| Phosphorus(P) is one of the important nutrients that plants need for growth and development which is involved in photosynthesis, respiration, glycometabolism and other important physiological processes. It is the key component of phospholipids, nucleic acid, and ATP. However, it is obvious that it is lack of phosphorus in rubber plantations in Hainan province, which has become one of the most important factors that limits latex yield increases. Chemical fertilizers applied to the soil can easily be fixed and a large number application will also cause the waste of phosphate and soil pollution. It is not a sustainable strategy to use a large number of fertilizer to improve the rubber tree on the absorption and utilization of phosphorus nutrition.Studies have shown that under the condition of low phosphorus stress, plants can increase the uptake and use efficiency of the phosphorus in the soil through changing the morphological structure, physiological and biochemical pathways. Therefore, it provides important clue through mining the key gene that the rubber trees efficiently absorb phosphorus under low phosphorus stress, and it is of great significance to increase the absorption of phosphorus to increase the latex yield. This research use expressed sequence tags(EST) which the project team has constructed suppression subtractive hybridization(SSH) libraries of Hevea under low P stress, cloning four related genes and analysising its expression characteristics under low phosphorus stress. The results are as follows:1. Based on subtractive hybridization library EST, through the method of RT-PCR and RACE, a hydrolase HbHyd gene was cloned from Hevea brasiliensis. The full-length is1860bp, the length of the open reading frame is1479bp, the longest encoding492amino acid protein whose molecular weight is predicted approximately56.1KD, and the theoretical isoelectric point is6.52. The gene homologize85%with a castor hydrolase gene nucleotide sequence, the comparison is also found84%homology with amino acid sequence. It is found that using real-time PCR to express for HbHyd gene under phosphorus deficiency stress which has a obvious up-regulated expression from day1to day8in rubber tree roots, while in leaves the expression increases only in day1and day8. The results showed that the HbHyd gene may involve in the rubber tree roots to low phosphorus stress responses. Under the GA treatment. HbHyd in roots and stems is significantly up-regulated, especially in24h the expression is highest in the root. The study results shows that the expression of HbHyd in rubber tree roots is induced by GA to some extent.2. Based on subtractive hybridization library EST, A Cysteine Desulfurase HbNfs genes was cloned from rubber tree. The full-length is1681bp, the length of the open reading frame is1182bp, the longest encoding395amino acid protein is predicted molecular weight of approximately44.9KD, and the theoretical isoelectric point is6.35. The gene homologize83%with a castor cysteine desulfurase gene nucleotide sequence, the comparison also is found95%homology with amino acid sequence. It is found that using real-time PCR to express for HbNfs gene under phosphorus deficiency stress has a obvious up-regulated expression from day1to8in rubber tree roots. It reaches the highest peak in the day6, leaves only in day6and8d increased apparently. It states that the initial process of HbNfs gene may be involved in the stress response of rubber tree root to low phosphorus. Under GA treatment, HbNfs in roots and stems have a significant up-regulated, especially in the root2h and8h expression. The study results showed that the expression of HbNfs in rubber tree roots is induced by GA to some extent.3. A fructose diphosphate aldolase gene HbAld was cloned from the rubber tree. The full-length is1553bp, open reading frame length is387bp, the longest length is to encode129amino acid protein is predicted molecular weight of approximately13.6KD, and the theoretical isoelectric point is9.41. The genes reach90%with a castor nucleotide sequence homology, homology of amino acid sequence also reaches95%with castor amino acid sequence coding of fructose diphosphate aldolase. It is found that using real-time PCR to express for HbAld gene under phosphorus deficiency stress,0d and2d of HbAld have the obvious up-regulated expression under low phosphorus treatment.In the leaf1d,2d and4d all have the up-regulated expression. It states that the HbAld gene may be involved in low phosphorus stress response process of rubber tree. Under the GA treatment of HbAld, roots in2h,8h and24h significantly regulate up, Stems is only in2h upregulated, while HbAld has no expression in the leaves. The study results shows that the expression of HbAld is induced by GA mainly in the roots of rubber tree. 4. A phosphate transporter2gene HbPT2is choned from the rubber tree. The foll-length is1981bp, open reading frame length is1143bp, the longest length is to encode382amino acid protein is predicted molecular weight of approximately41.8KD, and the theoretical isoelectric point is9.43. The genes with a castor nucleotide sequence homology reaches87%, it is alse discoved that also with castor amino acid sequence protein coding of fructose diphosphate aldolase, the homology reaches88%. It is found that using real-time PCR to express for HbPT2gene under phosphorus deficiency stress, HbPT2in1d have the obvious increased, the declined slightly in2-8d, it is still in the up-regulated expression then it tends to be steady. But in leaves don’t show increased expression of Id, followed by2-8d a clear up-regulated expression and the expression is maintained at a relatively high level. Under GA treatment, HbPT2is significantly up-regulated in the roots of1h.2h contrasting with lh decreases a little, the subsequent4h and8h compared to2h is incremental up-regulated, there is no obvious up-regulated in stems and leaves. The study results showed that the expression of HbPT2is induced by GA mainly in the roots of rubber tree. |
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