| Aluminum toxicity is the world’s biggest problem of forestry and environmental problems. Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) is one of the most important timber tree planted in southern China, is also facing the problem of aluminum toxicity. Early aluminum toxicity symptoms mainly inhibit plant root elongation, impact of the plant to absorb water and nutrients, futher affecting the production and quality of the wood. In recent years, the related research on aluminum toxicity of Chinese fir which involving the physiological aspect has been reported, because of the lack of genome sequence information, the molecular mechanism behind these physiological processes are unclear, especially combined with the level of ion flux and transcriptome to study the aluminum toxicity of Chinese fir has not been reported yet. In view of this, the clone of Chinese fir was used as experimental material in this study, with the help of noninvasive micro measurement technology and transcriptome sequencing and other physiological, biochemical and molecular biological research methods, we studied the effect of aluminum stress on the 6 kinds of mineral elements absorption in the ground and underground part of Chinese fir clone, the effect of aluminum stress on the chlorophyll fluorescence characteristics of Chinese fir clone, the effect of aluminum stress on the 4 kinds of ion absorption in the root of Chinese fir clone, and the transcriptome research of the root of Chinese fir clone which response to aluminum stress; Using real-time qPCR to study the differerntial expression of three selected representative genes which under aluminum stress, in order to reveal the molecular mechanisms of the clone of Chinese fir response to aluminum toxicity.In addition, we selected some differentially expressed genes which related to the element from transcriptome database of Chinese fir root under aluminum toxicity, and carried on the preliminary analysis. In order to provide theoretical basis and practical basis of cultivating the variety of Chinese fir with resistant ability of aluminum poison, expand and enrich the aluminum toxicity research in the field of Chinese fir, and provide a scientific basis and theoretical guidance for the forest production practice of Chinese fir which grown in the south acidic soil of China. The main results are followed below:(1) For the underground part of Chinese fir clone, the different aluminum stress concentration has the largest influence on the phosphorus, potassium, aluminum content of Chinese fir clone; and the different aluminum stress time has the biggest influence on the calcium, magnesium, iron content of Chinese fir clone. For the ground part of Chinese fir clone, the concentration of aluminum has the largest effect on the aluminum content of Chinese fir clone, while it is not obviously for other elements.(2) Different aluminum stress concentration and different aluminum stress time were affected the chlorophyll fluorescence parameters Fo, Fm, Fv, Fv/Fm of Chinese fir clone. With the increase of aluminum stress concentration and the extension of aluminum stress time, the chlorophyll fluorescence parameters Fo, Fm, Fv, Fv/Fm of Chinese fir clone are also with corresponding change subsequently. In general, with the increase of aluminum stress concentration and the extension of aluminum stress time, stress the chlorophyll fluorescence parameters Fo, Fm, Fv, Fv/Fm of Chinese fir clone firstly showed an increasing trend and then a decreasing trend in a short time aluminum stress. As stress time extended to 8 h, the chlorophyll fluorescence parameters Fo of Chinese fir clone showed an increasing trend, while the chlorophyll fluorescence parameters Fm, Fv of Chinese fir clone firstly showed a slight increasing trend and then decreasing trend. For the chlorophyll fluorescence parameters Fv/Fm of Chinese fir clone, with the increase of aluminum stress concentration, the chlorophyll fluorescence parameters Fv/Fm of Chinese fir clone was basic remained at around 0.85 in a short time (within 4 h) under aluminum stress, as further extension of aluminum stress time, the chlorophyll fluorescence parameters Fv/Fm value of Chinese fir clone showed a decrease trend in the low aluminum stress concentrations (0.5 mmol-L-1 and 1 mmol-L-1) after aluminum stress 16 h, while it showed a decrease trend in the high aluminum stress concentrations (2 mmol·L-1 and 4 mmol·L-1) after aluminum stress 8 h.(3) Different aluminum stress concentration and different aluminum stress time were affected the absorption of H+, K+、Ca2+ and Mg2+ ion flow in the root of Chinese fir clone. With the increase of aluminum stress concentration, there was a biggest influence of the absorption of H+、 K+ and Mg2+ ion flow in the root of Chinese fir clone after different aluminum concentration stress 1 h, and less effect on the absorption of Ca2+ ion flow in the root of Chinese fir clone; in which, the H+ ion flow in the root of Chinese fir clone was gradually from influx to efflux; the K+ ion flow in the root of Chinese fir clone was all efflux with high amplitude variation; Ca2+ ion flow in the root of Chinese fir clone was all influx after aluminum stress 1 h, and it was from influx to larger efflux in a long period of aluminum stress time. With the increase of aluminum stress concentration, there was a biggest influence of the absorption of H+、Ca2+ and Mg2+ ion flow in the root of Chinese fir clone after different aluminum concentration stress 32 h, and less effect on the absorption of K+ ion flow in the root of Chinese fir clone; in which, the H+ ion flow in the root of Chinese fir clone was gradually from influx to efflux; the K+ ion flow in the root of Chinese fir clone was from efflux to a small influx; Ca2+ ion flow in the root of Chinese fir clone was from influx to efflux and the efflux velocity decreases after aluminum stress 32 h, and Mg2+ ion flow in the root of Chinese fir clone was all efflux with a larger variation of efflux velocity. In general, with the increase of aluminum stress concentration and the extension of aluminum stress time, the absorption of H+ ion flow in the root of Chinese fir clone was gradually from influx into efflux; there is large variation in the K+ efflux, and the K+ efflux was gradually decreased in a long aluminum stress time; Ca2+ ion flow in the root of Chinese fir clone was all influx after aluminum stress 1 h, and it was from influx to larger efflux in a long period of aluminum stress time. There was the largest influence of different concentration of aluminum stress on Mg2+.(4) The clone of Chinese fir was used as experimental material in this study, using the Illumina HiSeqTM2500 sequencing platform for the studying the effect of aluminum stress on the root of Chinese fir clone from transcriptome level, sampled the root samples respectively after aluminum stress 1 h,4 h,8 h,16 h and 32 h, extracted the total RNA, then built two cDNA libraries by using the 2 RNA samples which mixed respectively from 15 total RNA samples of the control group and 15 total RNA samples of the aluminum stress group according to the quality of the total RNA, after transcriptome sequencing, we obtained a total of 24.23 Gb Clean data from the two root samples of Chinese fir, and got 66977 Transcripts and 118761 Unigenes after assembling the sequencing data, and obtained 28655 SNP and 2439 SSR markers after analyzing the data. The EBSeq software was used to analyze the differentially expressed analysis in this experiment, we obtained 3246 differentially expressed genes, after statistics found that compared with the control group of Chinese fir, and there were total 1856 differentially up-regulated genes and total 1390 differentially down-regulated genes in the aluminum stress group. On the whole grasp the expression patterns of the aluminum stress related differentially expressed genes by analyzing the function, metabolism and regulation pathway of the differentially expressed genes. The results showed that when the roots of Chinese fir suffering aluminum toxicity, the biological process, molecular function and cellular component in the root of Chinese fir that closely related to aluminum stress change obviously. After aluminum stress, there was a tremendous amount of differentially expressed genes in the roots of Chinese fir, involving amino acid transport and metabolism, carbohydrate transportation and generation, inorganic ion transport and metabolism, secondary metabolites synthesis, transport and catabolism and other multiple metabolic pathways, and these were associated with energy metabolism, these were provided energy for a series of physiological and biochemical reactions to resistant the aluminum stress damage on Chinese fir.(5) Aluminum stress has a different effect on the expression of three genes of Chinese fir root:aluminum stress significantly induced the expression of gene Cl-TSJTl in Chinese fir root, especially after aluminum stress 16 h reached the highest expression; 8 h aluminum stress significantly induced the expression of gene ClCa-uptakel; aluminum stress obviously inhibited the expression of gene ClPtl in the root of Chinese fir, the inhibition of 8 h aluminum stress is obviously.(6)Under the aluminum stress, there are 26 differentially expressed genes related to potassium in the transcriptome of Chinese fir root, including 16 up-regulated and 10 down-regulated; there are 13 differentially expressed genes related to calcium in the transcriptome of Chinese fir root, including 9 up-regulated and 4 down-regulated; there are 18 differentially expressed genes related to magnesium in the transcriptome of Chinese fir root, including 12 up-regulated and 6 down-regulated; there are 19 differentially expressed genes related to iron in the transcriptome of Chinese fir root, including 14 up-regulated and 5 down-regulated; there are 33 differentially expressed genes related to aluminum in the transcriptome of Chinese fir root, including 10 up-regulated and 23 down-regulated; there are 8 differentially expressed genes related to phosphorus in the transcriptome of Chinese fir root, including 5 up-regulated and 3 down-regulated;In conclusion, different aluminum stress concentration and different aluminum stress time were made the content of aluminum and magnesium increase in the underground part of Chinese fir clone, and the content of potassium and iron decrease in the underground part of Chinese fir clone, while the change of calcium content in the underground part of Chinese fir clone was not obvious; the content of aluminum and magnesium increase in the ground part of Chinese fir clone, and the content of potassium, iron and calcium in the ground part of Chinese fir clone were not obvious. And from the analysis of the change of the ion flow, the change of potassium, calcium and magnesium ion flow is consistent with the results. From the transcriptome analysis, the results showed that there was a tremendous amount of differentially expressed genes in the roots of Chinese fir, involving the inorganic ion transport and metabolism and energy metabolism, and further illustrated that aluminum stress affected the absorption and accumulation of various kinds of inorganic ions in the root of Chinese fir clone. The above researches can provide the theoretical foundation for studying the related research about aluminum toxicity of Chinese fir in the future. |
PDF Full Text Request