2 affects stress tolerance by interfering in the modulation of water potential.