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作物杂志,2020, 第5期: 41–47 doi: 10.16035/j.issn.1001-7283.2020.05.006

• 遗传育种·种质资源·生物技术 • 上一篇    下一篇

甜菜BvWRKY23基因的RNAi载体构建

李国龙1(), 吴海霞2, 孙亚卿1   

  1. 1内蒙古农业大学农学院,010018,内蒙古呼和浩特
    2内蒙古自治区水利科学研究院,010020,内蒙古呼和浩特
  • 收稿日期:2020-02-07 修回日期:2020-05-06 出版日期:2020-10-15 发布日期:2020-10-12
  • 作者简介:李国龙,主要从事甜菜抗逆机制及栽培技术研究,E-mail： lgl9@sina.com
  • 基金资助:
    国家自然科学基金(31760414);国家自然科学基金(31360355)

Construction of RNAi Expression Vector of BvWRKY23 Gene in Sugar Beet

Li Guolong1(), Wu Haixia2, Sun Yaqing1   

  1. 1College of Agronomy, Inner Mongolia Agricultural University, Hohhot 010018, Inner Mongolia, China
    2Inner Mongolia Research Institute for Water Conservancy, Hohhot 010020, Inner Mongolia, China
  • Received:2020-02-07 Revised:2020-05-06 Online:2020-10-15 Published:2020-10-12

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摘要：

WRKY转录因子家族成员在调控植物生长发育、应答生物与非生物胁迫等方面具有重要的生物学功能。以抗旱甜菜（Beta vulgaris L.）幼苗为材料,提取叶片总RNA并反转录为cDNA,通过RT-PCR方法扩增获得甜菜WRKY转录因子家族成员BvWRKY23基因的RNAi靶片段,以中间载体PBSK-RTM作为媒介,利用传统的“酶切-连接”法构建了含有CaMV 35S启动子、BvWRKY23基因片段反向重复序列的RNAi（RNA interference）植物表达载体pCambia2301ky-BvWRKY23-RNAi。

关键词: 甜菜, WRKY转录因子, RNA干扰, 抗旱

Abstract:

The WRKY transcription factor family members play an important role in regulating plant growth, development and responding to biotic and abiotic stress in plant. In our study, the leaves of sugar beet (Beta vulgaris L.) seedling were used as the material for RNA extraction, the total RNA was extracted and reverse transcribed into cDNA. The RNAi target fragment of BvWRKY23 gene in sugar beet was obtained by RT-PCR amplification. Based on the intermediate vector PBSK-RTM, the RNAi (RNA interference) plant expression vector pCambia2301ky-BvWRKY23-RNAi with inverted repeats of BvWRKY23 driven by the promoter CaMV 35S was successfully constructed by traditional restriction enzyme digestion and ligation methods.

Key words: Sugar beet, WRKY transcription factor, RNA interference, Drought stress

表1

RNAi片段及内含子扩增引物

引物类型Primer type 引物名称Primer name 序列(5'-3') Sequence (5'-3') 酶切位点Enzyme site
P1 BvWRKY23-F1 CGGGATCCGAACCATTGAAGATTGAA BamHI
BvWRKY23-R1 GCTCTAGAAGAGGTGGAGGGGTAGGA XbaI
P2 BvWRKY23-F2 CGAGCTCGAACCATTGAAGATTGAA SacI
BvWRKY23-R2 ATTTGCGGCCGCAGAGGTGGAGGGGTAGGA NotI
内含子Intron (RTM) RTM-F ACGTTGTAAGTCTATTTTTG
RTM-R TCTATCTGCTGGGTCCAAATC

图1

干扰载体构建策略

图2

不同水分胁迫对甜菜叶片BvWRKY23基因表达的影响

图3

PCR产物BvWRKY23基因的RNAi片段 M：DL2000,下同;1~2：RNAi片段产物

图4

中间载体PBSK-RTM-F阳性克隆菌落的PCR鉴定 1~8：PCR扩增干扰片段。下同

图5

PBSK-RTM-F阳性质粒测序 第一行为参考序列;第二行为测序序列。下同

图6

中间载体PBSK-RTM-FR阳性克隆菌落PCR 鉴定

图7

中间载体PBSK-RTM-FR阳性质粒测序

图8

pCambia2301ky-BvWRKY23-RNAi载体阳性重组PCR鉴定 1~8：不同转化子PCR鉴定

图9

pCambia2301ky-BvWRKY23-RNAi载体BamHⅠ/SacⅠ双酶切鉴定 1：BamHⅠ/SacⅠ双酶切pCambia2301ky-BvWRKY23-RNAi;2：BamHⅠ/SacⅠ双酶切空载质粒

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