生物技术通报 ›› 2022, Vol. 38 ›› Issue (2): 263-268.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0635
周承哲1,2,3(), 常笑君1,3, 朱晨1,2,3, 程春振1,3, 陈裕坤1,3, 赖钟雄1,3, 林玉玲1,3, 郭玉琼1,2()
收稿日期:
2021-05-14
出版日期:
2022-02-26
发布日期:
2022-03-09
作者简介:
周承哲,男,博士研究生,研究方向:茶树(茉莉花)生物技术;E-mail: 基金资助:
ZHOU Cheng-zhe1,2,3(), CHANG Xiao-jun1,3, ZHU Chen1,2,3, CHENG Chun-zhen1,3, CHEN Yu-kun1,3, LAI Zhong-xiong1,3, LIN Yu-ling1,3, GUO Yu-qiong1,2()
Received:
2021-05-14
Published:
2022-02-26
Online:
2022-03-09
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。
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.
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 |
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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