高表达花烟草防御素1基因K326烟草叶片总蛋白的提取及其生物活性分析  

Extraction and bioactivity analysis of total protein of K326 tobacco leaves with high expression of Nicotiala alata defensin 1 gene

作　　者：龚伟伟 黄小贞 赵懿琛[1,2] GONG Weiwei;HUANG Xiaozhen;ZHAO Yichen(Department of Bioengineering,College of Tea Sciences,Guizhou University,Guiyang 550025,Guizhou Province,China;不详)

机构地区：[1]贵州大学茶学院生物工程系,贵州贵阳550025 [2]贵州大学农业生物工程研究院山地植物资源保护与种质创新省部共建教育部重点实验室,贵州贵阳550025

出　　处：《中国生物制品学杂志》2023年第7期800-804,共5页Chinese Journal of Biologicals

基　　金：贵州省高层次创新型人才培养[黔科合人才(2016)4003号];中国烟草总公司贵州省公司科技项目(201608)。

摘　　要：目的 提取高表达花烟草防御素1(Nicotiana alata defensin 1,NaD1)基因的K326烟草叶片总蛋白,并分析其生物活性。方法 从花烟草花、野生型(WT)和高表达NaD1基因(转基因)K326烟草叶片中提取总蛋白,Bardford法测定蛋白浓度。滤纸片法检测总蛋白对真菌的抑菌活性,CCK-8法检测对癌细胞(HeLa细胞)的抑制活性。结果 花烟草花、WT及转基因K326烟草叶片总蛋白浓度分别为11.25、10.33和10.14 mg/mL。转基因K326烟草叶片总蛋白对白色念珠菌的抑菌活性为(85.68±3.08)%,分别为WT K326烟草叶片和花烟草花总蛋白的1.33和1.14倍(F=15 339,P <0.05);对尖孢镰刀菌的抑菌活性为(148.48±2.47)%,分别为WT K326烟草叶片和花烟草花总蛋白的1.09和1.08倍(F=4 927,P <0.05)。转基因K326烟草叶片总蛋白对HeLa细胞的IC_(50)最小(6.11 mg/mL),抑制活性分别为WT K326烟草叶片和花烟草花总蛋白的1.56和1.21倍(F=89 748,P <0.05)。结论 高表达NaD1基因的K326烟草总蛋白具有良好的抑菌和抗癌生物活性,本研究为以烟草作为生物反应器生产抑菌和抗癌生物制剂提供了实验依据。Objective To extract the total protein of K326 tobacco leaves with high expression of Nicotiana alata defensin 1(NaD1) gene and analyze its bioactivity.Methods Total proteins were extracted from Nicotiana alata flowers,wild type(WT) and K326 tobacco leaves(transgenic) with high expression of NaD1 gene,and determined for the concentrations by Bardford method,while for the antibacterial activity against fungi by filter paper method,and for the inhibition activity on cancer cells(HeLa cells) by CCK-8.Results The total protein concentrations of Nicotiana alata flowers,WT and transgenic K326 tobacco leaves were 11.25,10.33 and 10.14 mg/mL,respectively.The antibacterial activity of total protein from transgenic K326 tobacco leaves against Candida albicans was(85.68±3.08)%,which was 1.33 and 1.14 times that of total protein from WT K326 tobacco leaves and Nicotiana alata flowers,respectively(F=15 339,P 0.05);The antibacterial activity against Fusarium oxysporum was(148.48±2.47)%,which was 1.09 and 1.08 times that of total protein from WT K326tobacco leaves and Nicotiana alata flowers,respectively(F=4.927,P 0.05).The IC_(50) value of transgenic K326 tobacco leaf protein on HeLa cells was the smallest(6.11 mg/mL),and the inhibitory activity was 1.56 and 1.21 times that of total protein of WT K326 tobacco leaves and Nicotiana alata flowers,respectively(F=89.748,P 0.05).Conclusion The total protein of K326 tobacco with high expression of NaD1 gene has good antibacterial and anticancer bioactivities,which provides an experimental basis for producing antibacterial and anticancer biological agents with tobacco as bioreactor.

关 键 词：K326烟草 花烟草防御素1 转基因 生物活性 

分 类 号：Q512[生物学—生物化学]