【作者】 黄娟；

【导师】 张硕新； 安锋；

【作者基本信息】 西北农林科技大学 ， 生态学， 2011， 硕士

【摘要】 巴西橡胶树(Hevea brasiliensis)俗称橡胶树,是一种产胶植物,由它生产的胶乳制成的天然橡胶广泛运用于人们日常生活的各个方面并有着不可替代的作用。由于橡胶树对生长条件的要求较高,中国的植胶区主要集中在海南、云南和广东的热带和南亚热带地区,在植胶面积有限的情况下,提高橡胶树的产量就成为满足天然橡胶需求增长的重要途径。排胶作为限制橡胶树产量的主要因素,与胶乳的粘度和排胶时间的长短密切相关,而胶乳粘度又与胶乳中的含水量呈负相关。水通道蛋白(Aquaporin,AQP)作为植物体内水分跨膜运输的高效、专一的蛋白通道,普遍存在于植物体的各个器官中,在植物的水分代谢与运输中起着不可替代的作用。而关于橡胶树体内的水通道蛋白的研究成果表明,水通道蛋白无论是在水分运输方面还是在产量方面,都有很重要的作用。但这些研究只局限于部分水通道蛋白,需要理清的东西很多。因此,克隆和系统研究巴西橡胶树中水通道蛋白,对阐明橡胶树韧皮部水分平衡及其对水分胁迫等的响应机制,进而提高橡胶树的产量具有重要意义。本研究以橡胶树“热研7-33-97”为材料,克隆了2个水通道蛋白基因(HbPIP1;2和HbPIP2;2),并且对这两个基因编码的蛋白进行了生物信息学的分析和结构功能的预测。主要的研究结果如下:1根据GenBank中已经公布的不同植物的水通道蛋白基因的保守氨基酸序列,设计两对简并引物,然后利用RACE技术克隆得到了两个橡胶树树皮水通道蛋白基因的全长cDNA,分别命名为HbPIP1;2和HbPIP2;2,序列已提交至GenBank,登录号为HQ328777和HQ328778。HbPIP1;2编码273个氨基酸,分子量为29.15kD,等电点为8.55;HbPIP2;2编码278个氨基酸,分子量为29.78kD,等电点为8.20。2对HbPIP1;2和HbPIP2;2基因编码的氨基酸序列生物信息学分析表明,它们除了含有水通道蛋白基因家族的信号序列“SGXHXNPAVT”外,并且含有质膜水通道蛋白PIP的两个高度保守序列,“GGGANXXXXGY”和“TGI/TNPARSL/FGAAI/VI/VF/YN”。进一步在NCBI上对其进行Blast比对,结果表明HbPIP1;2属于PIP1亚类,HbPIP2;2属于PIP2亚类。3核苷酸和氨基酸序列比对表明,HbPIP1;2和HbPIP2;2均含有植物水通道蛋白典型的6个跨膜结构域;为疏水性蛋白;不存在信号肽,为非分泌性蛋白;对它们的磷酸化位点预测表明HbPIP1;2具有9个磷酸化位点,HbPIP2;2具有7个磷酸化位点;同时,HbPIP1;2和HbPIP2;2都具有2个二硫键。氨基酸序列的同源性分析表明HbPIP1;2和HbPIP2;2与同科蓖麻水通道蛋白基因RcPIP1-3和RcPIP2-1的同源性分别为91%和89%,与已经公布的HbPIP1,HbPIP2,HbPIP1;1,HbPIP2;1和HbTIP1;1的同源性分别为:61%,55%,57%,66%。38%和56%,58%,59%,61%,36%,并具有相同功能域。因此推测HbPIP1;2和HbPIP2;2都属于水通蛋白基因家族的新成员。4结构预测表明,HbPIP1;2和HbPIP2;2两个蛋白比较相似,随机卷曲在整个蛋白的二级结构中所占比例最大,而α-螺旋和延伸链所占比例相当,并且这两者分散于整个蛋白质中。通过PDB在线搜索水通道蛋白的同源模板,利用Swiss Mode Seve程序对HbPIP1;2和HbPIP2;2的三级结构进行了在线预测。更多还原

【Abstract】 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.更多还原