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Physiological Responses of Hevea Brasiliensis Seedlings to Low Potassium Stress and Analysis of Differentially Expressed Genes

Author: WuMin
Tutor: ChenShouCai
School: Hainan University
Course: Plant Molecular Genetics
Keywords: Hevea brasiliensis low K stress morphological and physiological responses SSH library oligonucleotide chip QPCR gene clone expressed analysis
CLC: S794.1
Type: PhD thesis
Year: 2011
Downloads: 18
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Abstract


Natural rubber is an important raw material for both industry and national defense. The natural rubber industry is the pillar industry of the agricultural development and economic growth in tropical regions. Potassium (K) is a kind of Mineral nutrition element which is greatly needed in the process of plant growth and plays a role in a range of physiological functions in plants:photosynthesis, enzyme activation, protein synthesis, osmotic potential. In Hevea brasiliensis (Hevea hereafter), the availability of K affects the characteristics of latex production, latex flow, wind resistance and cold hardiness, etc.China is short of K fertilizer resources. The situation that the supply of K fertilizer does not meet the demand becomes increasingly serious. The acreages of K-deficient soil are increasing rapidly in rubber plantations.In fact, the genetic improvement of plant nutrition traits which exist genotype differences in different plants is one of useful ways improving crop yields under poor soil nutrient environments.Recently, the researches of molecular response mechanisms of plant nutrient deficiencies becomes more and more common.However, in Hevea,similar research reports are almost blank, and the physiological researches of K nutrition are also scarce.The response mechanisms of Hevea seedlings to low K stress were investigated in the aspects of morphological and physiological characteristics. Furthermore, the differentially expressed Hevea seedlings genes to low K stress were firstly separated and screened by constructing the suppression subtractive hybridization (SSH) libraries and hybridizing the oligonucleotides chip. On the basis of these, the functions of the differentially expressed genes were preliminarily annotated by BlastX and BlastN, and the response characteristics of part differentially expressed genes to low K stress were verified by the real-time quantitative PCR(QPCR,hereafter). What’s more, the full-length or part-length cDNA of HbMPPl and HbCABl were cloned by the rapid amplication method of cDNA ends (RACE), the functional predictions and spatio-temporally expressed characteristics of two genes were done by Bioinformatics methods and QPCR, respectively.The major results were as follows.1.Morphological and physiological responses of Hevea seedlings to low k stressThe hydroponic culture was carried out to investigate the differences of morphological and physiological characteristics between the treatment of normal K supply and four treatments of low K supplies. The five K levels settled were OmgL-1,1mgL-1,10mgL-50mgL-1and150mgL-1(normal), respectively. The major results obtained were summarized as follows.(1)The low K supplies gradually decreased the plant height, root length, stem girth and biomass of the root, stem, leaf and whole plant.(2)Under low K stress, the accumulations of nitrogen and phosphorus in all Hevea seedling organs were decreased, but the N utilization efficiencies of the stem being increased.Moreover,the K contents and accumulations in all Hevea seedling organs were dramatically decreased, but the K utilization efficiencies, the most sensitive low K-responsive indicators, being significantly increased.(3)Under low K stress, the contents and the percentages of soluble phosphorus were decreased in different organs, but the insoluble phosphorus reversely being changed, and the ultimate P utilization efficiencies being decreased.(4)The low K supplies decreased the contents of chlorophyll a and chlorophyll b, but significantly increasing the ratio of chla/chlb. The contents of chlorophyll b were significantly different between the treatments of low K (below10mg L-1) supplies and the treatment of the normal K supply.(5) The soluble sugar contents (SSC) in the leaves and in the barks of the stem were increased in low K supplies of0and1mg L-1, but being decreased in the low K supplies of10and50mg L-1. However, all treatments of low K supplies decreased the SSC in roots, decreasing the soluble protein contents (SPC) in all organs.(6)The low K supplies irregularly decreased the superoxide dismutase (SOD) activities in different organs of Hevea seedlings.(7) In root,the low K supply of1mg L-1significantly increased the activities of peroxidase (POD),but other low K supplies having not significantly effects on the activities of POD.In leaves and barks of the stem,the activity diffirences of POD were little between the low K supplies and the normal K. supply.(8) The low K supplies significantly increased the activities of catalase (CAT), but the activities of CAT varied irregularly in different levels of low K supplies.(9) The low K supplies significantly decreased the activities of nitrate reductase(NR)in the leaves and the barks of the stem,but insignificantly decreasing the activities of NR in the roots.(10) The low K supplies affected little on the activities of the acid phosphatase.In general, low K stress suppressed the growth of Hevea seedlings, changing the characteristics of nutrient metabolism and decreasing the contents of chlorophyll, but also launching the systems of cell defenses,changing the contents of Osmotic regulation substances(such as SOC and SPC),regulating the activities of SOD,POD and CAT in anti-enzyme system.Thus, Hevea seedling can timely regulated the statuses of physiological metabolism to relieve the stress harm and to adapt the stress environment. 2. Seperation of low k-induced Hevea seedling genes(1)Four suppression subtractive hybridization (SSH) libraries firstly were constructed, including two leaf libraries (forward and reverse) and two root libraries (forward and reverse). The forward libraries of the leaf and root had432and1096cDNA clones, respectively. The reverse libraries of the leaf and root had377and1004cDNA clones, respectively. The clones revealed a recombination efficiency of95%, and containing cDNA inserts of200-500bp.(2)Most of the seperated unigenes of Hevea seedling under low K stress were low-abundantly expressed. The numbers and percentages of the low-abundantly expressed genes were111(75.00%),127(77.91%),85(57.23%) and55(56.12) in leaf forward, leaf reverse, root forward and root reverse library. So construction of Hevea SSH libraries under low K stress was useful for the seperation and identification of low-abundant differentially expressed genes.(3)The388Hevea seedling Unigenes in SSH libraries under low K stress were screened by oligonucleotidechip.The58gained genes were divided into12categories,including of15unkown functional genes(29.41%),7signal transduction (13.73%),7transcription factors(13.73%),5defence and stress(9.80),4secondary metablism(7.84),3protein synthesis(5.88%),3substance transportation(5.88%),3metablism of osmoregulation substance,l cell gowth(1.96%),1energy metablism(1.96%),1protein degradation(1.96%)andⅠ primary metabolism(1.96%).(4)The relative expressional characteristics of13genes in roots and leaves were studied by QPCR in the treatments of low K supplies,the genes including of soluble s tarch synthase,14-3-3protein,RNA-binding protein,serine/threonine phosphatase,chlorophy Ⅱ A-B binding protein,Photosystem I reaction center subunit N,lrr resistance protein,sen escence-associated protein、60S ribosomal protein L5,tubulin-specific chaperone A,zinc t ransporter3,cation efflux protein/zinc transporter)and ATPase. The results of QPCR in dicated the expressions of13genes were regulated by the low K stress, their expresse d patterns were consistent between QPCR and oligonucleotides, so the united applicati on of QPCR and oligonucleotides chip increased the reliability of screening low k-ind uced genes in Hevea seedling.3. Clone the Hevea seedling full-length cDNA sequences induced by low K stress(1)The full-length801bp cDNA sequence of HbMPPl firstly was cloned by meth od of rapid-amplification of cDNA ends(RACE,hereafter). The HbMPPl encoded a115amino acids(aa) protein whose molecular weight and isoelectric point were12.7Kda a nd5.72,respectively. The HbMPP1which most probably locates on nucleus has95%a nd94%amino acid sequence identities to the serine/threonine phosphoesterase family P rotein of Arabidopsis_thaliana and Oryza_sativa_Japonica_Group.Moreover, the HbMPP1has a conserved domain of MPP-superfamily superfamily between the first to66th a mino acid, having a active site/metal-binding site in the16th amino acid. The results o f QPCR indicated the low K stress from the first to the eighth day significantly incre ased the expressed levels of HbMPPl in root and stem,but decreasing the expressed levels in leaf.(2)The549bp cDNA sequence of HbCAB1firstly was cloned by RACE.The Hb CAB1probably encoded a protein composed of133amino acids(aa).The molecular we ight and isoelectric point of HbCAB1were13.9Kda and4.84,respectively.Moreover,the HbCAB1which most probably locates on chloroplast has95%and98%amino acid se quence identities to the Lhcbl-2of Selaginella tamariscina and the CAB of poplar.The results of QPCR indicated the expressed levels of HbCAB1significantly were increas ed in root after the fourth day of low K stress,but siginificantly being decreased in st em and leaf in five time points of the low k stress.This dissertation made a comprehensive study of the responses mechanisms of Hevea seedling to K-deficiency from the aspects of physiology and molecular biology, constructing4SSH libraries, further screening and validating the differentially expressed genes,cloning two low k-induced Hevea seedling genes.The results presented here would lay a basis for revealing the response mechanism in aspects of morphology,physiology and molecular biology,facilitate the rational application of K-fertilizer in rubber plantations, and provide a guidances for genetic modification of Hevea for improved K-nutrition.

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