棉花纤维素合酶基因GhcelA1克隆及其载体构建

 根据GeneBank 中GhcelA1（U58283）序列，利用RT-PCR技术，从棉纤维中克隆到GhcelA1 cDNA全长。运用基因重组技术，构建pCAM2300-35S-GhcelA1过表达载体；融合基因表达载体pCAM2300-35S-GhcelA1-EGFP，以及pCAM2300-35S-GhcelA1-RNAi载体，这些载体均通过限制性酶切鉴定和测序验证。农杆菌介导法将融合基因转化棉花子叶，通过激光共聚焦荧光显微镜，在蓝色激发光下观察诱导的愈伤，发现在转化的活体细胞核与质膜内侧有绿色荧光，说明融合基因载体EGFP构建正确，可以在棉花中正常表达。所构建的系列载体可用于转化棉花，促进纤维素在棉纤维中合成与积累。

Cloning and Construction of GhcelA1 Gene in Cotton Fiber

GhcelA1 cDNA full length which reported in GeneBank (U58283) was cloned from Coker 312 (Gossypium hirsutum) fiber at 20 DPA (days after anthesis) for the first time using RT-PCR method. A serial of plant expression vectors for over-expression, fusion gene with EGFP (Enhanced Green Fluorescent Protein) and RNAi (interference) by recombination technology were constructed and confirmed by molecular and sequence methods. Expression of EGFP in infected cells was clearly evident in confocal microscopy (Zeiss LSM-510) experiments. This showed that the GhcelA1-EGFP fusion in correct construction and could express in cotton cells successfully. Clone and construction were the precondition of transformation for culture improved variety and studying on the pivotal function of cellulose synthetase in the second cell wall thicken period of cotton fiber.