| The Hevea brasiliensis is commonly called rubber tree, it can produce natural rubber, which is widely used in all aspects of people's daily life and cannot be replaced. Due to rubber tree can only grow well under superior condition, the mainly planting regions of China are Hainan, Yunnan, Guangdong tropical and Subtropical regions, since the rubber cultivation areas are very limited, increasing of the yield of rubber tree has become a major way to meet the nature rubber demand. Latex yield depends on the characteritics of latex flow, including its velocity and duration, latex viscosity which is inversely related to its water content. Aquaporin, as the specific and efficient transmembrane water channel of plant, is widespread exist in all organts of plant and cannot be replaced in water metabolism and transport. Researches on the aquaporins of rubber tree showed that they played a very important role in both water transportation and latex yield. However, the researches were only limited to partial aquaporins, of which there have been some questions to be answered. So, clonging and system research on the aquaporins of rubber tree will be helpful to clarify the water balance in phloem and the mechanism of water stress, and then important for improving latex yield.In this study, we have cloned two aquaporin genes(HbPIP1;2 and HbPIP2;2)from the bark of rubber tree (CATAS7-33-97) and analyzed the characteristics of the encoding proteins with bioinformatic method, then predicted the functions. The main results are as follows:1. Degenerate primers were designed based on the conserved amino acid sequence of the published AQPs from different plant species, the method of RACE was adopted and two full-length cDNAs encoding aquaporin genes were cloned from the bark of rubber tree, named HbPIP1;2 and HbPIP2;2, their sequences have been deposited in GenBank, with accession numbers were HQ328777and HQ328778. The HbPIP1;2 encoding 273 amino acids with molecular weight 29.15kD and theoretical pI 8.55; and the HbPIP2;2 encoding 278 amino acids with molecular weight 29.78kD and theoretical pI 8.20.2. Bioinformatic analysis on the encoding amino acid sequence of HbPIP1;2 and HbPIP2;2 showed that both HbPIP1;2 and HbPIP2;2 possess the MIP family signal consensus sequence"SGXHXNPAVT"and the highly conservative sequences"GGGANXXXXGY"and"TGI/TNPARSL/FGAAI/VI/VF/YN"of the higher plant are the typical structure of plasma intrinsic proteins. Comparison of their Blast based on the NCBI, showed that HbPIP1;2 belongs to PIP1 subfamily and HbPIP2;2 to PIP2 subfamily.3. The analysis on the nucleic acid and amino acid sequence homology showed that both HbPIP1;2 and HbPIP2;2 contained six transmembrane areas, and were predicted hydrophobic protein, without signal peptide and secretory proteins. HbPIP1;2 has nine phosphorylation sites and HbPIP2;2 seven phosphorylation sites. Meanwhile, both of them have two disulfide bonds. The amino acid homology of HbPIP1;2 and HbPIP2;2 reaches more than 91% and 89% with Ricinus communis (RcPIP1-3 and RcPIP2-1), the HbPIP1;2 and HbPIP2;2 have 61%,55%,57%,66%。38% and 56%,58%,59%,61%,and 36% homology respectively compared with the published aquaporin genes of the HbPIP1,HbPIP2,HbPIP1;1,HbPIP2;1 and HbTIP1;1. Therefore, the HbPIP1;2 and HbPIP2;2 are new members of plant aquaporin family.4. The predicted secondary structure of HbPIP1;2 and HbPIP2;2, demonstrated that the random coil is the main motif the two proteins. Alpha helix and extended strand spread to the whole secondary structure of proteins and share the equal ratio. By searching the homologous template of aquaporins with Swiss Mode Seve software online the predictedtertiary structure were given. |
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