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作物学报 ›› 2023, Vol. 49 ›› Issue (7): 1758-1768.doi: 10.3724/SP.J.1006.2023.24197

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

谷子毛状根诱导方法的建立与优化

万夷曼(), 肖圣慧, 白依超, 范佳音, 王琰, 吴长艾*()   

  1. 山东农业大学生命科学学院, 山东泰安 271000
  • 收稿日期:2022-08-26 接受日期:2022-11-25 出版日期:2023-07-12 网络出版日期:2022-12-06
  • 通讯作者: *吴长艾, E-mail: cawu@sdau.edu.cn
  • 作者简介:E-mail: wanyiman123@163.com
  • 基金资助:
    本研究由国家重点研发计划项目(2018YFD1000704);本研究由国家重点研发计划项目(2018YFD100070);山东省重点研发计划(农业良种工程)项目(2021LZGC006)

Establishment and optimization of a high-efficient hairy-root system in foxtail millet (Setaria italica L.)

WAN Yi-Man(), XIAO Sheng-Hui, BAI Yi-Chao, FAN Jia-Yin, WANG Yan, WU Chang-Ai*()   

  1. School of Life Science, Shandong Agricultural University, Tai’an 271000, Shandong, China
  • Received:2022-08-26 Accepted:2022-11-25 Published:2023-07-12 Published online:2022-12-06
  • Contact: *E-mail: cawu@sdau.edu.cn
  • Supported by:
    The National Key Research and Development Program of China(2018YFD1000704);The National Key Research and Development Program of China(2018YFD100070);The Key Research and Development Program (Agricultural Fine Seed Project) of Shandong Province(2021LZGC006)

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摘要：

为建立一种快速鉴定谷子基因功能的技术体系, 本研究通过比较谷子外植体、品种、乙酰丁香酮、菌液浓度和共培养时间对发根农杆菌K599介导的毛状根诱导效率的影响, 发现发根农杆菌浓度在OD600为0.5、以谷子芽尖为外植体、在含有100 μmol L-1乙酰丁香酮的毛状根诱导培养基上共培养3 d时, 可使毛状根诱导率高达80.24%。将GFP基因进行毛状根遗传转化, 通过GFP基因的PCR扩增和GFP荧光观察结果分析, 发现谷子毛状根的转基因效率大于70%。利用该体系对谷子SiDVL1和SiDVL3的亚细胞定位和SiNHX2、SiCBL4和SiCBL7基因功能进行了分析和鉴定。结果表明SiDVL1和SiDVL3在谷子毛状根中的亚细胞定位与在烟草叶片中的一致; SiNHX2、SiCBL4和SiCBL7转基因谷子的存活率显著高于空载体转化谷子。说明本研究建立了一种高效快速鉴定谷子基因定位和功能的方法。

关键词: 发根农杆菌, 毛状根, 谷子, 转基因效率, 基因功能鉴定

Abstract:

To establish a system for rapid identification of millet gene functions, in this study, we compared the effects of explants from different foxtail millet cultivars, the concentrations of acetyleugenone, bacterial solution, and co-culture time on the hairy root induction mediated by Agrobacteria rhizogenes strain K599. When the shoot tips were infected with bacterial solution 0.5 optical density (OD) and containing 100 μmol L-1 acetyleugenone, and co-cultured for three days, the induction efficiency of hairy roots reached to 80.24%. Using this system, we analyzed the transformation efficiency of the hairy roots, the subcellular localization of SiDVL1and SiDVL3, and gene functions of SiNHX2, SiCBL4, and SiCBL7. The PCR results of GFP gene and GFP fluorescence microscopic observation indicated that the transformation efficiency of the hairy roots was over 70%. The subcellular localization of SiDVL1 and SiDVL3 was similar in millet hairy-root and tobacco epidermal cells. Simultaneously, the survival rates of SiNHX2, SiCBL4, and SiCBL7 transgenic millet were significantly higher than that of empty vector transformed millet. Therefore, our study established an efficient and rapid method to analyze the subcellular location and to identify the function of genes from foxtail millet.

Key words: Agrobacterium rhizogenes, hairy roots, foxtail millet (Setaria italica), transformation efficiency, gene function identification

图1

不同外植体对诱导毛状根的影响 A和B分别为发根农杆菌K599侵染谷子胚性愈伤组织和芽尖外植体诱导10 d和5 d时毛状根发生情况; C: 培养14 d诱导产生毛状根的根长; D: 培养14 d诱导产生毛状根的诱导效率。3次生物学重复, n = 30。采用单因素方差分析, 不同字母表示差异显著(P < 0.05)。"

图2

不同谷子品种的芽尖外植体对毛状根诱导的影响 A~E: 分别为Ci846 (A)、xiaomi (B)、吨谷(C)、龙珠1号(D)和豫谷1号(E)芽尖外植体对毛状根诱导的影响; F: 不同品种来源的芽尖外植体诱导毛状根的根长; G: 不同品种来源的芽尖外植体诱导毛状根的诱导效率; H: 不同品种来源的芽尖外植体诱导毛状根的平均生根数。3次生物学重复, n = 30。采用单因素方差分析, ns表示在0.05概率水平无显著差异。"

图3

乙酰丁香酮对谷子毛状根诱导的影响 A~E: 分别为0、50、100、150和200 μmol L-1 AS对诱导毛状根的影响; F: 0、50、100、150和200 μmol L-1 AS诱导毛状根的根长; G: 0、50、100、150和200 μmol L-1 AS诱导毛状根的诱导效率; H: 0、50、100、150和200 μmol L-1 AS诱导毛状根的平均生根数。3次生物学重复, n = 30。采用单因素方差分析, 不同字母表示差异显著(P < 0.05)。"

图4

菌液浓度对谷子毛状根诱导的影响 A~F: OD600分别为0.1、0.2、0.3、0.5、0.8和1.0对诱导毛状根的影响; G: 不同菌液浓度诱导产生毛状根的根长; H: 不同菌液浓度诱导产生毛状根的诱导效率; I: 不同菌液浓度诱导产生毛状根的平均生根数。3次生物学重复, n = 30。采用单因素方差分析, 不同字母表示差异显著(P < 0.05)。"

图5

共培养时间对谷子毛状根诱导的影响 A~E: 分别为共培养1、2、3、4和5 d对诱导毛状根的影响; F: 共培养1、2、3、4和5 d诱导毛状根的根长; G: 共培养1、2、3、4和5 d诱导毛状根的效率; H: 共培养1、2、3、4和5 d诱导毛状根的平均生根数。3次生物学重复, n = 30。采用单因素方差分析, 不同字母表示差异显著(P < 0.05)。"

表1

芽尖外植体诱导毛状根的数据统计"

试验编号
Test ID		 侵染的芽尖总数
Total number of
infected shoot tips		 诱导发根的芽尖数
Number of bud tips to induce rooting		 发根总数
Total bud tip rooting number		 平均发根数
Average rooting number		 发根率
Rooting rate
(%)
1 19 17 63 4 89.47
2 22 17 44 3 77.27
3 21 14 44 3 66.67
4 21 21 65 3 100.00
5 21 17 64 2 80.95
6 25 19 57 3 76.00
7 20 17 55 3 85.00
8 22 18 66 3 81.82
9 18 14 41 5 77.78
10 21 17 50 3 80.95
11 20 13 29 2 65.00
12 23 19 38 2 82.61
总计 Total 253 203 616 3 80.24

图6

部分转基因发根的PCR验证结果 M: DNA marker; 1: 基因表达载体质粒(阳性对照); 2~33: 基因表达载体转化毛状根的DNA; WT: 空载体转化毛状根DNA。"

图7

发根农杆菌介导的谷子DVL基因定位分析"

图8

SiNHX2、SiCBL4和SiCBL7转基因谷子幼苗对盐胁迫的响应 A: SiNHX2、SiCBL4和SiCBL7转发根谷子幼苗在含有175 mmol L-1 NaCl的MS培养基(MS培养基为对照)上生长7 d的表型; B: SiNHX2、SiCBL4和SiCBL7转基因谷子幼苗在含有175 mmol L-1 NaCl的MS培养基(MS培养基为对照)上生长7 d的存活率统计。3次生物学重复, n = 30。ns表示在0.05概率水平无显著差异, ***表示在0.01概率水平显著相关。"

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