The Physiologic Response Of Hevea Brasiliensis And The Ssh Construction And Analysis Under P Dificiency

Nature rubber is important for both industry and national defense. As a resource constraint industry, it was mainly produced by Hevea brasiliensis (Hevea hereafter). Phosphorus (P) plays an important role in Hevea growth and development. P deficiency occurs commonly in rubber plantations, and constitutes a restricting factor of rubber yield. Thereby, the researches on the physiological and molecular mechanisms of Hevea P nutrition, and screening of the key genes involved in phosphorus high efficiency utilization, are very important to improve the rubber yield and the P utilization efficiency of Hevea.The experiments were carried out under low P treatments with different levels and days. The research explored the response mechanisms of Hevea underlying the low P stress by investigating the morphological and physiological changes. Furthermore, we firstly constructed the suppression subtractive hybridization (SSH) libraries of Hevea under low P stress. On the basis of this, we preliminarily explored the gene expression of P deficiency, by sequencing and profiling part of the ESTs.The results were shown below:1. The morphological response of Hevea to P deficiencyComparing with the CK(normal P),the P deficiency significantly decreased the plant height, total biomass and leaf biomass, but no obvious change occurred in the root biomass,tap root length and stem girth and the biomass.The P deficiency decreased the dry matter partitioning of leaf, but increased the dry matter partitioning of root, meanwhile no obvious changes of dry matter partitioning occurred in the stem.2. The N,P,K nutrition response of Hevea to P deficiencyUnder the low P stress, the N content and accumulation in the leaf,root and stem were significantly decreased. Therefore the N utilization efficiency of the Hevea was increased. N and P have a good collaborative effect.The K accumulation in the leaf,root and stem were significantly decreased with the P deficiency, but the K content in the leaf and root haven't obvious change. The P deficiency hasn't a significantly influence to K utilization efficiency.The P deficiency significantly decreased the P accumulation,the P content and the soluble P content in all the organs. Thereinto, the soluble P content in the root and stem were significantly higher than the leaf. The P utilization efficiency was significantly improved under P deficiency. 3. The physiological response of Hevea to P deficiencyAfter three months of low P stress, the soluble sugar content(SSC) in root,stem and leaf decreased with the P level. On the point of the treatment days,the SSC in the leaf is significantly lower than the CK except the 56th; the SSC in the stem is significantly higer than the CK at 28th, but significantly lower after 42th; the SSC in root is significantly lower than the CK after the 42th day, but higher before the 42th day.The soluble protein content (SPC) of leaf and root increased under P deficiency. But the SPC in stem was higher than the CK before the 42th day, and hasn't obvious change after the 42th day.After 56 days low P stress, the acid phosphatase activity(APA) in the root,stem and leaf are significantly higher than the CK.Mild P deficiency has a little effect to chlorophyll content of Hevea; however, with the stress deepen, the chlorophyll content increased significantly. In the point of treatment days, the chlorophyll content under P deficiency was slightly higher than CK before the 42th day, and was significantly higher after the 42th day.The nitrate reductase(NR) activity and root activity were decreased with the P level. Under the P deficiency, they both were significantly lower than the CK.4. The protective enzyme system response of Hevea to P deficiencyThe malondialdehyde (MDA) content of leaf,stem and root were increased with the decreasing of the P level. With the prolongation of treatment days, the MDA content of the root increased gradually, during the whole experiment, the MDA content of root under P deficiency was significantly higher than the CK.The superoxide dismutase (SOD) activities of leaf,stem and root were increased with the decreasing of the P level. With the prolongation of treatment days, the SOD activities in all the organs under P deficiency increased gradually; however, the CK hasn't obviously changes. During the whole experiment, the SOD activity under P deficiency was significantly higher than the CK.With the decrease of P level, the activities of peroxidase (POD) in leaf increased firstly, get the maximum at 10mg/L P, then decreased gradually, get the minimum at 0mg/L P. The activities of POD in the stem and root decreased gradually with the decrease of P level. At the beginning of the treatment, the POD activities of all the organs under P deficiency were higher than the CK; however, those were significantly lower than the CK after the 70th days. With the decrease of the P level, the activities of catalase (CAT) of leaf and root increased gradually. The the activities of CAT in all the organs under heavy low P stress were significantly higher than the CK. At the beginning of the experiment, the activities of CAT in leaf,stem and root under P deficiency increased rapidly till 42 days; then decreased gradually. But during the whole experiment, the activities of CAT of all the organs were significantly higher than the CK.5. To identify differently expressed genes in rubber tree (Hevea brasiliensis) under P deficiency, four cDNA libraries were constructed using suppression subtractive hybridization (SSH) method. The results were as followed:(1) After clustering and assembly sequences of SSH libraries,94 genes(EST) were up-regulated in root of rubber tree under P deficiency; 45 genes(EST) were down-regulated in the root; 64 genes(EST) were up-regulated in leaf; 166 genes (EST) were down-regulated in the leaf.(2) GO (Gene Ontology) analysis showed that:cellular component mainly containplastid part, ribosome, intracellular part, thylakoid membrane, photosynthetic, intrinsic to membrane, cytoplasmic part, ribonucleoprotein, membrane; molecular function mainly contain cation binding, translation factor activity, transferase activity, ligase activity, hydrolase activity, DNA binding, RNA binding, transferring glycosyl, chlorophyll binding, dioxygenase activity; biological process mainly contain cellular response to stress, establishment of protein localization, intracellular transport, ion transport, protein metabolic, nucleotide and nucleic acid metabolic, carbohydrate metabolic, organic acid metabolic process, amine metabolic process, cellular biosynthetic process, photosynthesis, macromolecule biosynthetic and so on.(3) In the root of forward and reverse libraries,7 ribosomal protein genes were regulated by P deficiency, among them,5 genes were up-regulated,2 genes were down-regulated; meanwhile, an AP2 domain-containing transcription factor (9211496),cation efflux protein/zinc transporter(Contig132),bile acid:Na+ symporter family protein gene (Contig216) were also up-regulated under P deficiency.(4) In the leaf of forward and reverse libraries, ribosomal protein genes showed differently expressed,6 of them were up-regulated by P deficiency; Photosynthesis-related genes showed different expressed level under P deficiency, among them, a thylakoid membrane phosphoprotein, chlorophyll a/b-binding protein and photosystemâ… reaction center subunit N were up-regulated; but another three genes related to photosynthesis were down- regulated by P deficiency. Moreover, cation efflux protein/zinc transporter were up-regulated by P deficiency.(5) The expression level of four genes from the the libraries was further examined by real-time PCR. The four genes were E3 ubiquitin protein ligase, hydrolase, cytochrome P450-like TBP protein and ribosomal protein L2. Expression level of them showed differently under the different time of P deficiency, and the expression patterns were consistent with the result of SSH libraries.The results lay a foundation of researching the morphorlogical,physiological and molecular response of Hevea under P deficiency, provide a basis for exploring the high P efficiency characteristic, screening the high P efficiency resource and breeding high P efficiency clone of Hevea.