Cloning Hbpht1 Gene From Hevea Brasiliensis And Constructing The Ssh Cdna Libraries Of The Roots And Leave By Pi Deficiency-induced

Phosphorus(Pi) is one of the essential macronutrients required by the roots of plants. Phosphorus(Pi) deficiency in soils is a major yield-limiting factor of Hevea in Hainan province. The genetic improvement of Pi efficiency of Hevea is an effective way to relieve the lack of Pi resources, improve the rubber yield and the utilization efficiency of Pi fertilizer.Pi uptake systems consist of two components, that is low-affinity and high-affinity transport systems. Under Pi deficiency, the high-affinity transport system is considered to be the major mechanism for Pi uptake by roots. Genes encoding high affinity Pi transporters have been isolated from many plant species including Lycopersicon esculentum, Arabidopsis thaliana and so on. However, there are not any report about the genes and function of Hevea Pi transporters. Recently, differentially expressed genes of rubber trees carried out step by step. However, by the way of the stress of phosphorus and phosphorus nutrition-related studies of differentially expressed genes is still not reported.In this paper, HbPhtl, a novel phoshpate transporter was cloned for the firsrt time from Hevea. by the techniques of suppression subtractive hybridization (SSH), some express sequence tags (ESTs) were identified as Pi deficiency stress induced. The main results as follows:1 By the techniques of degenerate RT-PCR and RACE, a phoshpate transporter gene was cloned for the first time from Hevea brasiliensis latex, named as HbPhtl (GenBank accession number:HM015901). The full-length cDNA of this gene is 1084bp, the length of 5'untranslated region is 65bp,3'untranslated region is 128bp.2 HbPhtl ORF encods a polypeptide with 536 amino acid residues, predicted molecular mass and isoelectric point are 59 KDa and 8.766 respectively. Hydropathy analysis of the deduced polypeptides showed that the HbPhtl is highly hydrophobic and contains 12 putative membrane-spanning regions. The phosphorylation sites for protein kinaseC (TAR), casein kinase II (SLEE) and the N-glycosylation site (NATT) areconserved among high affinity Pi transporters from other eukaryotes.3 The sequence of phoshpate transporter was highly conserved, which shares more than 70%amino acid identity with some other high-affinity phoshpate transporters of plants. The result of phylogenetic tree indicated that HbPht1 was included in Phtl family, displayed a high degree of sequence similarity to PtrPHT1-4, and belongs to high-affinity Pi transport. 4 The prokaryotic expression of HbPhtl was carried on in Escherichia coli. Recombinant plasmid pET-HbPhtl was structured successfully.5 Two forward subtracted cDNA libraries were constructed via suppressive subtractive hybridization(SSH), the roots and leave cDNA from low-phosphorus stressing Hevea sapling was utilized as driver while tester is cDNA derived from the roots and leave of regular phosphorus treated Hevea sapling. Ohter two reverse subtracted cDNA libraries were constructed simultaneously, the roots and leave cDNA from low-phosphorus stressing Hevea sapling was utilized as tester while driver is cDNA derived from the roots and leave of regular phosphorus treated Hevea sapling. Some phosphorus nutrition-related express sequence tags (ESTs) were obtained from these four SSH cDNA libraries.