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林业科学 ›› 2021, Vol. 57 ›› Issue (8): 195-204.doi: 10.11707/j.1001-7488.20210820

• 研究简报 • 上一篇    

农杆菌介导的楸树遗传转化体系

岑云昕1,刘佳1,陈发菊1,杨敬元2,刘强2,王韬2,梁宏伟1,*   

  1. 1. 三峡区域植物遗传与种质创新重点实验室(三峡大学) 三峡大学生物技术研究中心 宜昌 443002
    2. 神农架国家公园管理局 神农架 442421
  • 收稿日期:2020-10-16 出版日期:2021-08-25 发布日期:2021-09-30
  • 通讯作者: 梁宏伟
  • 基金资助:
    2019年湖北省技术创新专项重大项目(2019ACA162);神农架金丝猴保育生物学湖北省重点实验室开放课题(SNJGKL202103)

Agrobacterium-Mediated Genetic Transformation System of Catalpa bungei

Yunxin Cen1,Jia Liu1,Faju Chen1,Jingyuan Yang2,Qiang Liu2,Tao Wang2,Hongwei Liang1,*   

  1. 1. Key Laboratory of Plant Genetics and Germplasm Innovation in the Three Gorges Region(China Three Gorges University) Biotechnology Research Center, China Three Gorges University Yichang 443002
    2. Shennongjia National Park Administration Shennongjia 442421
  • Received:2020-10-16 Online:2021-08-25 Published:2021-09-30
  • Contact: Hongwei Liang

摘要:

目的: 以楸树胚性愈伤组织为受体,建立有效的楸树遗传转化体系,为今后楸树性状的遗传改良奠定基础。方法: 通过农杆菌EHA105介导以胚性愈伤组织作为外植体进行遗传转化,通过正交试验获得最优的遗传转化条件,进而将外源基因转入到楸树基因组中。结果: 在1/2 MS培养基中添加不同梯度浓度的卡那霉素(Kana)进行选择压力筛选,在添加了60 mg·L-1 Kana的1/2 MS培养基中,楸树胚性愈伤组织的分化率为0.00%,存活率仅为5.71%,因此确定60 mg·L-1为遗传转化的选择压。采用正交设计L18(37)进行农杆菌介导的楸树遗传转化试验,通过GUS化学组织染色统计瞬时表达率,正交试验直观分析和单因素方差分析结果表明:在预培养时间为2天,采用农杆菌菌株EHA105、菌液浓度OD600值为0.7、添加乙酰丁香酮(AS)浓度为300 μmol·L-1、侵染时间为10 min,共培养时间为5天的条件下,农杆菌介导的转化效率最高,且对转化效率影响最大的2个因素是乙酰丁香酮浓度和预培养时间。对浸染后的胚性愈伤组织进行8个月的筛选培养,共获得32个抗性组织团,对其中15个增殖较多的抗性愈伤组织进行PCR检测,表明86.67%的抗性组织团中有外源基因整合到楸树基因组中。内源激素水平会对植物体细胞胚分化产生影响,细胞分裂素(CTK)和脱落酸(ABA)促进体胚发生,生长素(IAA)和赤霉素(GA)对体胚发生有抑制作用。通过测定内源激素可知,转基因的抗性组织中内源CTK和ABA水平显著低于野生型的楸树胚性愈伤组织,而内源IAA和GA则显著高于野生型胚性愈伤组织,推测内源激素水平可能是转基因抗性组织体胚分化能力比较差的原因。结论: 建立了农杆菌介导的楸树胚性愈伤组织的遗传转化体系,对筛选获得的15个抗性愈伤组织进行PCR检测,其中13个抗性愈伤组织中有外源基因的整合。内源激素水平的变化可能是导致楸树转基因抗性愈伤组织难以分化的原因。

关键词: 农杆菌介导, 楸树, 遗传转化, 内源激素

Abstract:

Objective: Embryogenic callus of Catalpa bungei was used to establish an effective genetic transformation system, in order to provide a basis for the genetic improvement of C. bungei in the future. Method: Agrobacterium-mediated genetic transformation was carried out with embryogenic callus as explants. The optimal genetic transformation conditions were obtained through orthogonal test, and then exogenous genes were transferred into C. bungei. Result: Selective pressure screening was performed in 1/2 MS medium with different gradient concentrations of kanamycin. The differentiation rate of embryogenic callus was 0.00%, and the survival rate was only 5.71% in 1/2 MS medium supplemented with 60 mg·L-1 Kana. Therefore, 60 mg·L-1 Kana was identified as the selective pressure for genetic transformation of C. bungei. The orthogonal design was carried out by L18(37) table in Agrobacterium-mediated genetic transformation experiments. The transient expression rate was obtained by GUS histochemical staining. The intuitive analysis of the orthogonal test and one-way ANOVA were performed to the data obtained from the experiment. The results showed that Agrobacterium-mediated transformation efficiency was the highest under the conditions of 2 days of pre-culture, EHA105 of Agrobacterium strain, OD600 0.7 of liquid concentration, 300 μmol·L-1 of acetyleugenone(AS), 10 min of infection, and 5 days of co-culture time. And the two factors that have the biggest influence on the transformation efficiency are AS concentration and pre-culture time. A total of 32 resistant tissue groups were obtained by screening and culture of the infected embryogenic callus for 8 months. PCR detection was conducted on 15 resistant callus with high proliferation. The results showed that 86.67% resistant tissue groups had exogenous genes integrated into the genome of C. bungei. Endogenous hormone levels affect somatic embryo differentiation, cytokinin(CTK) and abscisic acid(ABA) promote somatic embryogenesis, and auxin(IAA) and gibberellin(GA) inhibit somatic embryogenesis. According to the determination of endogenous hormones, the levels of CTK and ABA in transgenic resistant tissue were significantly lower than those in the embryogenic callus of wild-type, while the levels of IAA and GA were significantly higher than those in the wild-type callus. Therefore, the level of endogenous hormones may be the reason for the poor somatic embryo differentiation ability of transgenic resistant tissue of C. bungei. Conclusion: Through Agrobacterium-mediated transformation of the embryogenic callus of C. bungei, PCR detection was conducted on 15 screened resistant callus, among which 13 resistant callus had integration of exogenous genes. The somatic embryo differentiation ability of transgenic resistant tissues is poor, and the change of endogenous hormone level may be the reason that the resistant callus is difficult to differentiate.

Key words: Agrobacterium-mediated, Catalpa bungei, genetic transformation, endogenous hormones

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