油棕病毒诱导的基因沉默体系的建立及优化
CSTR:
作者:
作者单位:

1.海南大学热带作物学院, 海口570228;2.海南大学三亚南繁研究所,三亚572024

作者简介:

李萌晗,E-mail: 1184660256@qq.com

通讯作者:

李东栋,E-mail: lidd@hainanu.edu.cn

中图分类号:

S565.9

基金项目:

海南省重点研发项目(ZDYF2022XDNY148)


Establishment and optimization of oil palm virus-induced gene silencing system
Author:
Affiliation:

1.College of Tropical Crops, Hainan University, Haikou 570228, China;2.Sanya Nanfan Research Institute, Sanya 572024, China

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    摘要:

    为开展油棕油脂代谢调控相关基因鉴定研究,以油棕八氢番茄红素脱氢酶基因(phytoene desaturase gene,PDS)作为报告基因,探索以病毒载体TRV为载体在油棕胚状体上应用病毒诱导基因沉默(virus-induced gene silencing,VIGS)的可能性,并对油棕胚状体VIGS体系的相关参数进行优化。结果显示:以EHA105为菌种、侵染菌液OD600 = 0.5、侵染时间5 min、乙酰丁香酮(AS)质量浓度20 mg/L、共培养48 h、侵染后培养时间为12 d能取得最佳的基因沉默效果。在此基础上,利用优化后的VIGS体系对油棕二酰甘油酰基转移酶基因(diacylglycerol acyltransferase gene,DGAT)进行沉默,取得了预期的基因沉默效果。

    Abstract:

    The phytoene desaturase gene (PDS) as a reporter gene was used to study the feasibility of applying virus-induced gene silencing (VIGS) on oil palm embryoid using the TRV as a vector, and to optimize the relevant parameters. The results showed that the effect of gene silencing was the best when EHA105 was used as the strain under the condition that the OD600 of infected bacterial solution was 0.5, with infection time of 5 min, acetosyringone (AS) mass concentration of 20 mg/L, co-cultivation of 48 h, and post-infection incubation time of 12 d. On this basis, the VIGS system optimized was used to silence the diacylglycerol acyltransferase gene (DGAT), and the expected gene silencing effect was achieved.

    图1 初步建立的油棕VIGS体系结果Fig.1 Experimental results of preliminary establishment of oil palm VIGS system
    图2 侵染的菌液浓度与时间(A)、共培养时间(B)、烟草介导(C)、不同农杆菌菌株(D)、AS浓度(E)、培养时间(F)对油棕的VIGS体系转化效率的影响Fig.2 Effects of concentration and time (A), co-culture time (B), tobacco-mediated (C), different agrobacterium strains (D), concentration of AS (E) and culture time (F) on the transformation efficiency of oil palm VIGS system
    图3 油棕胚性愈伤组织中EgDGAT1表达量的检测Fig.3 Detection of EgDGAT1 expression in oil palm embry-type callus of experimental group and control group
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李萌晗,王威,邹积鑫,李东栋.油棕病毒诱导的基因沉默体系的建立及优化[J].华中农业大学学报,2023,42(2):259-264

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  • 收稿日期:2022-06-15
  • 在线发布日期: 2023-03-31
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