Cloning And Preliminary Expression Analysis Stimulated By Ethephon On Aquaporin Genes From Hevea Brasiliensis

At present, China has become the largest consumption country of natural rubber in the world. Yet the self-sufficiency rate of natural rubber in China is less than 1/3 and areas in tropical region of China for rubber cultivation are very limited, hence increasing of the yield of rubber tree has become a major issue to be solved in natural rubber latex industry. The production of latex is mainly affected by the mechanisms of latex regeneration, latex flow and laticifer differentiation, which are closely related with water balance in phloem. As the specific and efficient bi-directional transmembrane water channel in plasma and vacuole membrane, aquaporin could quickly adjust the water flowing in cells and regulate the growth and metabolism of plant. Therefore, the water-specific protein-AQP in protoplast and vacuole membrane may be closely related with latex production. However, there are only HbPIP2; 1 and HbTIP1; 1 cloned in rubber tree PB217 and limited to non-tapping trees and part of aquaporin genes. In this study, two aquaporin genes from Hevea brasiliensis (CATAS7-33-97) were cloned and a preliminary analysis on their expression was measured. The main results are as follows:1. Pairs of degenerate primers were designed according to the conserved regions of amino acids which enode the aquaporin genes. By the methods of PCR and RACE, two aquaporin genes were cloned from Hevea brasiliensis (CATAS7-33-97), named HbPIP1 and HbPIP2. The accession numbers were GQ479823 and GQ479824 in GenBank. HbPIP1 and HbPIP2 encode 287 amino acids and 278 amino acids respectively.2. HbPIP1 and HbPIP2 both possess the MIP family signal consensus sequence- SGXHXNPAVT/SFG and the highly conservative sequence-GGGANXXXXGY, TGI/TNPARSL/FGAAI/VI/VF/YN of the higher plant, which are the typical structure of plasma intrinsic proteins. HbPIP1 and HbPIP2 both exhibit six transmembrane domains, containing the highly conserved unit-EXXXXTXXF/L. The nucleic acid and amino acid sequence homology analysis shows that HbPIP1 belongs to PIP1 subfamily and HbPIP2 to PIP2 subfamily.3. The amino acid homology of HbPIP1 and HbPIP2 reaches more than 95% with ricinus communis, more than 88% with gossypium hirsutum, populus tremula, vitis vinifera, arabidopsis thaliana, juglans regia, zea mays. The nucleotide sequence homology of HbPIP1 and HbPIP2 with HbPIP2; 1 and HbTIP1; 1 is 69%, 72% and 44%, 49%. HbPIP1 and HbPIP2 are new members of aquaporin family.4. Real-time PCR analysis shows that the expression of HbPIP1 and HbPIP2 triggered by ethephon were significant time-effectiveness. The expressions of both HbPIP1 and HbPIP2 genes were up-regulated to their peak afrer 6 hours stimulation. Then they were sharply down-regulated below the general level CK, with slight recovery.