百合GBSS基因RNAi载体构建

doi:10.11923/j.issn.2095-4050.cjas2020-0211

农学学报 ›› 2022, Vol. 12 ›› Issue (7): 69-73.doi: 10.11923/j.issn.2095-4050.cjas2020-0211

所属专题：生物技术；园艺

• 农业工程/农业机械/生物技术/食品科学 • 上一篇下一篇

百合GBSS基因RNAi载体构建

张进忠()

广西农业科学院花卉研究所,南宁 530007

收稿日期:2020-09-24 修回日期:2021-02-07 出版日期:2022-07-20 发布日期:2022-09-20
作者简介:张进忠,男,1979年出生,副研究员,博士,研究方向为作物遗传育种。E-mail： jzzhang@foxmail.com。
基金资助:
广西科技计划项目“隆林县绿色生态农业一体化科技创新创业与服务”(桂科AD19110114);广西农业科学院基本科研业务专项“百合组培鳞茎培育研究”(桂农科2019M26)

RNAi Vector Construction of Lilium GBSS Gene

ZHANG Jinzhong()

Flowers Research Institute of Guangxi Academy of Agricultural Sciences, Nanning 530007, Guangxi, China

Received:2020-09-24 Revised:2021-02-07 Online:2022-07-20 Published:2022-09-20

摘要/Abstract

摘要：

为研究GBSS基因在百合鳞茎形成及发育过程中影响鳞茎淀粉合成代谢的作用机理,构建干扰GBSS基因的RNAi载体为其在转录水平沉默表达提供材料。从百合鳞片克隆GBSS基因300 bp的保守序列,通过BP与LR反应将该序列正反向连接到干扰载体pJawohl8-RNAi中,经过酶切反应鉴定所构建RNAi表达载体的正确性。实验成功构建pDONR221-GBSS入门克隆与pJawohl8-RNAi-GBSS表达载体,为在百合鳞茎淀粉合成代谢途径中研究GBSS基因功能及对鳞茎发育的影响提供了材料与思路。

关键词: 百合, GBSS基因, 鳞茎淀粉, 代谢, 机理, 颗粒结合淀粉合酶, RNAi, 表达载体, 鳞茎发育

Abstract:

To study the mechanism of GBSS gene affecting the synthesis and metabolism of bulb starch during the formation and development of lily bulbs, a RNAi vector interfering with GBSS gene was constructed to provide materials for GBSS silencing expression at the transcriptional level. The conserved sequence of 300 bp of GBSS gene was cloned from lily scales. The sequence was positively and inversely connected to the interference vector pJawohl8-RNAi by BP and LR reaction. pDONR221-GBSS entry clone and pJawohl8-RNAi-GBSS expression vector were successfully constructed, which could provide materials and reference for the study of GBSS gene function and its influence on bulb development in the synthesis and metabolism pathway of lily bulb starch.

Key words: Lilium, GBSS gene, bulb starch, metabolism, mechanism, granule-bound starch synthase, RNAi, expression vector, bulb development

中图分类号:

Q812

张进忠. 百合GBSS基因RNAi载体构建[J]. 农学学报, 2022, 12(7): 69-73.

ZHANG Jinzhong. RNAi Vector Construction of Lilium GBSS Gene[J]. Journal of Agriculture, 2022, 12(7): 69-73.

图/表 5

图1. 目的基因GBSS干扰片段 M为分子量标记,1~2为目的序列

图2. BP反应后菌液PCR结果 M为分子量标记,1~3为目的序列

图3. 干扰表达载体pJawohl8-RNAi-GBSS酶切结果 M为分子量标记,1.目的载体pJawohl8-RNAi,2.目的载体pJawohl8-RNAi的NheI与SpeI双酶切,3.目的载体pJawohl8-RNAi的Acc65I酶切, 4.干扰表达载体pJawohl8-RNAi-GBSS的NheI与SpeI双酶切,5. 干扰表达干扰载体pJawohl8-RNAi-GBSS的Acc65I酶切

图4. 最终构建的pJawohl8-RNAi-GBSS质粒图谱

图5. 目的干扰片段测序结果

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百合GBSS基因RNAi载体构建

RNAi Vector Construction of Lilium GBSS Gene

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