茶树原位转化方法的建立

doi:10.13560/j.cnki.biotech.bull.1985.2021-0635

摘要/Abstract

摘要：

解决茶树缺乏高效、稳定的遗传转化体系问题,建立了一种基于根癌农杆菌介导的茶树原位转化转基因方法,为茶树基因功能研究和种质创新奠定坚实的基础。以茶树品种‘铁观音’、‘白叶1号’和‘龙井43’的实生幼苗为受体材料,进行去顶芽和腋芽处理。利用根癌农杆菌菌液侵染实生苗伤口,通过抗性筛选获得再生芽,经分子生物学鉴定后,采用短穗扦插法获得茶树转基因植株。结果表明,再生芽中GUS（β-glucuronidase）和HYG（hygromycin）标记基因经多次PCR检测均为阳性,经测序验证PCR产物序列与标记基因序列一致。‘铁观音’、‘白叶1号’和‘龙井43’的转化率分别为8.14%、2.99%和2.53%。建立了不依赖茶树组织培养的原位转化转基因体系,具有操作简便、转化率高、成本低、试验周期短的特点。

关键词: 茶树, 原位转化, 根癌农杆菌

Abstract:

To address the lack of an efficient and stable genetic transformation system for tea trees,an in planta transformation transgenic method based on Agrobacterium tumefaciens-mediated transformation of tea trees was established,laying a solid foundation for gene function research and germplasm innovation of tea tree. The seedlings of tea cultivar ‘Tieguanyin’,‘Baiye1’,and ‘Longjing43’ were used as receptor plants,and the apical bud and axillary bud were removed. Using the A. tumefaciens broth infecting the wounds of the seedling,regenerative buds were obtained through resistance screening. After molecular biological identification,transgenic plants were obtained by short-ear cutting method. The results demonstrated that the GUS（-glucuronidase）and HYG（hyglymycin）labeled genes were tested to be positive with multiple PCR,and the PCR product sequence was consistent with the labeled gene sequence. The transformation efficiency of ‘Tieguanyin’,‘Baiye1’,and ‘Longjing43’ was 8.14%,2.99%,and 2.53%,respectively. This study establishes an in planta transformation method of tea plant that does not rely on tissue culture,and the transformation protocol characterizes of simple operation,high transformation rate,low cost,and short experiment cycle.

Key words: Camellia sinensis, in planta transformation, agrobacterium tumefaciens

周承哲, 常笑君, 朱晨, 程春振, 陈裕坤, 赖钟雄, 林玉玲, 郭玉琼. 茶树原位转化方法的建立[J]. 生物技术通报, 2022, 38(2): 263-268.

ZHOU Cheng-zhe, CHANG Xiao-jun, ZHU Chen, CHENG Chun-zhen, CHEN Yu-kun, LAI Zhong-xiong, LIN Yu-ling, GUO Yu-qiong. Establishment of an Efficient in planta Transformation Method for Camellia sinensis[J]. Biotechnology Bulletin, 2022, 38(2): 263-268.

图/表 3

表1 引物序列信息

Table 1 Primer sequenle information

引物名称Primer name	引物序列Primer sequence（5'-3'）	退火温度Annealing temperature/℃	引物用途Purpose
GUS-F	ACGTCCTGAAGAAACCCCAACC	55.0	阳性转基因植株筛选 Screening of positive transgenic plants
GUS-R	TCCCGGCAATAACATACGGCGT	55.0
HYG-F	CTATTTCTTTGCCCTCGGACGAG	55.2
HYG-R	GAATCGGTCAATACACTACATGGC	53.7

图1 茶树转基因植株的获得 A：茶树实生苗;B：去顶芽和腋芽的茶树实生苗;C：根癌农杆菌侵染茶树顶芽和腋芽伤口;D：利用封口膜包裹伤口保湿;E-H：经过HYG抗性筛选后腋芽处长出抗性芽;I和J：转基因植株扦插

Fig. 1 Process of obtaining transgenic tea plants A：Tea seedlings. B：Tea seedlings without apical and axillary buds. C：Agrobacterium tumefaciens-mediated transformation through infection of wounds. D：Wound moisturized using sealing film. E-H：Resistant buds sprouted from axillary buds after HYG resistance screening. I and J：Cuttings of transgenic plants

图2 转基因植株的标记基因检测 A：转基因植株的GUS标记基因（675 bp）检测;B：转基因植株的HYG标记基因（675 bp）检测;M：DNA marker;CK：阳性对照;H2O：阴性对照

Fig. 2 Detection of marker genes in the transgenic plants A：GUS marker gene（675 bp）detection in transgenic plants. B：HYG marker gene（675 bp）detection in transgenic plants. M;DNA marker. CK：Positive control. H2O：Negative control

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