【作者】 贺亚军；

【导师】 张天真；

【作者基本信息】 南京农业大学 ， 生物技术， 2008， 博士

【摘要】 棉花是世界重要的经济作物之一。棉纤维是棉子上的一种表皮毛,也是重要的经济价值之所在,是纺织工业的原料。优良的纤维品质是世界棉花育种的主要目标之一。因此,从分子水平阐明棉纤维发育的机理,具有重要的理论价值与现实意义。本研究的目的即利用基因芯片方法筛选与棉纤维品质相关的EST,并分离和鉴定部分棉纤维发育相关基因,这些研究将有助于我们进一步探讨形成优质纤维的分子机理。构建高强纤维种质系7235开花后5-25天棉纤维伸长、次生壁加厚期间的cDNA文库,随机大规模测序,获得1436条EST,制成基因芯片。提取7235和纤维品质一般的陆地棉遗传标准系TM-1不同发育时期的mRNA与之杂交,筛选到31条差异表达EST,主要是参与代谢、次级代谢、胁迫与抗逆、转录等相关基因。通过RT-PCR验证,23条差异表达EST与基因芯片实验结果一致。为了进一步定位这些差异表达EST并分析其与纤维品质性状的相关性,我们分析了所有差异表达EST在7235与TM-1间的多态性。结果表明,三条EST在7235与TM-1间存在多态。利用(7235×TM-1)RIL群体,对这三条EST进行染色体定位及单标记分析,结果表明,两条EST(分别编码LTP基因与ADH基因)与纤维品质性状极显著相关。由于大部分基因在7235与TM-1间不存在差异,所以,我们利用[(TM-1×Hai 7124)×TM-1]群体对其进行定位。结果表明,13条差异表达EST被定位到四倍体棉花遗传图谱的相应染色体上,其中8条EST定位在品质相关QTL区间或附近。通过基因芯片技术,我们鉴定了一条可能编码谷氨酰胺合成酶的EST。Northern杂交验证了该基因在7235与TM-1开花后8天的胚珠和纤维中表达有显著差异。随后,利用7235纤维cDNA文库测序,结合5′RACE技术从7235纤维中获得了全长谷氨酰胺合成酶基因。序列分析结果表明,该基因与已报道的其他植物的细胞质谷氨酰胺合成酶(GS1)基因具有较高氨基酸序列同源性,命名为GhGS。Southern blot分析结果表明,GhGS在陆地棉基因组的A、D亚基因组可能各存在一个拷贝。之后,从不同材料7235、TM-1、非洲棉、雷蒙德氏棉基因组中分离到GhGS基因序列。根据7235与TM-1基因组GhGS序列的SNP,将该基因在(7235×TM-1)RIL群体中进行定位并分析基因与纤维表型的相关性,结果发现,GhGS与纤维强度存在相关,但由于该RIL图谱上的位点较少,GhGS基因不能整合到该图谱上。随后,利用[(TM-1×Hai 7124)×TM-1]群体,将GhGS基因定位在四倍体棉花遗传图谱的D7染色体上。另外,我们测定了棉纤维发育不同时期胚珠和纤维的GS活力和总蛋白含量。测定结果表明,在开花后5天和8天的7235胚珠GS活力显著高于TM-1相应时期的胚珠GS活力。同时发现,开花后11天的7235胚珠总蛋白含量和胚珠重量都显著高于同时期TM-1胚珠总蛋白含量和重量。这一结果暗示GS可能通过提供N而促使棉花种子的形成。而在纤维中的测定结果表明,在7235中GS活力和总蛋白含量都低于TM-1中的GS活力和总蛋白含量。这一结果暗示GS在胚珠和纤维中行使的功能可能并不完全相同。GS在纤维发育过程中的可能功能还需进一步探讨。另一个克隆的基因为脂肪酶基因。通过对7235纤维cDNA文库测序,获得一个可能编码脂肪酶的cDNA片段。随后,利用RACE技术获得该cDNA全长并命名为GhLipase。序列分析结果表明,GhLipase全长1286bp,包含一个编码368个氨基酸的开放读码框。BLAST分析表明,它与已报道的其他植物的脂肪酶基因具有较高的氨基酸序列同源性。从表达特征看,GhLipase在胚珠和纤维细胞优势表达,而且在开花后5-17天的纤维中表达水平较高。Southern blot结果表明,GhLipase基因在陆地棉基因组中可能存在两个拷贝。将该基因定位在四倍体棉花遗传图谱的A13染色体上。第三个利用7235cDNA文库分离的基因为木聚糖水解酶基因。该cDNA全长1790bp,ORF编码560个氨基酸。CDS分析表明,GhXyl基因推导的氨基酸含有糖基水解酶家族10的保守区域,具有典型的糖基水解酶家族10基因结构,BLAST结果表明其与来源于其他植物的木聚糖水解酶有较高的同源性,命名为GhXyl基因。RT-PCR和Northern blot分析结果表明,GhXyl属纤维细胞优势表达,且在开花后5-8天的纤维中表达水平最高。Southern杂交结果表明木聚糖水解酶基因在陆地棉基因组中存在两个拷贝。将该基因定位在四倍体棉花遗传图谱的A3染色体上。本研究利用7235纤维cDNA文库,还分离到六个小分子质量的热激蛋白基因,分别命名为LMWHSP1、LMWHSP2、LMWHSP3、LMWHSP4、LMWHSP5、LMWHSP6。分析它们的序列结构,发现它们分别属于三种不同类型的小热激蛋白,LMWHSP1、LMWHSP2、LMWHSP4、LMWHSP6都与细胞质Ⅰ类热激蛋白序列同源性较高,而LMWHSP3与细胞质Ⅱ类热激蛋白序列同源性较高,LMWHSP5与线粒体热激蛋白序列同源性较高。对这六个小分子质量热激蛋白基因进行转录表达分析,结果表明,它们在棉花体内具有不同的表达特征,LMWHSP2是组成型表达的基因,LMWHSP 1、LMWHSP 4、LMWHSP5、LMWHSP6都在开花当天的胚珠中表达量最高,而LMWHSP3在棉花叶片中优势表达。这一结果暗示,线粒体小热激蛋白和细胞质Ⅰ类小热激蛋白基因可能受纤维启始和分化的调控,而细胞质Ⅱ类小热激蛋白与棉花叶片的生长发育相关。利用本实验室的四倍体棉花遗传图谱,对这六个小热激蛋白基因进行定位,其中三个基因LMWHSP1、LMWHSP2、LMWHSP3分别被定位在A4、D8和A6染色体上。更多还原

【Abstract】 Cotton is one of the most important economic crop in the world.Cotton fiber,which plays an important role in global economy and as a raw material in the textile industry,is a single-celled hair or trichome that develops from individual epidermal cells on the outer integument of cotton ovules.The super quality fiber is one of the major goals in the cotton breeding all over the world.So,it is important to elucidate fiber development process and major factors related to fiber quality by molecular biology.This study served to screen ESTs associated with cotton fiber quality by using cDNA microarray,to isolate and identify some genes related to fiber quality.This would help us to understand the heredity mechanism underlying super quality fiber and lay a basis for further study.A cDNA library was constructed using 7235 fibers at 5 to 25 DPA.At this time,the fiber cells were in the process of elongation and synthesis of secondary cell wall cellulose. Random sequencing generated 1436 ESTs and a microarray containing the 1436 ESTs was subsequently identified by reciprocal hybridization using 7235 fiber mRNAs,a superior fiber quality property germplasm line,and TM-1,a genetic standard in G.hirsutum. Thirty-one ESTs involved in metabolism,defense,secondary metabolism,transcription,and unknown function were found differentially expressed in the two varieties.In order to further confirm the data from the microarray analysis,RT-PCR was performed for these 31 differentially expressed ESTs and we found 23 of 31 ESTs expressed differently between 7235 and TM-1.To map the differentially expressed ESTs and analyze the correlation of DNA polymorphism and phenotypic effect for fiber quality,all differentially expressed ESTs were chosen to conduct a 7235 and TM-1 polymorphism analysis.Between the two varieties,three ESTs exhibited polymorphisms.Using a RIL population derived from 7235×TM-1,the three ESTs were detected by single-marker analysis and the results revealed that two ESTs coded an ADH and LTP gene,which were significantly correlated with fiber quality traits.Since polymorphisms between 7235 and TM-1 are less and most of the differentially expressed genes can not be mapped,the[(TM-1×Hai 7124)×TM-1] population comprised of 140 individuals was subsequently used to map these genes. Thirteen of these selected ESTs were mapped on the chromosomes by using the BC1 population from the cross[(TM-1×Hai 7124)×TM-1].Eight of these ESTs are mapping in the QTL intervals or near the QTLs for fiber quality traits.An EST encoding glutamine synthetase(GS) was identified by microarray-based hybridization.Northern blot analysis verified transcript accumulation differences in 8 DPA fibers(including ovules) in the two varieties.Then,the full-length cDNA encoding GS in 7235 was isolated and named GhGS.Sequence analysis revealed the GhGS was similar to cytosolic GS.Southern blot analysis showed tetraploid cotton contained at least one copy of the A sub-genome and the D sub-genome.Genomic GhGS sequences were subsequently isolated from different varieties,TM-1,7235 and two diploid progenitor cottons,G. herbaceum(A-genome) and G.raimondii(D-genome).Molecular mapping and single-marker analysis revealed the GhGS was significantly correlated with fiber strength and was mapped to chromosome D7.Additionally,GS activities and total protein of the ovules and the fibers were assayed.The results showed significantly higher GS activity in 7235 seeds than TM-1 seeds at 5 and 8 DPA.Also significant differences in total protein content and seed weight at 11 DPA were found.This suggested GS promoted the seed-forming process by providing N.On the other hand,in fibers,GS activity and total protein assay indicated the lower total GS activity and longer fiber elongation period in 7235.These results suggest the respective roles of the GS in ovules and fibers do not completely overlap.However,further experimentation is necessary to understand the mechanism of GhGS playing a role in fiber.The second gene we cloned was GhLipase gene.The cDNA library from fibers of 7235 was randomly sequenced and an cDNA fragment encoding lipase was obtained. Subsequently,the full-length cDNA encoding lipase was cloned from 7235 using the rapid amplification of cDNA ends(RACE) technique and named the resulting gene GhLipase. Sequence analysis showed the full-length cDNA was 1286 bp which contains a major open reading frame of 368 amino acids.BLAST analysis indicated that it shared a high homological identity with other lipase from other plants.From its expression characters,the GhLipase gene is dominantly expressed in ovule and fiber cells,and its expression quantity is higher in 5-17DPA fiber ceils.Southern blot analysis showed that there were two copies of the GhLipase gene in the genome of G.hirsutum.By using[(TM-1×Hai7124) TM-1] population,GhLipase gene was mapped on chromosome A13.Sequenced the cDNA library,The third gene was isolated and designated as GhXyl. The full-length cDNA is 1790 bp which contains a major open reading frame of 560 amino acids.CDS analysis showed the amino acids sequence contained a conserved domain of Glycosyl hydrolase family 10.BLAST analysis indicated that it shared a high homological identity with other xylanse from other plants.RT-PCR analysis and Northern blot analysis revealed GhXyl gene was specifically expressed in fiber cells,and its expression quantity was higher in 5-8DPA fiber cells;it was not expressed in root,stem and leaf.Southern blot analysis showed that there were two copies of the GhXyl gene in the genome of G.hirsutum. By using[(TM-1×Hai7124) TM-1]population,GhXyl gene was mapped on chromosome A3.Using the cDNA library from fibers of 7235,six low-molecular-weight heat-shock proteins(HSP) genes were also isolated and designated as LMWHSP1,LMWHSP2, LMWHSP3,LMWHSP4,LMWHSP5 and LMWHSP6,respectiively.Sequence analysis showed they belonged to three different types of low-molecular-weight HSP.LMWHSP1, LMWHSP2,LMWHSP4 and LMWHSP6 all belonged to cytosolic class I low molecular weight HSP,LMWHSP3 belonged to cytosolic classⅡlow molecular weight HSP,and LMWHSP5 belonged to mitochondrial low molecular weight HSP.RT-PCR analysis revealed they exhibited differently transcripional profile in cotton.LMWHSP2 was expressed in all cotton tissues,however,LMWHSP 1,LMWHSP 4,LMWHSP5 and LMWHSP6 possesed higher transcript accumulations in cotton ovules at 0DPA,and LMWHSP3 genes was expressed preferentially in cotton leaves.These result indicated the mitochondrial and cytoplasmic I HSP genes appeared to be under developmental control, with higher transcript accumulation occurring during fiber initiation,however, cytoplasmicⅡHSP genes were more closely correlated with the growth and development of cotton leaves.Using genetic map of the tetraploid cotton constructed by our lab,three of these genes,LMWHSP1,LMWHSP2 and LMWHSP3,were mapped on the cotton chromosomes A4,D8 and A6,respectively.更多还原