Crops ›› 2020, Vol. 36 ›› Issue (5): 41-47.doi: 10.16035/j.issn.1001-7283.2020.05.006

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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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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

Table 1

Amplifying primers of RNAi fragments and intron"

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

Fig.1

The construction strategy of RNAi expression vector"

Fig.2

Effects of water stress on BvWRKY23 gene expression in sugar beet leaves"

Fig.3

PCR products of BvWRKY23 gene RNAi fragments M: DL2000 Marker, the same below; 1-2: The products of RNAi fragments"

Fig.4

The PCR identification of positive clones for intermediate vector PBSK-RTM-F 1-8: The PCR products of interference fragment. The same below"

Fig.5

Sequencing analyses of positive clones PBSK-RTM-F The first line is the reference sequence, the second line is the sequencing. The same below"

Fig.6

The PCR identification of positive clones for intermediate vector PBSK-RTM-FR"

Fig.7

Sequencing analysis of positive clones of intermediate vector PBSK-RTM-FR"

Fig.8

The identification of positive recombinant clone for pCambia2301ky-BvWRKY23-RNAi by PCR 1-8: The PCR identification of different transformants"

Fig.9

The identification of plasmid pCambia2301ky-BvWRKY23-RNAi vector by double digestion with BamHⅠ/SacⅠ 1: Double enzyme digestion products of pCambia2301ky-BvWRKY23-RNAi by BamHⅠ/SacⅠ; 2: Double enzyme digestion products of pCambia2301ky by BamHⅠ/SacⅠ"

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