农杆菌介导的山苍子遗传转化体系的构建

Establishment of Agrobacterium Mediated Genetic Transformation System of Litsea cubeba

[目的]筛选适合山苍子愈伤组织分化的激素比例,研究其对抗生素的耐受性,构建山苍子遗传转化体系。[方法]以山苍子无菌苗茎段诱导的愈伤组织为试验材料,研究不同激素浓度配比对山苍子愈伤组织诱导不定芽及不定芽生根的影响,探讨愈伤组织对潮霉素和头孢霉素的临界筛选浓度,并通过农杆菌介导法将外源基因导入山苍子愈伤组织中。[结果]筛选出诱导愈伤组织不定芽分化培养基为:MS+2.0 mg·L^(-1)6-BA+0.01 mg·L^(-1)IBA+0.05 mg·L^(-1)TDZ,分化率为16.67%~36.67%;不定芽生根培养基为1/2MS+0.5 mg·L^(-1)IAA,生根率为97.33%。遗传转化初期筛选抗性愈伤组织的潮霉素浓度为5 mg·L^(-1)(约7~10 d),通过逐步增加潮霉素浓度至30 mg·L^(-1)进行筛选培养,头孢霉素最适浓度为300 mg·L^(-1)。采用农杆菌介导法将外源基因转入愈伤组织中,并设计PCR引物进行鉴定,共检测16株抗性苗均含有目的条带,表明目的基因已插入山苍子基因组中,转化率为0.67%。通过Southern检测表明,目的片段已插入山苍子愈伤组织中。[结论]初步建立山苍子再生及遗传转化体系,且已获得多个基因的抗性愈伤组织,为进一步开展基因功能研究及遗传改良提供技术支持。

[Objective]To screen the hormone proportion suitable for callus differentiation of Litsea cubeba and clarify its tolerance to antibiotics,and preliminarily establish the genetic transformation system of L.cubeba.[Method]The effects of different hormone concentrations on adventitious bud induction and adventitious bud rooting of L.cubeba callus were studied.The critical screening concentrations of hygromycin and cefotaxime were discussed,and the foreign gene was introduced into L.cubeba callus by Agrobacterium mediated method.[Result]The optimum medium for inducing adventitious bud differentiation of callus was MS+2.0 mg·L^(-1)6-BA+0.01 mg·L^(-1)IBA+0.05 mg·L^(-1)TDZ,and the differentiation rate was 16.67%~36.67%;The optimum medium for adventitious bud rooting was 1/2MS+0.5 mg·L^(-1)IAA,and the rooting rate was 97.33%.The initial concentration of hygromycin for resistant callus screening was5 mg·L^(-1)(about 7~10 days),and then the critical screening culture was carried out by gradually increasing the hygromycin screening concentration to 30 mg·L^(-1).the optimum concentration of cephalosporin was300 mg·L^(-1).Finally,foreign gene was transferred into the callus by Agrobacterium mediated method,and PCR primers were designed for identification.A total of 16 resistant seedlings contained the target band,indicating that the target gene had been inserted into the L.cubeba genome,with a transformation rate of0.67%.In addition,our research group obtained the resistant calli of multiple genes through this method.Southern detection showed that the target fragment had been inserted into the calli of L.cubeba.[Conclusion]The regeneration and genetic transformation system of L.cubeba has been preliminarily established,and the resistant calli of multiple genes have been obtained,which provides technical support for further gene function research and genetic improvement.The next step is to optimize the genetic transformation system and improve its transformation efficiency.