| Nitrogen and potassium are the essential macro nutrient elements for the growth and development of plants, and they are mainly obtained from the soil through the root system. The contents of potassium and nitrogen in the cultivated soils are obviously insufficient in our country. The long-term application of large amounts of nitrogen and potassium fertilizer taken mainly to meet the needs of the growth and development of plants in agriculture, could lead to pollutions of soil and water resources, as well as other environmental problems. The rich-potassium genotypic tobacco, Zhongyan-98, was screened out from74tobacco cultivar in our experiment and it was used to identify its low potassium-responsive genes by using suppression subtractive hybridization (SSH). It helped clarify the resistant mechanism to low-potassium at molecular level, and find new gene resources for genetic engineering in improving low-potassium-resistance of plants. In addition, the objective of the present study is to over-express the genes related to nitrogen metabolism and potassium absorption in tobacco leaves for exploring the absorption and utilization mechanism of nitrogen and potassium. It could be provide the technical support for the research of acquiring the nitrogen-or nutrition-rich crops using the genetic engineering technology. The main results in our studies are as followings:Firstly,74tobacco varieties treated with three different potassium concentrations (20mM,2.5mM and50μM) under the aseptic condition were carried out to study the potassium nutrition characteristics of tobacco and screen out potassium-rich genotype varieties which were resistant to low-potassium conditions. The growth of tobacco was obviously limited, and the fresh weight and dry weight of tobacco were decreased sharply under the low potassium condition. The potassium contents of tobacco leaf under the high conditions were more different in different varieties ranging from2.921to3.520%and the difference value was0.053. The potassium contents of tobacco leaf were reduced obviously with the decrease of potassium applied. The potassium contents of low-potassium conditions were only ranged from0.618to 1.241%, and the difference value increased to0.623. The cluster analysis of74tobacco genotypes showed that22were rich potassium nutrition varieties,45were high potassium nutrition varieties and7were low potassium nutrition varieties. Although potassium contents of different genotypic flue-cured tobacco in the same potassium supply conditions were different, it was positively correlated with the root activity. Low-potassium concentration helped to screen the rich-potassium tobacco genotypes. In addition, the physiological changes in Zhongyan-98under low-potassium stress were analyzed in our experiment. The content of potassium ion and H2O2in roots and the soluble protein content in leaves showed significant decrease in the early stage of low-potassium. Meanwhile, the contents of soluble sugar, proline and MDA were significantly elevated. Zhongyan-98, a rich potassium genotypic flue-cured tobacco variety, showed a better tolerance to low-potassium stress, so it was selected as the material to construct the forward SSH cDNA library of tobacco roots under low-potassium stress.Secondly, a forward SSH cDNA library for Zhongyan-98roots under50μM K+was constructed. After232clones greater than300bp were selected to sequence,203expressed sequence tag (ESTs) were generated. The sequence repetitive analysis showed that32.6%of the EST being only one repeat,67.4%of the EST being2-5repeat. The ESTs of highest frequency were cytochrome P450, phospholipase Al, metallothionein, heat shock protein and nitrate reductase. Blastx analysis indicated that107were non-redundant genes among all203ESTs, of which50ESTs showed similarity to proteins of known function and57ESTs were of unknown function. The50ESTs with known functions were classified into six categories. The genes involved in stress and cell death, metabolism, signal transduction and transcription factor and so on. To validate the SSH library data, the expression profiles of16genes including calreticulin, heat shock protein, ethylene-responsive-element-binding factor an so on under low potassium stress was investigated by semi-quantitative RT-PCR analysis. The results showed that the expression of all these genes was increased to various degrees. These genes might be providing new genetic resources for further study of plant potassium nutrition genetic engineering. The information obtained from the SSH library established an important foundation for clarification of the low potassium mechanism in tobacco at molecular level.Thirdly, overexpressing of potassium ion transporter cDNA (AtKUPl) of Arabidopsis thanliana was driven by P35S and potassium ion channel cDNA (AKT2) of Arabidopsis thanliana was driven by PrbcS in transgenic tobacco plants using plant genetic engineering in the present study. The changes in contents of some physiological index and in ability of low-potassium tolerances in transgenic tobacco lines were compared. The results showed that the selected transgenic tobacco plants overexpressing AtKUP1or AKT2did not have significant differences compared with wild-type tobacco under rich-potassium conditions. K+uptake regulation mechanism of plant might be activated to control the absorption amount of K+during providing the excessive potassium nutrient. However, transgenic tobacco under low-potassium conditions showed significant growth advantages, such as plant height, fresh weight and dry weight of shoot, and leaf surface area. In addition, the expression level of exogenous gene, the contents of chlorophyll, soluble protein, glucose, sucrose, potassium ion and the activity of SOD were induced by low-potassium while the significant decrease in the contents of proline, malondialdehyde and peroxide hydrogen related to stress tolerance. The results suggested that the transferred exogenous AtKUP1or AKT2gene might be promoter the absorption and transport of potassium ion of plants under low-potassium stress, and reduce the impairment of plants.Finally, one plant expression vector (pK2-35S-Dofl) about the transcription factor Dofl from Arabidopsis thanliana using the constitutive promoter CaMV35S (35S), and three plant expression vectors (pPZP221-PrbcS-Dofl, pH2-35S-PrbcS-GS1and pK2-35S-PrbcS-*T-GS2) about Dofl, chloroplastic (GS2) and cytosolic (GS1) glutamine synthetase under the control of the light inducible promoter of PrbcS were constructed. Eight kinds of transgenic tobacco lines including WD, WRD, WG1, WG2, WG12, WDG1, WDG2and WDG12, were generated by overexpressing signal gene and co-overexpressing two or three genes simultaneously. Indexes of growth characteristics and the levels of some metabolites in tobacco leaves were measured to investigate the plant’s tolerance to low-nitrogen conditions. There were a certain level of expression of the exogenous Dofl, GSI or GS2in the selected transgenic lines. They showed better growing characteristics, and an increase in the enzyme activities of GS, NR, PK and PEPC which were the key enzymes in the nitrogen assimilation and carbon metabolism. The contents of some intermediate metabolites, such as glucose, sucrose, and most of amino acids, increased significantly, whereas the contents of nitrate, malic acid and citric acid declined. The nitrogen utilization efficiency (NUE) of eight transgenic tobacco lines were significantly higher than that of the wild-type, and the most significant change were obtained in the transgenic lines co-overexpressing Dofl and GS. It suggested that the overexpression of exogenous Dofl and/or GS genes could improve the level of nitrogen assimilation and utilization, and promote the growth of tobacco plants. Transgenic lines co-overexpressing DofI and GS showed better growth than single transgenic lines. The strategy of co-overexpressing Dofl and GS might be providing a new pathway to enhance the low-nitrogen tolerance of plants. |
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