发根农杆菌介导的桑遗传转化体系的优化  

Optimization of Agrobacterium rhizogenes-Mediated Genetic Transformation System of Morus alba L.

作　　者：吴敏 廖扬振 皮继岚 刘尚煜 魏渊[1] 欧阳臻[1] Wu Min;Liao Yangzhen;Pi Jilan;Liu Shangyu;Wei Yuan;Ouyang Zhen(School of Pharmacy,Jiangsu University,Zhenjiang 212013,China;School of Food and Biological Engineering,Jiangsu University,Zhenjiang 212013,China)

机构地区：[1]江苏大学药学院,江苏镇江212013 [2]江苏大学食品与生物工程学院,江苏镇江212013

出　　处：《山东农业科学》2025年第3期27-36,共10页Shandong Agricultural Sciences

基　　金：国家自然科学基金项目“桑叶DNJ类多羟基生物碱生物合成途径中关键羟基化过程解析”(82274040);“桑叶DNJ类多羟基生物碱生物合成关键酶基因挖掘和功能研究”(81872961)。

摘　　要：本研究在已有桑遗传转化体系的基础上对其进行优化,以提高转化效率,为后续桑基因功能研究及分子育种奠定基础。首先分别从发根农杆菌种类、外植体类型、预培养时间、侵染时间和共培养时间五个方面对农杆菌介导的桑遗传转化体系进行优化,再构建MaCYP90A-pBI121质粒转入农杆菌诱导发根以验证优化体系的稳定性。结果表明,优化后的桑遗传转化条件为:采用OD_(600)为0.6的农杆菌LBA9402菌液侵染预培养2 d的无菌桑苗真叶10 min,共培养时间为2 d。在此优化条件下,毛状根诱导率可达到84.51%。同时利用该体系将重组质粒MaCYP90A-pBI121转化至桑叶中,经荧光定量PCR分析,阳性转基因毛状根中MaCYP90A基因表达量为空白对照的39.97倍。综上,本研究成功对已有桑遗传转化体系进行了优化,毛状根诱导率相比优化前(30.21%)提高了1.8倍,可为后续桑基因功能研究、次生代谢物合成途径解析以及分子育种提供技术支撑。On the basis of the existing genetic transformation system,the genetic transformation system was optimized in this study to improve the transformation efficiency and lay a foundation for the subsequent research on gene function and molecular breeding of mulberry(Morus alba L.).Firstly,the Agrobacterium-mediated mulberry genetic transformation system was optimized from five aspects:Agrobacterium rhizogenes species,explants types,pre-culture time,infection time and co-culture time;then the MaCYP90A-pBI121 plasmid was constructed and transported into Agrobacterium rhizogenes to induce hairy roots.The results showed that the optimized genetic transformation conditions were as follows:using Agrobacterium LBA9402 with OD_(600)=0.6 to infect the true leaves of sterile mulberry seedlings pre-cultured for 2 d for 10 min,and coculturing for 2 d.In the optimized conditions,the induction rate of hairy roots reached 84.51%.The recombinant plasmid MaCYP90A-pBI121 was transformed into mulberry leaves,and the positive transgenic efficiency was 81.84%after hydomycin resistance screening and PCR identification.qRT-PCR results showed that the expression level of MaCYP90A gene in the positive transgenic hairy roots was 39.97 times higher than that of the blank control.Based on the established mulberry genetic transformation system,this study successfully optimized the system,and the conversion efficiency was 1.8 times higher than that(30.21%)of the system before optimization,which could provide technical supports for the follow-up study of mulberry gene function,analysis of secondary metabolite synthesis pathway and molecular breeding.

关 键 词：桑 遗传转化体系 发根农杆菌 毛状根诱导 转基因 

分 类 号：S888.2[农业科学—特种经济动物饲养]