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利用同源克隆方法从大豆中克隆到一个耐盐基因HAL3,命名为GmHAL3a,组织表达分析发现GmHAL3a基因在大豆根中表达量最高,荚中次之,叶中表达量最低;非生物胁迫实验表明该基因受NaCl、LiCl和山梨醇诱导表达;通过生物信息学的方法分析发现该基因具有2个跨膜螺旋区域,可能定位于叶绿体中;同源序列比对证实该基因氨基酸序列具有与其他物种相似的保守位点:黄素单核苷酸(FMN)结合位点和磷酸泛酰半胱氨酸(PPC)脱羧酶活性位点。同时扩增了这个基因的2个干扰片段,用Gateway技术将扩增的片段通过BP反应连接到入门载体pDONR221,测序后又通过LR反应分别将目的片段插入到RNAi载体pB7GWIWG2(Ⅱ),再转入根癌农杆菌EHA105中。这2个载体的构建对于进一步研究大豆中GmHAL3a基因的功能具有重要意义。
HAL3, named as GmHAL3a, was cloned from soybean by homologous cloning method. The expression of GmHAL3a gene was the highest in soybean root, followed by the pod, and the lowest in leaves. The abiotic stress experiment showed that The gene was induced by NaCl, LiCl and sorbitol. Bioinformatics analysis showed that the gene has two transmembrane helix regions, which may be located in the chloroplast. The homologous sequence alignment confirmed that the amino acid sequence of the gene has the same sequence with other species Similar conserved sites: flavin mononucleotide (FMN) binding site and phosphopitoacylcysteine (PPC) decarboxylase active site. At the same time, two interfering fragments of this gene were amplified. The amplified fragment was linked to the entry vector pDONR221 by the Gateway reaction using Gateway technology. After sequencing, the target fragment was inserted into the RNAi vector pB7GWIWG2 (Ⅱ) Agrobacterium tumefaciens EHA105. The construction of these two vectors is of great significance to further study the function of GmHAL3a gene in soybean.