果蔗SoSgt1基因的克隆表达与植物反义表达载体的构建  

Cloning of SoSgt1 from Chewing Cane and Construction of Antisense Vector

作　　者：林生[1,2] 潘大仁[1,2] 周以飞[1,2] 陈观水[1] 张绪璋[2] 吴子峰[1] 

机构地区：[1]福建省作物分子与细胞生物学重点实验室福建农林大学,福建福州350002 [2]作物遗传育种与综合利用教育部重点实验室福建农林大学,福建福州350002

出　　处：《热带生物学报》2010年第1期1-7,共7页Journal of Tropical Biology

基　　金：国家自然科学基金(30370900);福建省自然科学基金(2003N001)

摘　　要：利用不同作物的Sgt1基因的SGS保守区域的氨基酸保守位点设计简并引物,从果蔗的cDNA中克隆分离出果蔗SoSgt1基因片段。以果蔗SoSgt1基因片段为探针,利用电子克隆和序列拼接方法并通过RT-PCR验证获得了一个全长1438bp的cDNA序列。通过ORF软件分析,预测得到的蛋白质具有362个氨基酸。利用NCBI数据库中的Protein Blast分析SoSgt1蛋白氨基酸序列,得出含有SGT1蛋白的3个典型的保守结构域(TPR,CS和SGS),与其他作物的SGT1蛋白比较具有较高的相似性。利用梢腐病病原Gibberella fujikuroi接种福安果蔗叶片,检测到SoSgt1基因的转录水平逐渐提高,并用果蔗SoSgt1基因的cDNA片段设计2个酶切位点,反向插入到植物真核表达载体pCAMBIA1301,构建反义表达载体pCAM-SGT。Based on SGS conservative amino acid region of Sgtl gene from different crops, degenerate primers were designed and RT-PCR was used to amplify SoSgtl cDNA fragment of chewing cane, The results indicated that SoSgtl full length cDNA from chewing cane includes 1438 bp, 362 amino acids. Protein Blast from NCBI database was used to analysis its amino acid sequence, and the results showed that the SoSgtl protein have three SGT1 typical conserved domains eg. TPR, CS and SGS, and the similarity among SGTI of other crops were very high. The expression levels of SoSgtl increased after inoculating with Gibberella fujikuroi. Based on SoSgtl cDNA , two restriction sites were designed and antisense expression vector pCAM-SGT were constructed, in whichplant eukaryotic expression vector, pCAMBIA1301, were used as backbone.

关 键 词：果蔗 SoSgt1基因 克隆 半定量RT-PCR 载体构建 

分 类 号：Q781[生物学—分子生物学]