植物学报 ›› 2022, Vol. 57 ›› Issue (3): 327-339.DOI: 10.11983/CBB21192
翟琼, 陈容钦, 梁晓华, 曾楚淳, 胡博, 李玲, 李晓云(
)
首页收稿日期:2021-11-13
接受日期:2022-03-03
出版日期:2022-05-01
发布日期:2022-05-18
通讯作者:
李晓云
作者简介:* E-mail: 20185131@m.scnu.edu.cn基金资助:
Qiong Zhai, Rongqin Chen, Xiaohua Liang, Chuchun Zeng, Bo Hu, Ling Li, Xiaoyun Li()
Received:2021-11-13
Accepted:2022-03-03
Online:2022-05-01
Published:2022-05-18
Contact:
Xiaoyun Li
About author:First author contact:† These authors contributed equally to this paper
摘要： 遗传转化是植物基因工程的重要手段。快速、高效地将目的基因导入植物细胞, 并缩短获得转基因后代的时间是遗传转化的关键。花生(Arachis hypogaea)是我国重要的油料及经济作物。目前花生的遗传转化体系尚未完善, 制约着花生的基因功能解析和分子育种进程。该文建立了一套快速、稳定的花生遗传转化体系。通过将农杆菌注射于花生第2茎节的切面获得转化植株, 再将阳性植株进行移栽和回土, 采摘注射点以上的荚果进行后续鉴定与分析。结果表明, 利用该方法可获得40%以上的T0代嵌合体植株, 约5个月可收获T0代花生种子, 其中约有9%的T1代花生植株为非嵌合体的杂合体。针对部分转基因植株结实少的问题, 进一步提出了将快速转化体系与传统组培方法相结合的优化方案。构建的快速转化方法对大蒜(Allium sativum)、马铃薯(Solanum tuberosum)和香雪兰(Freesia refracta)的遗传转化具有潜在应用价值, 对其它植物的遗传转化也有重要参考价值。
翟琼, 陈容钦, 梁晓华, 曾楚淳, 胡博, 李玲, 李晓云. 一种花生快速遗传转化方法的建立与应用. 植物学报, 2022, 57(3): 327-339.
Qiong Zhai, Rongqin Chen, Xiaohua Liang, Chuchun Zeng, Bo Hu, Ling Li, Xiaoyun Li. Establishment and Application of a Rapid Genetic Transformation Method for Peanut. Chinese Bulletin of Botany, 2022, 57(3): 327-339.
图1 农杆菌转化所用载体
Figure 1 Vectors used for Agrobacterium transformation (A) pDR5:RUBY; (B) p35S:GFP; (C) p35S:GUS (A) pDR5:RUBY; (B) p35S:GFP; (C) p35S:GUS
图2 花生快速遗传转化过程 (A) 在第2茎节处剪去顶端; (B) 转化前准备(菌液、活性剂和注射器, 活性剂为100 μmol∙L-1乙酰丁香酮); (C) 用注射器吸取农杆菌; (D) 将农杆菌注射至花生第2茎节处; (E) 暗培养3-5天; (F) 新芽生长; (G) 剪去侧芽; (H) 正常光照培养; (I) 移栽和回土, 以土壤盖过注射点为宜; (J) 采摘注射点以上的荚果(白色箭头示注射点)。Bars=1 cm
Figure 2 The process of rapid genetic transformation in peanut (A) Cut off the top at the second stem node; (B) Preparation before transformation (bacteria, active agent and syringe, active agent is 100 μmol∙L-1 acetosyringone); (C) Syringe draws Agrobacterium; (D) Agrobacterium is injected at the second stem node of the peanut; (E) Dark culture for 3 to 5 days; (F) New buds grow; (G) Cut off the lateral bud; (H) Normal light culture; (I) Transplanting and replanting, the injection point should be covered with the soil; (J) Pick pods above the injection point (the white arrow indicated the injection point). Bars=1 cm
图3 农杆菌注射花生部位的选择 (A) 随机选择实验田的120棵粤油13花生进行果针发生部位统计; (B) 对花生各茎节徒手切片后, 进行碘液染色和拍照(bars=1 mm); (C) 各个茎节的淀粉含量(*表示与第1茎节比较差异显著(P<0.01)); (D) 农杆菌注射花生第2茎节后及新芽生长情况(bar=1 cm)。SN1代表第1茎节, 即2片子叶发生的茎节, SN2代表子叶结节向上的第2个茎节, 以此类推。
Figure 3 Selection of injection site for Agrobacterium in peanut (A) The 120 peanuts were randomly selected to calculate the occurrence site of gynophores in experimental farm; (B) Peanut stems were hand out sliced and stained with iodine solution, then take photos (bars=1 mm); (C) Measurement of starch content in each stem node (* represent the significant differences when compared with the first stem node (P<0.01)); (D) New shoots growth after Agrobacterium injection (bar=1 cm). SN1 represents the first stem node that occurred with two cotyledons, SN2 represents the second stem node that above the cotyledons, and so on.
图4 快速转基因花生植株的筛选与鉴定 (A) PCR鉴定pDR5:RUBY转基因花生的电泳结果(R1-R5代表独立的转基因株系; Mock只注射不含pDR5:RUBY载体的对照菌, 下同); (B) 体视显微镜下观察对照(Mock)和pDR5:RUBY转基因植株花生叶片(bars=5 mm); (C) T0代转基因花生表型, 分别为对照(Mock)、pDR5:RUBY以及p35S:GFP的花生苗(bars=1 cm); (D) PCR鉴定p35S:GFP转基因花生的电泳结果(1-15代表独立的转基因株系); (E) 体视荧光显微镜下观察T0代转基因花生叶片中GFP蛋白的荧光(绿色), Mock为对照植株叶片, p35S:GFP为GFP过表达植株(bars=50 μm); (F) 卡纳抗性筛选p35S:GFP的T1代转基因花生苗(bar=1 cm); (G) 利用GFP抗体, Western Blot鉴定p35S:GFP转基因花生(G1-G7代表独立的转基因株系); (H) 采用共聚焦显微镜观察p35S:GFP的T1代花生原生质体中的GFP蛋白荧光(绿色), 红色为叶绿素a的自发荧光(bars=5 μm)。
Figure 4 Screening and identification of rapidly-transformation peanuts (A) The electrophoresis result is identification of pDR5:RUBY transgenic peanut plants through PCR using specific primers (R1-R5 represents independent transgenic lines; Mock, injected only with empty bacteria, the same as below); (B) Control (Mock) and pDR5:RUBY transgenic peanut leaves were observed by stereomicroscopy (bars=5 mm); (C) Phenotypic observation of T0 generation transgenic peanut, including control (Mock), pDR5:RUBY and p35S:GFP plants (bars=1 cm); (D) The electrophoresis result is the identification of p35S:GFP transgenic peanut plants using specific PCR primers (1-15 represent independent transgenic lines); (E) The fluorescence of GFP protein (green) were observed by stereomicroscopy in leaves of T0 p35S:GFP transgenic peanut, Mock is control plant, p35S:GFP is GFP overexpression plant (bars=50 μm); (F) Screening of T1 p35S:GFP transgenic peanuts using Kan resistance (bar=1 cm); (G) Identification of p35S:GFP transgenic peanut plants through Western Blot using GFP antibody (G1-G7 represents independent transgenic lines); (H) The fluorescence of GFP protein (green) were observed in T1 p35S:GFP protoplast by Confocal microscopy, red is chlorophyll a spontaneous fluorescence (bars=5 μm).
| Gene (species) | Generation | Total identified plants | Positive transgenic plants | Transformation rate (%) |
|---|---|---|---|---|
| p35S:GFP | T0 | 30 | 18 | 60 |
| pDR5:RUBY | T0 | 21 | 9 | 42.8 |
表1 花生快速遗传转化率
Table 1 The ratio of rapid transformation in peanut
| Gene (species) | Generation | Total identified plants | Positive transgenic plants | Transformation rate (%) |
|---|---|---|---|---|
| p35S:GFP | T0 | 30 | 18 | 60 |
| pDR5:RUBY | T0 | 21 | 9 | 42.8 |
图5 快速转基因花生植株的纯化与扩繁 (A) 用潮霉素B抗性筛选pDR5:RUBY转基因花生T1代的幼胚, 并将有抗性的胚诱导愈伤组织和丛生芽, 通过丛生芽扩繁转基因植株(箭头示pDR5:RUBY高表达部位); (B) 将T0代植株上具有除草剂(Basta)抗性的叶片进行传统花生组织培养, 筛选、纯化和扩繁转基因植株(图(a)中的*表示具有除?剂抗性的叶片)。Bars=1 cm
Figure 5 Purification and propagation of rapidly-transformation peanuts (A) Screening the embryo of T1 generation pDR5:RUBY transgenic peanut using hygromycin B resistance, then the callus and clustered shoots were induced from these resistance embryos, the transgenic plants were propagation using clustered shoots (the arrow indicated the high expression of pDR5:RUBY); (B) Transgenic plants were screening, purifying and propagating using tissue culture, these tissues came from peanut leaves that displayed the Basta resistance T0 plants (the * in (a) showed the Basta-resistance leaves). Bars=1 cm
图6 对大蒜、香雪兰和马铃薯芽切面进行p35S:GUS快速转化 (A1) 大蒜芽点切片示意图(左)以及注射后新长出的大蒜(右) (bars=1 cm); (A2) 对照(Mock)及p35S:GUS转化苗的GUS检测(bars=1 mm); (B1) 香雪兰芽点切片示意图(左)以及注射后长出的新芽(右) (bars=1 cm); (B2) 对照(Mock)及p35S:GUS转化苗的GUS检测(bars=1 mm); (C1) 马铃薯注射后长出的新芽(bars=1 cm); (C2) 对照(Mock, bar=1 cm)以及p35S:GUS转化苗(bars=1 mm)的GUS检测。
Figure 6 Rapidly-transformation of p35S:GUS in sprout sections from garlic, freesia and potato (A1) Garlic bud point section (left) and newly grown garlic after injection (right) (bars=1 cm); (A2) GUS detection of control (Mock) and p35S:GUS transgenic lines (bars=1 mm); (B1) Shoot spot section of freesia (left), and the new shoots after injection (right) (bars=1 cm); (B2) GUS detection of control (Mock) and p35S:GUS transgenic lines (bars=1 mm); (C1) The shoots of potato after injection (bar=1 cm); (C2) GUS detection of control (Mock, bar=1 cm) and p35S:GUS transgenic lines (bars=1 mm).
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