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以圆果黄麻(Corchorus capsularis L.)“179”茎部韧皮为材料,利用同源克隆和RACE技术,克隆黄麻纤维素合成酶基因CcCesA1 5′端500 bp序列外的全部cDNA。其序列长度为2529 bp,编码627氨基酸残基,经Blast基因比对和蛋白质结构分析,确定是黄麻纤维素合成酶基因。半定量RT-PCR分析表明,CcCesA1在植株不同部位表达量具组织差异性,依次为茎部韧皮>根>叶>顶芽>麻骨。利用CcCesA1基因部分cDNA序列和3′UTR区,构建黄麻CcCesA1基因反义载体,用测序验证的阳性质粒转化模式植物拟南芥。Southern杂交结果表明,外源基因以单拷贝方式整合进入基因组,转基因拟南芥生长严重受阻,植株变得矮小且茎部易弯曲倒伏,角果数量变少,长度变短,纤维素含量有不同程度的降低,本研究结果表明,所克隆的黄麻CcCesA1基因除了参与植物其他生理代谢过程外,还参与纤维素的生物合成。
Using the stem phloem of Corchorus capsularis L. “179 ” as material, homologous cloning and RACE technique were used to clone all the cDNAs outside the 500 bp sequence of 5 ’end of CcCesA1. Its sequence length was 2529 bp, encoding 627 amino acid residues. The Blast gene alignment and protein structure analysis confirmed that it was a jute cellulose synthase gene. Semi-quantitative RT-PCR analysis showed that there were differences in tissue expression of CcCesA1 in different parts of the plants, which were in turn stem phloem> root> leaf> top bud> numb bone. The cDNA encoding CcCesA1 gene and 3’UTR region were used to construct the antisense vector of CotA gene of Jute CotA and the model plant Arabidopsis thaliana was transformed with the positive plasmid verified by sequencing. Southern hybridization results showed that exogenous genes were integrated into the genome by single copy. The transgenic Arabidopsis thaliana grew severely, the plants became short and the stems were easily bent and lodging, the number of pods reduced, the length decreased, and the content of cellulose varied The results showed that the cloned jcCcAA1 gene involved in other physiological metabolic processes in plants, but also involved in cellulose biosynthesis.