| The world population is going to boom from around 7 to 9 billion in the next 50 years. At the same time, the extreme climate events happened frequently. The arable land is on the decrease because of the increasing of human activities, which leads to insufficient food supply for the increasing human populations. How to secure the staple crops in the world is the primary task for the government, and it is also one of most important state security strategies. Stress(both biotic and abiotic stress) is one of the most important limitation factors for the agriculture. As sessile organism, plants are subjected to various unfavorable environmental conditions during their growth and development, which causes the development retard, even defect, leading to loss of production. Drought, salt, nutrient inbalance(mineral toxicity and mineral deficiency) and extreme temperature(low and high temperature) are major environmental factors causing loss of the global crop production. Salt and alkaline, drought, and low temperature account for more than 20% of the loss of total global crop production. Among them, drought and salt are most severe limitation factors leading to crop production loss. Investigation on the mechanisms and signaling pathways of drought and salt resistance in plants would contribute to the understanding of plant response to abiotic stress, which is important for both basic research and applied sciences.Halophyte four wing saltbush(Atriplex canescens) is widely spread in arid and semi-arid area in west America. And it is also stored as food for the livestock in the United States. It’s an elite plant for plant biology research, particularly for understanding the abiotic stress resistance in plants, since it has endurable traits for salt, drought and low temperature stresses. What’s more, current plant abiotic stress research is mainly focusing on the herbaceous plants, and the investigation of abiotic stress resistance mechanism is still missing. Focusing on the investigation of abiotic stress mechanism in halophyte A. canescens would provide new insights into abiotic stress resistance mechanism in woody plants and provide more elite resistance genes for plant engineering science.In this study, several experiments were performed to isolate and functionally characterize resistance related genes from A. canescens. Subsequently, the functions of several transportors(Ac NIP5;1, Ac PIP2, Ac HMA1) in A. canescens were investigated:1. In this study, we employed the Superscript full length library construction kit II to generate a 400 m M Na Cl treated A. canescens full-length uncut c DNA library, and the c DNA library was subjected to EST sequencing and sequence analyses. At the same time, the primay c DNA library was transferred to the secondary c DNA library using LR recombination reaction using p YES-DEST52 yeast expression vector, which would be used for yeast transformation and expression. A total of more than 105 yeast transformants were subsequently subjected to the functional screening on SC-U media with 2M Na Cl. Totally, 53 transformants were selected based on the primary screening.2. After primary screening, selected transformants were inoculated into SC-U liquid media, respectively. After miniprep of corresponding plasmids, DNA information in each transformant was obtained through DNA sequencing and bioinformatics analyses. Then a total of 28 salt stress related genes were selected and their transcriptional expression profile was investigated by using q RT-PCR in A. canescens. The results indicated that yeast expression and functional screening system could be a time-saving and robust method to screen resistance related genes at a relatively high throughput level. And researches on real funtions and underlying mechanisms in real plants of these stress related genes isolated by yeast functional expression are still further needed.3. From previous EST sequencing, a novel member in MIP superfamily, Ac NIP5;1(A. canescens nodulin 26-like intrinsic protein 5;1) was isolated. q RT-PCR results in A. canescens showed that Ac NIP5;1 was transcriptionally up-regulated by 20% PEG6000 in leaves tissue. However, it’s down-regulated by application of 400 m M Na Cl at transcriptional level. Meanwhile, transgenic Arabidopsis overexpressing Ac NIP5;1 was generated. Results for abiotic stress assay demonstrated that overexpression of Ac NIP5;1 in Arabidopsis could increase the resistance to drought stress and sensitivity to salt stress treatment in pot experiment. What’s more, overexpression of Ac NIP5;1 could enhace enzymatic activities of SOD and CAT in Arabidopsis, as well as enhance the water retention and stomatal closure in dehydrate conditions. Ac NIP5;1-e GFP fusion proteins were localized to the plasmembrane when transiently expressed in Nicotiana benthamiana.4. From previous work on the EST sequencing in the lab, an aquaporin c DNA sequence, Ac PIP2, from PIP2 subfamily was isolated. Transgenic Arabidopsis by overexpressing Ac PIP2 exhibited accelerated growth rate and transpiration rate. And overexpression of Ac PIP2 in Arabidopsis could enhance the resistance to salt stress and compromise the drought stress resistance. The transgenic Arabidopsis showed a quick water loss phenotype under dehydrate conditions, as well as more accumulation of ROS in transgenic plants. Overexpression of Ac PIP2 could also decrease the stomatal frequency in Arabidopsis leaves. Subcellular localization results showed that Ac PIP2-e GFP fusion proteins localized to the plasma membrane when transiently overexpressed in N. benthamiana.5. From A. canescens c DNA EST sequencing, a heavy metal associated protein, Ac HMA1, was isolated. Detailed sequence analysis showed that Ac HMA1 contains two heavy metal associated domain. Exogenous application of heavy metals(Fe, Cu, Ni, Cd or Pb), PEG6000 and Na HCO3 could transcriptionally up-regulate Ac HMA1 in A. canescens. However, Ac HMA1 was down-regulated by Na Cl and low temperature stress treatment. Overexpression of Ac HMA1 in yeast could significantly enhance the tolerance to excess iron in yeast. Ac HMA1 also contributed to the tolerance to Na Cl, alkaline, drought and oxidative stress in yeast. Subcellular localization of Ac HMA1-e GFP fusion protein in N. benthamiana provided the idea that Ac HMA1-GFP fusion proteins localized to the plasmembrane. These results suggested that Ac HMA1 encoded a protein localized to the plasmembrane and involved in heavy metal stress resistance. Furthermore, Ac HMA1 might also be involved in resistance to multiple abiotic stresses in plants.Isolation and characterization of novel resistance related genes and investigation of regulatory networks in plant abiotic stress response signaling pathway is a key process to understand the underlying mechanisms for the interactions between plant and environmental factors. Development of a robust functional genomics screening system and isolation of resistance related genes are urgently required from both basic research and applied sciences perspective. How do plants response to salt, drought and heavy metal stress is complicated traits controlled by multiple genes. Isolation and functional characterization of novel transporters Ac NIP5;1, Ac PIP2, Ac HMA1 from elite halophyte A. canescens could also provide new insights into plant resistance to abiotic stresses in woody plants. And the results in this study could also provide new directions for plant genetics engineering in abiotic stress and novel genes for plant breeding. |
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