巴西橡胶树死皮相关基因Hbf的克隆及表达

【作者】 傅湘辉；

【导师】 陈守才；

【作者基本信息】 华南热带农业大学 ， 作物遗传育种， 2001， 硕士

【摘要】 死皮给橡胶种植业带来了严重危害，研究和阐明橡胶死皮发生的分子机制，具有重大的理论和实际意义。在前期工作中，我们对5个无性系的健康和死皮树胶乳和树皮进行DDRT-PCR（Differential Display Reverse Transcription-Polymerase Chain Reaction），鉴定了一个在健康树中表达活跃，在死皮树中表达受到严重抑制的差异cDNA片段Hb-1（721bp）（其相应基因命名为Hbf）。经Genbank检索，Hb-1为新基因片段，Northern杂交证实其为真实的差异带。 在此基础上，本研究进一步运用5’RACE方法扩增得到了Hbf cDNA。该cDNA共1204个核苷酸，含有完整的阅读框架，编码310个氨基酸。包含一个45bp的5’非编码区和一个229bp的3’非编码区。将cDNA序列和推导的氨基酸序列进行BLAST分析，结果表明它与拟南芥的转录激活子F16M14.2具有较高的同源性。在氨基酸水平上，其同源性为59％。同时，它与Ibbf、Mybltf、MybStl、Ibbfl、F16M14.2等DNA结合蛋白具有相同的HTH DNA结合域。蛋白质结构域预测结果表明在全长氨基酸序列中含有一个可能的N糖基化位点(¹⁶⁰NISP)、三个可能的cAMP/cGMP依赖性蛋白激酶糖基化位点(¹²⁰KPTT、¹⁹³RRSS、²¹⁶KKSS)、一个可能的PKC磷酸化位点(¹⁶⁷TTR)、9个可能的酷蛋白激酶Ⅱ磷酸化位点(³⁰TPEE、⁶³TVGD、⁷⁹SDIE、⁹²SSSD、¹²⁸SEQE、¹³⁹TEEE、¹⁹⁶SIHD、²¹²SPDE、²¹⁸SSPD)、5个可能的N—豆蔻基化位点(¹¹⁸GGKRTT、¹³⁵GVPWTE、²³⁷GMAKGL、²⁵¹GGGAAG、²⁸⁸GTLPGY)、一个可能的胺基化位点(¹¹⁸GGKR)及一个可能的ATP/GTP结合位点(⁵⁶AAVIPGKT)。蛋白质定位信号分析表明Hbf基因表达产物极有可能是一种核蛋白。 根据Hbf cDNA序列，设计一对特异引物，经PCR扩增得到了全长为1277bp的DNA序列。序列分析表明，该DNA除包含有HbfcDNA编码区外，还含有一个344bp的内含子。它是一个典型的核mRNA内含子，包含明显的5’端的供体剪切位点、3’端的受体剪切位点和分支点序列。 Southern杂交和酶切位点分析表明，Hbf为低拷贝基因，只有2个拷贝，且橡胶各无性系及健康树与死皮树之间并不存在拷贝数的差别。 Northern杂交表明，Hbf基因在各无性系的健康树的表达远高于其在死皮树中的表达；从无性系间进行比较，越耐死皮的无性系，其表达量越高；Hbf基因的表达在不同组织中存在表达量的差别，树皮最高、乳管次之、叶片最低；Hbf基因在死皮树不同部位的乳管中也存在着差异表达，即割线处表达量最低，阴刀处次之，而距离割线的较远处（未发生死皮）表达量最高。 利用 GenomeW8lker的方法获得 Hbf基因的 5’前导序列，序列分析表明，它可能包含启动子区域，并对其调控元件进行了分析。 用定向克隆方法把带有起始密始子的成熟Hbf基因插入pBllZI质粒，构建了Hbf的植物表达载体pBIH。更多还原

【Abstract】 CIoning and Expression of Hbf associated with TappingPanel Drvness of H6vea brasmensiSAbstractIn previous study, a cDNA fragment Hb-1 of H6vea brasgfen5iS was obtained byDDRT-PCR.The objective of this study was to cIone and express this cDNA.Degenerate primers were designed based on the sequence of Hb-1 to amplify thecorresponding fuII cDNA of Hb-1 by 5’-RACE. The fuII-length cDNA, called Hbf (for HE veabrasHfensiS faCtor), was 1204 bp in Iength with a 930 bp open reading frame. A5’-UTR(untransIated region) of 45 bp and a 3’-UTR of 209 bp was fOund. It deduced aminoacid sequence shows similarlties 59% with F16N14.2 and pre5ents a common DNA bindingmotif with F16M14.2, Ibbf, Ibbfl, Mybltf and MybSt1. The resuIts of protein domain predictionindicate that there may be a N-glycosyIation site, three CAMP- and cGMP-dependent proteinkinase phosphorylation sites, a Protein kinase C phosphorylation site, nine Casein kinase IIphosphoryIation sites, flve N-myristoyIation sites, an Amidation site and an ATP/GTP-bindingsite. The resuIt of PSORT (PrediCtion of Protein LocaIization Sites) suggests that hbf mayencode a nucIear protein.The DNA sequence of Hbf was obtained using PCR. The product was 1277 bp in lengthwith a M4 bp intron, which contain apparently a donor splice site, an acceptor splice site anda branch point.The result5 of Southern anaIysis indicated that Hbf has Iow copies in the H6veabrasHtensiS gename and there was no diff6rence in copies between the heaIthy trees and theTPD trees.The resuIts of Northern analysis indicated that Hbfwas expressed significantIy higher inheaIthy trees than in TPD trees and it was expressed highest in bark (near cambium) andlowest in Ieaf. Moreover, Hbfwas expressed f8r Iower in the Iatex of the tapping line than inthose of f8r from the tapping Iine. Those f8Cts indicated that the leveI of expression Hbf isrelated to TPD of Hevea brasHfen5iS.The 5’ Ieader sequence of Hbfwas obtained by using GenomeWaIker. It might contain apromoter region.The gene fragment of Hbf was obtained by PCR method and inserted into pBI121 vectorat the site of Xba I and Sac I to construCt an expression vector.更多还原