【作者】 王瑞；

【导师】 朱伟； 杨作仁；

【作者基本信息】 河南农业大学 ， 作物遗传育种， 2017， 硕士

【摘要】 棉花是世界上重要的经济作物之一,棉纤维是纺织工业的重要原料。目前,陆地棉是最主要的栽培品种之一,其棉纤维产量高,但纤维品质有待改良。提高纤维品质是棉花的主要育种目标,因此,对陆地棉纤维发育相关基因的挖掘及调控机制的探究具有重要意义。棉纤维由胚珠外表皮细胞发育而来,是最长的植物细胞之一。扩张蛋白是一类参与细胞扩大、细胞壁修饰过程的细胞壁蛋白,几乎参与植物的整个发育进程。扩张蛋白在棉纤维细胞发育中的作用仍不清楚。前期研究通过分析棉花纤维短缩突变体pag1的转录组数据,发现扩张蛋白基因GhEXPA8的表达量在纤维伸长期显著下调^[73]。为探讨扩张蛋白在棉纤维细胞发育中的作用机制,本论文开展了陆地棉纤维发育相关基因GhEXPA8的克隆与功能分析研究,结果如下:1.GhEXPA8基因的克隆和生物信息学分析根据中国农科院棉花研究所发布的陆地棉基因组中的核苷酸序列（CotAD₂9957,Expansin2）,从陆地棉中克隆了该基因cDNA编码框全长并将其命名为GhEXPA8。生物信息学分析显示GhEXPA8基因的CDS长762bp,编码253个氨基酸,1-25个氨基酸为信号肽,含有DPBB₁与pollen_allerg₁两个结构域。信号肽和两个保守的结构域都是α扩张蛋白的典型特征。进化树分析进一步表明该基因编码的蛋白隶属于扩张蛋白α亚家族。2.GhEXPA8基因的功能分析荧光定量PCR实验检测GhEXPA8在棉花的不同组织及纤维的不同发育阶段的相对表达量,结果显示GhEXPA8在根、茎、叶、胚珠中表达量较低,在纤维发育过程中呈先上升后下降的趋势,在开花后10天的纤维中表达量最高,在开花后15天的纤维中表达量趋于下降。GhEXPA8启动子驱动GUS基因在拟南芥中表达发现GUS染色出现在转基因植株的各个器官,尤其在根毛、根尖、茎尖较等幼嫩部位。维管组织例如叶脉、叶柄等部位GUS信号也很明显。拟南芥的毛状体发育和棉纤维发育机制类似,GhEXPA8启动子GUS活性在拟南芥根毛处检测到,暗示该基因在棉花中可能与纤维发育有关。GhEXPA8在棉花和转基因拟南芥中的表达模式说明它可能在棉花纤维伸长过程中发挥作用。通过GhEXPA8基因在拟南芥中过表达分析发现拟南芥幼苗根明显变长。这表明GhEXPA8是一个有功能的α扩张蛋白,也暗示了GhEXPA8可能在棉纤维发育中发挥调控作用。另外,上述组织表达模式分析也暗示该基因可能在纤维伸长过程中起作用。综合起来,推测GhEXPA8可能参与纤维发育调控过程。3.以中棉所24下胚轴作为外植体材料,利用根癌农杆菌介导的棉花遗传转化方法获得GhEXPA8转基因棉花T0代转化体24个,为深入解析GhEXPA8基因的功能打下了基础。更多还原

【Abstract】 Cotton is one of the most important economic crops in the world and cotton fiber is important raw material for textile industry.At present,upland cotton is one of the main cultivated species whose fiber yield is high;however fiber quality needs to be improved.High fiber quality is the major goal of the cotton breeding.It is of great significance to discover genes related to land cotton fiber development and to explore their action mechanism on fiber elongation.As one of the longest plant cells,Cotton fiber is developing from ovule outer epidermal cells.Expansin is cell wall proteins that involved in cell expansion and cell wall modification process.It is involved in almost the whole process of the plant development.The role of expansin in cotton fiber cells development is not clear.Gh EXPA8,an α expansin gene,was down-regulated significantly in pag1 mutant with reduced fiber lengthwhen analyzing the transcriptome data of rapidly elongating fibers.In order to reveal the role of expansin in cotton fiber cells development,Gh EXPA8 gene related to fiber development was clonedand transformed to Arabidopsis thaliana by inflorescence infiltration procedure to analyze its function characterization.The results are as follows:1.The Gh EXPA8 gene cloning and bioinformatics analysisBased on the nucleotide sequences（Cot AD₂9957,Expansin2）submitted by Institute of cotton research of CAAS,the c DNA of the gene were cloned by RT-PCR and renamed as Gh EXPA8.Bioinformatics analysis showed that the CDS of Gh EXPA8 is 762 bp long,encoding 253 amino acids.The 1-25 amino acids encode the signal peptide.It contains DPBB₁ and pollen_allerg₁ structural domain.The signal peptide and two conserved domains are typical features of α expansin.Evolutionary tree analysis further showed that Gh EXPA8 encodes an α expansin.2.The functionanalysis of Gh EXPA8 geneThe relative expression level of Gh EXPA8 in different tissue and fiber of different development phase was analyzed by q PCR.The q PCR resultsalso showed that Gh EXPA8 gene was lowly expressed in the root,stem,leaf and ovule,while highly expressed in the 10 DPA fiber.The transcript level peaked at 10 DPA and thereafter declined sharply at 15 DPA in the fiber development process.The expression of PROGh EXPA8: GUS fusion was detected in Arabidopsis thaliana.Histochemical staining of GUS can be detected in various organs of transgenic plants,especially in young developing organs such as root hairs,root tip and stem tip.GUS activity was also evident in vascular tissue such as the veins and petioles.Arabidopsis trichome has a similar development mechanism with cotton fiber.GUS activity of Gh EXPA8 promoter can be detected in root hairs of Arabidopsis which suggested that the gene may be play a role in cotton fiber development.The expression patterns of Gh EXPA8 gene in cotton and transgenic Arabidopsis suggested that it may play a role in fiber elongation.Gh EXPA8 gene was also overexpressed in Arabidopsis thaliana.Phenotypic analysis showed that it increases Arabidopsis root length significantly.It suggested that Gh EXPA8 is really a functionalα expansin which most likely play a role in fiber development.In addition,the expression pattern suggest that the gene play a role in the process of fiber elongation.These results indicate that Gh EXPA8 is likely to be involved in fiber development process.3.We chose the hypocotyls of CRRI 24 as explants and conduct the transformation experimentof Gh EXPA8 gene by the method of Agrobacterium-mediated genetic transformation.The transgenic plants of T0 generation of Gh EXPA8 gene were obtained,which benefit the further study of Gh EXPA8 gene function.更多还原