【作者】 王晟；

【导师】 杜雄明；

【作者基本信息】 中国农业科学院 ， 作物种质资源学， 2008， 博士

【摘要】 本研究以亚洲棉(Gossypium arboreum L.)种质DPL971及其无短绒有纤维突变体(DPL972)为材料。从亚洲棉中分离得到腺苷酸环化酶结合蛋白(CAP)的cDNA表达序列及基因组DNA序列。为深入研究CAP基因对棉纤维发育的作用,对克隆的CAP基因的cDNA序列进行了表达和蛋白表达纯化,还分析了CAP基因在棉纤维早期发育阶段的表达水平及蛋白性质。主要研究结果包括:1.对253条棉纤维(表皮毛)发育相关基因在DPL971和DPL972基因组中的基因一级结构进行了比较。寻找到了与这对材料表型性状差异紧密相关的目标基因——CAP基因。2.以DPL971和DPL972为材料,利用RT-PCR技术克隆了亚洲棉的全长CAP基因-GaCAP和GaCAPm。该基因的开放阅读框(ORF)有1416 nt,共编码一个471氨基酸的蛋白质,分子量为50.6kDa。核苷酸序列及氨基酸序列的同源性比较表明:GaCAP及GaCAPm与已经报道的其它物种的CAP基因具有有较高的同源性。3.对CAP基因在野生型和突变体中的表达谱进行了RT-PCR和Northern杂交分析。结果显示,CAP基因在野生型和突变体材料的棉纤维发育早期阶段(-1～4 DPA)的表达信号稳定。但在突变体材料的4～9 DPA阶段,CAP基因的表达量随着时间的推移呈下降趋势,尤其是在9 DPA时,CAP基因仅有痕量表达。在野生型及突变体材料的叶片、下胚轴和根等组织器官中,均检测到了CAP基因一定丰度的表达信号。上述试验结果显示,CAP基因在棉花各组织器官中均有表达,尤其在棉纤维发育早期阶段的胚珠中表达信号较高;比对CAP基因在野生型及突变体胚珠不同发育阶段的表达信号,推测CAP基因参与了亚洲棉短绒纤维发育的起始过程。4.按所分离的棉花腺苷酸环化酶结合蛋白cDNA序列设计、合成特异引物,以亚洲棉叶片DNA为模板,通过PCR扩增分离获得棉花GaCAP基因DNA序列。利用DNAStar对该基因的序列进行分析表明:GaCAP基因编码区含有10个外显子和9个内含子,内含子的大小在100～1000 bp范围内不等。CAP蛋白在棉花和拟南芥中的序列长度一致(分别为471和476个氨基酸残基),但其在棉花(序列长度约4kb)和拟南芥(序列长度与2.4kb)基因组中的存在情况不同,说明棉花CAP基因内含子的复杂程度较高。5.多序列比对结果显示:GaCAP蛋白的C-末端与拟南芥、苜蓿和水稻的相似度达到83%,而N-末端的序列相似性分别为74%、73%和63%。表明GaCAP蛋白的C-末端较N-末端更为保守;CAP蛋白的特征序列:RLE重复基序(RLE repeats)、Actin结合域(actin binding domain)、SH3基序(SH3 motif)和多聚结合域(multimerization domain)均定位在预测的GaCAP蛋白序列之中;GaCAPm编码的蛋白序列的第44位是苏氨酸(极性氨基酸),这与GaCAP编码蛋白序列的同一位置(丙氨酸,非极性氨基酸)不同,而且在不同物种的CAP蛋白中,该位点是极为保守的,因此,这一位点的突变可引起GaCAPm蛋白的结构和功能变化。6.以GaCAP基因的cDNA序列为探针与基因组DNA酶切片段进行Southern blotting杂交分析,结果显示棉花基因组中仅含有一个CAP基因的拷贝。7.构建了GaCAP原核表达载体,在E.coli中进行表达,并纯化目的蛋白,进行理化分析。结果显示,GaCAP蛋白的最适反应温度为36℃,并且可以在一个较宽的温度范围(30～60℃)内保持其高结合活性(＞60%);该蛋白的最适pH值在5.0～6.0之间,在pH=4.0的缓冲液中处理30min后,GaCAP的蛋白活性仍然有80%,说明该蛋白有一定程度的耐酸性。更多还原

【Abstract】 Two cotton germplasm(Gossypium arboreum L.),DPL971 and its lint-fuzzlessmutant(DPL972), were used as materials in this study.The cDNA and DNA sequences of adenylyl cyclase-associated protein(CAP)were obtained from DPL971 and DPL972.To gain insight on the CAP role in cotton fiber development,the cloned CAP cDNA was expressed and purified.The expression level in earlier stages of fiber development and protein characters of this CAP gene were analyzed in this research. The main results are as follows:1.The primary structure of 253 fiber(trichome)development associated-genes were compared between fuzzless mutant(DPL972)and wild-type(DPL971),and the target gene(cap)closely related to the fuzzless mutant was found.2.The whole cDNA sequence of the cap genes(GaCAP/GaCAPm)were cloned between DPL971 and DPL972 by RT-PCR technology.This gene possess an open reading frame(ORF)of totally 1416 nt encoded a protein of 471 amino acids,and the molecular weight of its protein is 50.6kDa.The deducible nucleotide sequence and amino acid sequence of GaCAP/GaCAPm are highly homologous to the CAP genes of other species.3.The expression patterns of GaCAP and GaCAPm were identified in different tissues from DPL971 and DPL972 using semi quantitative reverse transcriptase PCR(RT-PCR)and confirmed by Northern blot analysis.The results showed that CAP transcripts maintained a persistent high level during the early ovule development(from -1 to 4 DPA)in both DPL971 and DPL972.While the level of CAP transcripts in the mutant decreased gradually as the ovule developed(4 to 9 DPA)and hardly any transcripts were detected in the 9 DPA ovule.In addition,a moderate level of CAP was detected in leaves,hypocotyls,and roots during the different developmental stages.These results indicated the CAP gene specifically expressed in ovules during the fiber developmental stages compared with that in other tissues over various vegetative periods.The comparison of gene expression profiles between wild type and fuzzless mutant also provided the evidence that CAP was involved in fuzz formation.4.The specific primers were designed by the GaCAP cDNA sequence isolated from cotton.Using the leaf DNA of Gossypium arboreum L.as template,GaCAP DNA sequence was obtained by PCR. The gene’s sequence was analyzed by means of DNAStar.The coding region of the GaCAP gene contains ten exons and nine introns.The intron size is approximately ranged from 100 to 1000 bp.The CAP proteins in G.arboreum(471 amino acids)and A.thaliana(476 amino acids)are similar in size. However,the genome sequence of GaCAP(～4kb)was much larger than AtCAP gene(～2.9kb)which indicated that the sequence of intron is more complex in G.arboreum.5.The alignment of the amino acid sequences between G.arboreum CAP and the CAPs from other species showed that the identity between the C-terminal domain of G.arboreum L.CAPs and those of A. thaliana,Medicago truncatula and Oryza sativa CAP is 83%,while the N-terminal domain of G arboreum L.CAP is 74%,73%and 63%identical with the corresponding regions of A.thaliana,M. truncatula and O.sativa CAP,respectively.So the C-terminal domain of G.arboreum L.CAP shows higher homology with other plant CAPs than the N-terminal domain.The CAP key motifs(e.g.Arginine, Leucine and Glutamic acid repeats motif,actin binding domain,SH3 motif and multimerization domain) were consistently found in the putative GaCAP protein sequence.Intriguingly,the nonpolar amino acid Alanine was substituted by a polar amino acid Threonine at conserved position 44 of GaCAPm.This may result in the different structure and function of GaCAPm from other CAP homologs.6.To determine the copy number of GaCAP gene homologue in cotton genomic DNA,Southern blotting hybridization was conduced by applying GaCAP cDNA probe.The result indicated that there was only one copy in cotton genome.7.The Prokaryota expression vector was constructed,the protein was expressed in vitro and purified to study the characterization of objective protein.The optimal temperature of the recombinant CAP was 30-60℃with a best temperature at 36℃,and its optimal pH was 5.0～6.0.The GaCAP maintained 80%activity after treated 30 min in the buffer with pH 4.0,and this reveal the protein is acid tolerance.更多还原