Abstract: To study the expression and function of GhCAD6 gene in transgenic cotton fibers, the transgenic material was obtained by Agrobacterium-mediated transformation into Xinluzao 36, and the T6 generation plant material was obtained after successive generations of selection. The genomic DNA from cotton leaf of the T6 generation material plants was detected by PCR, and the positive plants were identified. The genomic DNA of the positive plants were hybridized by Southern blotting. The results showed that GhCAD6 was integrated into the cotton genome in different genes loci in the form of single copy. Total RNA from cotton fiber was extracted by hot borate-proteinase K method. The method for detecting the expression of GhCAD6 gene in cotton fiber was established by quantitative real-time PCR. The expression of GhCAD6 gene in cotton fiber at different developmental stages was determined by the method. The expression of GhCAD6 in transgenic plant fiber was lower than that in control samples at the same developmental stage. The expression of GhCAD6 was inconsistent with that in the control samples at 15 DPA (day post-anthesis), and the other periods were approximately the same. The change of GhCAD6 gene expression at 15 DPA may be the main reason for the variation of fiber quality of transgenic cotton. The results of this research could provide theoretical basis for further analysis of the mechanism of GhCAD6 gene in improving cotton fiber quality.

Key words: cotton, GhCAD6 gene, Southern blotting, quantitative real-time PCR

摘要： 为研究GhCAD6基因在转基因棉花纤维中的表达及功能，通过农杆菌介导法获得新陆早36号转GhCAD6基因材料，经过连续多代的选择得到T6代植株材料，对该T6代材料植株叶片基因组DNA进行PCR检测，确定其阳性植株，并对阳性植株叶片基因组DNA进行Southern杂交，结果发现GhCAD6以单拷贝的形式整合到受体棉花基因组中。利用热硼酸-蛋白酶K法提取出棉花纤维RNA，结合实时荧光定量PCR建立了棉花纤维中GhCAD6基因的表达量的检测方法，并测定了GhCAD6基因在不同发育阶段棉纤维中的表达量。转基因植株纤维中GhCAD6的表达量低于相同发育阶段对照样品。GhCAD6的表达量在转基因植株和其对照在15 DPA(开花后天数)时变化趋势不一致，其他时期大致相同。15 DPA中GhCAD6基因的表达量的变化可能是造成转基因棉花纤维品质变化的主要原因。研究结果可为深入分析GhCAD6基因在改良棉花纤维品质的作用机理提供理论依据。

关键词: 棉花, GhCAD6基因, Southern杂交, 实时荧光定量PCR