| Rubber tree(Hevea brasiliensis Muell,Arg)is the main source of production of natural rubber(NR).Tapping is the only way to obtain NR,as well as a kind of damage to rubber tree.Previous studies have showed that concentration equilibrium of copper ion in latex plays an important role in stress tolerance and rubber biosynthesis,while copper transporter(COPT)is the key to this balance mechanism.Copper ion(Cu)can induce the production of endogenous ethylene and change the quality of latex.As the key enzyme to the equilibrium mechanism,copper transporter is regulated by Ctr/COPT gene family.The biological function of copper transport protein in rubber tree is studied by expression of COPT gene and the physiological function of copper under different stimulating intensitiesaim,which also revealed the relationship between the stimulus intensity and the copper homeostasis of latex,then provide the theoretical basis for the physiological index of stimulating intensity.The content of Cu in latex was significantly higher after ethylene stimulation in 24 hours and reached the highest point at 24h,indicating that copper content in latex significantly increased along with stimulated time in a certain period of time.Higher strength of ethylene gas stimulate was more significant than ethephon stimulation to improve the concentration of copper in latex.It was showed that preliminary showed that within a certain range of stimulus intensity,the stimulation intensity is positively correlated with the concentration of copper in latex.COPT plays a key role in the process of copper transport.In present study,9 genes of rubber tree copper transporter(COPT)belonging to the Ctr family,were cloned and named as HbCOPTl-9.Bioinformatics analysis showed that HbCOPTs are composed of 1 conserved amino acid residues of Ctr transporters and 2-3 transmembrane domains.Amino acid sequence analysis showed that HbCOPT1,HbCOPT2 and HbCOPT3 had high identity with AtCOPT5;HbCOPT4,HbCOPT5 and HbCOPT6 had high identity with AtHbCOPT6;HbCOPT7,HbCOPT8 and HbCOPT9 had high identity to AtHbCOPT3.Phylogenetic analysis revealed that the COPT genes could be divided into 3 clades(?-?).HbCOPT4 belonged to ? subgroup;HbCOPT5-6 belonged to ?subgroup;HbCOPT1-3 belonged to ? subgroup.The above results indicate that the 9 genes belonged to the HbCOPT genes which were associated with copper transport.HbCOPT genes expressed in different tissues of rubber tree by fluorescent quantitative PCR,except that HbCOPT4 and HbCOPT9 did not expressed in rubber latex.HbCOPT1,HbCOPT4,HbCOPT7,HbCOPT8 and HbCOPT9 had the highest expression in root,and HbCOPT4 was a root-specific cexpressed gene.HbCOPT2,HbCOPT5 and HbCOPT6 had the highest expression level in leaves,and HbCOPT3 had the highest expression level in latex.The expression of HbCOPT1,HbCOPT2,HbCOPT3,HbCOPT6 and HbCOPT8 were upregulated by ethylene stimulation,and the expression of HbCOPT gene reached the highest at 12 h after treatment,indicating that HbCOPT was regulated by ethylene.Increased stimulation intensity the amount of HbCOPT gene was up-regulated and expressed significantly higher than ethephon stimulation,except that HbCOPT7 and HbCOPT8 was down-regulated after ethylene treatment.The results of functional complementary experiments of yeast mutants showed that defective yeast mutants(ctr1?ctr3?and ctr1?ctr2?ctr3?)could grow on non-1 fermented carbon source(YPEG)medium after remedied by HbCOPT and HbCOPT1-3 and HbCOPT7-8 had the physiological function of copper transport in yeast.It was showed that these HbCOPT genes can encode proteins with copper transport function,among them HbCOPT1 and HbCOPT2 were high affinity copper ion transport proteins.This study indicated that the strength of stimulation induced the expression of HbCOPT gene,HbCOPT protein had transport function of copper.Within a certain range of stimulus intensity,the stimulation intensity is positively correlated with the concentration of copper in latex,it was showed that stimulus intensity was closely related to latex copper homeostasis. |
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