谷子茎尖体外遗传转化体系的建立与优化

doi:10.11983/CBB20119

摘要/Abstract

摘要： 以谷子(Seteria italica)豫谷一号为实验材料, 建立了一套简便、稳定的体外茎尖遗传转化体系。通过根癌农杆菌(Agrobacterium tumefaciens)介导的茎尖转化法, 对转化受体采取不同的处理方式, 待拟转化株长到三叶期后进行PCR鉴定。探明了草丁膦(Basta)喷施处理用于谷子转基因幼苗筛选的最适浓度, 以及2种不同检测方式(直接PCR和喷施Basta+PCR)鉴定转基因植株的效果。在上述基础上, 对影响谷子遗传转化体系的多种因素进行优化。结果表明, 菌液浓度(OD₆₀₀)=1.4、侵染液中乙酰丁香酮浓度为800 μmol∙L ^-1、侵染压强为0.05 MPa、侵染40分钟有利于谷子茎尖的遗传转化。同时, 采用上述优化系统获得谷子转SiCBL4基因植株, 通过喷施草丁膦和实时荧光定量PCR对T₂代转基因植株进行遗传稳定性分析, 可节约检测时间。综上, 该研究初步建立了稳定的谷子体外茎尖遗传转化体系, 并开发了一种便捷的检测后代转基因植株的组合方法。

关键词: 谷子, 遗传转化, 草丁膦, 体外, 茎尖

Abstract: In this study, a simple and stable genetic transformation system of foxtail millet (Seteria italica) was established and optimized, in which shoot tips were used as the explant. We transformed Yugu 1, an elite millet cultivar, by Agrobacterium-mediated transformation, and tested different treatments to boost transformation efficiency. We used a PCR-based assay to screen transformants in third-leaf stage seedlings. We determined an optimal lethal concentration of glufosinate (Basta) when sprayed to millet seedlings, and tested the different PCR-based genotyping methods with or without Basta spary. Using the newly established pipeline, we further optimized various crucial factors that affect genetic transformation efficiency. We found that an optimal concentration of bacterial culture was OD₆₀₀=1.4, an optimal concentration of acetolsyringone was 800 μmol∙L ^-1. We also obtained high transformation efficiency with an infecting pressure at 0.05 MPa, and an infecting time of 40 min. We used the above-mentioned transformation method to transform a Seteria italica calcineurin B-like protein 4 (SiCBL4) overexpression construct. Genetic stability analysis on T₂ generation transformed plants was performed by the combination assay of Basta resistance and real-time quantitative fluorescence RT-PCR, which can save the time of genotyping. Altogether, this study establishes a shoot tip-based stable genetic transformation system for foxtail millets, and also develops a robust pipeline to detect transgenic offsprings.

Key words: foxtail millet, genetic transformation, glufosinate, in vitro, shoot tip

杨澜, 刘雅, 项阳, 孙秀娟, 颜景畏, 张阿英. 谷子茎尖体外遗传转化体系的建立与优化. 植物学报, 2021, 56(1): 71-79.

Lan Yang, Ya Liu, Yang Xiang, Xiujuan Sun, Jingwei Yan, Aying Zhang. Establishment and Optimization of a Shoot Tip-based Genetic Transformation System for Foxtail Millet. Chinese Bulletin of Botany, 2021, 56(1): 71-79.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB20119

https://www.chinbullbotany.com/CN/Y2021/V56/I1/71

图/表 8

图1 pCUN-NHF质粒T-DNA区段示意图 LB: LB T-DNA重复; Ubi promoter: 玉米Ubiquitin1启动子; HA: HA标签; MCS: 多克隆位点; 35S promoter: CaMV35S启动子; Bar: 抗草铵膦基因; RB: RB T-DNA重复

Figure 1 The T-DNA region of plasmid pCUN-NHF LB: LB T-DNA repeat; Ubi promoter: Zea mays Ubiquitin1 promoter; HA: HA tag; MCS: Multiple cloning site; 35S promoter: CaMV35S promoter; Bar: Herbicide bialaphos-resistance gene; RB: RB T-DNA repeat

图2 谷子转基因植株鉴定 (A) 谷子幼苗GUS染色图(bar=0.1 cm); (B) PCR鉴定(1: 阳性对照; 2: 阴性对照; 3: 切一刀的植株叶片; 4: 切两刀的植株叶片; 5: 不做切除处理的植株叶片)

Figure 2 Identification of transgenic foxtail millet (A) Image from GUS staining of foxtail millet seedlings (bar=0.1 cm); (B) PCR identification (1: Positive control; 2: Negative control; 3: A leaf with one wound cut; 4: A leaf with two wounds cut; 5: A leaf without wound)

图3 谷子茎尖遗传转化体系的建立 (A) 待萌发的种子; (B) 萌发3天后的种子; (C) 转化5天后的幼苗; (D) 拔节期植株; (E) 抽穗期植株; (F) 成熟种子。(A), (C), (E) Bars=1.0 cm; (B) Bar=0.5 cm; (D) Bar=10.0 cm; (F) Bar=5.0 cm

Figure 3 Establishment of the genetic transformation system for foxtail millet using shoot tip (A) Seeds to be germinated; (B) Seeds after germination for 3 d; (C) Seedlings after transformation for 5 d; (D) Plants at jointing stage; (E) Plants at heading stage; (F) Mature seeds. (A), (C), (E) Bars=1.0 cm; (B) Bar=0.5 cm; (D) Bar=10.0 cm; (F) Bar=5.0 cm

图4 不同浓度的草丁膦对谷子幼苗存活率的影响 (A) 不同浓度的草丁膦处理谷子的表型图(bar=5.0 cm); (B) 不同浓度草丁膦处理下谷子的存活率。数据为平均值±标准差(n=3)。Student’s t-test, ** P<0.01, *** P<0.001

Figure 4 Effects of glufosinate concentrations on survival rate of foxtail millet seedlings (A) Phenotypes of foxtail millet seedlings treated with glufosinate at different concentrations (bar=5.0 cm); (B) The survival rate of foxtail millet seedlings treated with different concentrations of glufosinate. Data are means±SD (n=3). Student’s t-test, ** P<0.01, *** P<0.001

图5 两种不同方式检测转基因植株的转化率数据为平均值±标准差(n=3)。Student’s t-test, * P<0.05

Figure 5 Transformation rate obtained using two different methods Data are means±SD (n=3). Student’s t-test, * P<0.05

图6 农杆菌浓度(A)、乙酰丁香酮浓度(B)、侵染压强(C)、侵染时间(D)及同时改变4个变量(E)对谷子茎尖转化率的影响数据为平均值±标准差(n=3)。Student’s t-test, * P<0.05。不同小写字母表示差异显著(P<0.05)。

Figure 6 Effects of Agrobacterium concentration (A), acetosyringone concentration (B), infecting pressure (C), infecting time (D) and changing four variables (E) on transformation rates for foxtail millet using shoot tip Data are means±SD (n=3). Student’s t-test, * P<0.05. Different lowercase letters indicat significant differences (P<0.05).

表1 初步建立的体系和优化后的体系条件对比

Table 1 Comparison of the conditions of the initially established system and the optimized system

	Agrobacterium concentration (OD₆₀₀)	Acetosyringone concentration (μmol?L^-1)	Pressure (MPa)	Time (min)
Before optimization	1.0	400	0.04	20
After optimization	1.4	800	0.05	40

图7 qRT-PCR分析谷子转基因植株中SiCBL4基因表达量数据为平均值±标准差(n=3)。Student’s t-test, * P<0.05, ** P<0.01

Figure 7 qRT-PCR analysis of the expression of SiCBL4 in transgenic foxtail millet Data are means±SD (n=3). Student’s t-test, * P<0.05, ** P<0.01

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