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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

[1] Ishiguro S, Nakamura K. Characterization of a cDNA encoding a novel DNA-binding protein,SPF1,that recognizes SP8 sequences in the 5’ upstream regions of genes coding for sporamin and beta-amylase from sweet potato. Molecular Genetics and Genomics, 1994,244(6):563-571.
[2] Chen W Q, Provart N J, Glazebrook J, et al. Expression profile matrix of Arabidopsis transcription factor genes suggests their putative functions in response to environmental stresses. The Plant Cell, 2002,14(3):559-574.
doi: 10.1105/tpc.010410 pmid: 11910004
[3] Xu X P, Chen C H, Fan B F, et al. Physical and functional interactions between pathogen-induced Arabidopsis WRKY18,WRKY40,and WRKY60 transcription factors. The Plant Cell, 2006,18(5):1310-1326.
doi: 10.1105/tpc.105.037523 pmid: 16603654
[4] Ramamoorthy R, Jiang S Y, Kumar N, et al. A comprehensive transcriptional profiling of the WRKY gene family in rice under various abiotic and phytohormone treatments. Plant and Cell Physiology, 2008,49(6):865-879.
doi: 10.1093/pcp/pcn061 pmid: 18413358
[5] Christian R, Shen Q X. The WRKY gene family in rice (Oryza sativa). Journal of Integrative Plant Biology, 2007,49(6):827-842.
doi: 10.1111/j.1744-7909.2007.00504.x
[6] Ning P, Liu C C, Kang J Q, et al. Genome-wide analysis of WRKY transcription factors in wheat (Triticum aestivum L.) and differential expression under water deficit condition. PeerJ, 2017,5:e3232.
doi: 10.7717/peerj.3232 pmid: 28484671
[7] Mangelsen E, Kilian J, Berendzen K W, et al. Phylogenetic and comparative gene expression analysis of barley (Hordeum vulgare) WRKY transcription factor family reveals putatively retained functions between monocots and dicots. BMC Genomics, 2008,9(1):194.
doi: 10.1186/1471-2164-9-194
[8] Wei K F, Chen J, Chen Y F, et al. Molecular phylogenetic and expression analysis of the complete WRKY transcription factor family in maize. DNA Research, 2012,19(2):153-164.
doi: 10.1093/dnares/dsr048
[9] Yang Y, Zhou Y, Chi Y, et al. Characterization of soybean WRKY gene family and identification of soybean WRKY genes that promote resistance to soybean cyst nematode. Scientific Reports, 2017,7(1):17804.
doi: 10.1038/s41598-017-18235-8 pmid: 29259331
[10] Dellagi A, Birch P R J, Heilbronn J, et al. A potato gene,erg-1,is rapidly induced by Erwinia carotovora ssp. atroseptica,Phytophthora infestans,ethylene and salicylic acid. Journal of Plant Physiology, 2000,157(2):201-205.
doi: 10.1016/S0176-1617(00)80191-1
[11] 祖倩丽, 尹丽娟, 徐兆师, 等. 谷子WRKY36转录因子的分子特性及功能鉴定. 中国农业科学, 2015,48(5):851-860.
doi: 10.3864/j.issn.0578-1752.2015.05.03
[12] 孔维龙, 于坤, 但乃震, 等. 甜菜WRKY转录因子全基因组鉴定及其在非生物胁迫下的表达分析. 中国农业科学, 2017,50(17):3259-3273.
doi: 10.3864/j.issn.0578-1752.2017.17.002
[13] Yao D X, Zhang X Y, Zhao X H, et al. Transcriptome analysis reveals salt-stress-regulated biological processes and key pathways in roots of cotton (Gossypium hirsutum L.). Genomics, 2011,98(1):47-55.
doi: 10.1016/j.ygeno.2011.04.007
[14] Ülker B, Somssich I E. WRKY transcription factors:from DNA binding towards biological function. Current Opinion in Plant Biology, 2004,7(5):491-498.
doi: 10.1016/j.pbi.2004.07.012
[15] Rushton P J, Somssich I E, Ringler P, et al. WRKY transcription factors. Trends in Plant Science, 2010,15(5):247-258.
doi: 10.1016/j.tplants.2010.02.006
[16] Jiang Y J, Liang G, Yu D Q. Activated expression of WRKY57 confers drought tolerance in Arabidopsis. Molecular Plant, 2012,5(6):1375-1388.
doi: 10.1093/mp/sss080
[17] Chen H, Lai Z R, Shi J W, et al. Roles of Arabidopsis WRKY18,WRKY40 and WRKY60 transcription factors in plant responses to abscisic acid and abiotic stress. BMC Plant Biology, 2010,10:281.
doi: 10.1186/1471-2229-10-281 pmid: 21167067
[18] Pandey S P, Roccaro M, Schon M, et al. Transcriptional reprogramming regulated by WRKY18 and WRKY40 facilitates powdery mildew infection of Arabidopsis. The Plant Journal, 2010,64(6):912-923.
doi: 10.1111/j.1365-313X.2010.04387.x pmid: 21143673
[19] Schon M, Toller A, Diezel C, et al. Analyses of wrky18 wrky40 plants reveal critical roles of SA/EDS1 signaling and indole-glucosinolate biosynthesis for Golovinomyces orontii resistance and a loss-of resistance towards Pseudomonas syringae pv. tomato AvrRPS4. Molecular Plant-Microbe Interactions, 2013,26(7):758-767.
doi: 10.1094/MPMI-11-12-0265-R pmid: 23617415
[20] Babitha K C, Ramu S V, Pruthvi V, et al. Co-expression of AtbHLH17 and AtWRKY28 confers resistance to abiotic stress in Arabidopsis. Transgenic Research, 2013,22(2):327-341.
doi: 10.1007/s11248-012-9645-8
[21] Wu X L, Shiroto Y, Kishitani S, et al. Enhanced heat and drought tolerance in transgenic rice seedlings overexpressing OsWRKY11 under the control of HSP101 promoter. Plant Cell Reports, 2009,28(1):21-30.
doi: 10.1007/s00299-008-0614-x
[22] Tao Z, Kou Y J, Liu H B, et al. OsWRKY45 alleles play different roles in abscisic acid signalling and salt stress tolerance but similar roles in drought and cold tolerance in rice. Journal of Experimental Botany, 2011,62(14):4863-4874.
doi: 10.1093/jxb/err144
[23] Shen H S, Liu C T, Zhang Y, et al. OsWRKY30 is activated by MAP kinases to confer drought tolerance in rice. Plant Molecular Biology, 2012,80(3):241-253.
doi: 10.1007/s11103-012-9941-y
[24] Raineri J, Wang S H, Peleg Z, et al. The rice transcription factor OsWRKY47 is a positive regulator of the response to water deficit stress. Plant Molecular Biology, 2015,88(4):401-413.
doi: 10.1007/s11103-015-0329-7
[25] Lee H, Cha J, Choi C, et al. Rice WRKY11 plays a role in pathogen defense and drought tolerance. Rice, 2018,11(1):5.
doi: 10.1186/s12284-018-0199-0 pmid: 29330772
[26] Gao H M, Wang Y F, Xu P, et al. Overexpression of a WRKY transcription factor Tawrky2 enhances drought stress tolerance in transgenic wheat. Frontiers in Plant Science, 2018,9:997.
doi: 10.3389/fpls.2018.00997 pmid: 30131813
[27] Wang C T, Ru J N, Liu Y W, et al. Maize WRKY transcription factor Zmwrky106 confers drought and heat tolerance in transgenic plants. International Journal of Molecular Sciences, 2018,19(10):3046.
doi: 10.3390/ijms19103046
[28] 李大红, 王春弘, 刘喜平, 等. 大豆GmWRKY35基因的克隆及其增强烟草耐旱能力研究. 大豆科学, 2017,36(5):685-691.
[29] 韩凯虹, 张继宗, 王伟婧, 等. 水分胁迫及复水对华北寒旱区甜菜生长及品质的影响. 灌溉排水学报, 2015,34(4):63-68.
[30] 李国龙, 吴海霞, 温丽, 等. 甜菜苗期抗旱鉴定指标筛选及其综合评价. 干旱地区农业研究, 2011,29(4):69-74.
[31] 王伟伟, 刘妮, 陆沁, 等. RNAi技术的最新研究进展. 生物技术通报, 2017,33(11):35-40.
[32] Fire A, Xu S, Montgomery M K, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998,391(6669):806-811.
doi: 10.1038/35888 pmid: 9486653
[33] Wang L Q, Li Z, Lu M Z, et al. ThNAC13,a NAC transcription factor from Tamarix hispida,confers salt and osmotic stress tolerance to transgenic Tamarix and Arabidopsis. Frontiers in Plant Science, 2017,8:635.
doi: 10.3389/fpls.2017.00635 pmid: 28491072
[34] Xu H J, Xue J, Lu B, et al. Two insulin receptors determine alternative wing morphs in planthoppers. Nature, 2015,519(7544):464-467.
doi: 10.1038/nature14286 pmid: 25799997
[35] Tang B, Wei P, Zhao L N, et al. Knockdown of five trehalase genes using RNA interference regulates the gene expression of the chitin biosynthesis pathway in Tribolium castaneum. BMC Biotechnology, 2016,16:67.
doi: 10.1186/s12896-016-0297-2 pmid: 27596613
[36] Hu Z, Lin Q, Chen H, et al. Identification of a novel cytochrome P450 gene,CYP321E1 from the diamondback moth,Plutella xylostella (L.) and RNA interference to evaluate its role in chlorantraniliprole resistance. Bulletin of Entomological Research, 2014,104(6):716-723.
doi: 10.1017/S0007485314000510
[37] Guo Z J, Kang S, Zhu X, et al. The novel ABC transporter ABCH1 isa potential target for RNAi-based insect pest control and resistance management. Scientific Reports, 2015,5:13728.
doi: 10.1038/srep13728 pmid: 26333918
[38] Li L, Li Q Y, Bao Y H, et al. RNAi-based inhibition of porcine reproductive and respiratorysyndrome virus replication intransgenic pigs. Journal of Biotechnology, 2014,171:17-24.
doi: 10.1016/j.jbiotec.2013.11.022
[39] Linke L M, Wilusz J, Pabilonia K L, et al. Inhibiting avian influenza virus shedding using a novel RNAi antiviral vector technology:proof of concept inan avian cell model. AMB Express, 2016,6(1):16.
doi: 10.1186/s13568-016-0187-y pmid: 26910902
[40] Wu S H, Wen F F, Li Y Y, et al. PIK3CA and PIK3CB silencing by RNAi reverse MDR and inhibit tumorigenic properties in human colorectal carcinoma. Tumor Biology, 2016,37:8799-8809.
doi: 10.1007/s13277-015-4691-5 pmid: 26747178
[41] Wang F J, Chen W C, Liu P F, et al. Lentivirus-mediated RNAi knockdown of LMP2A inhibits the growth of the Epstein-Barrassociated gastric carcinoma cell line GT38 in vitro. Experimental and Therapeutic Medicine, 2017,13:187-193.
doi: 10.3892/etm.2016.3954 pmid: 28123488
[42] Weinsteina S, Toker I A, Emmanuel R, et al. Harnessing RNAi-based nanomedicines for therapeutic gene silencing in B-cell malignancies. Proceedings of the National Academy Sciences of the United States of America, 2015,113(1):E16-E22.
[43] 柴晓杰, 王丕武, 关淑艳, 等. 玉米淀粉分支酶基因反义表达载体的构建和功能分析. 作物学报, 2005,31(12):1654-1656.
[44] Chen S B, Pattavipha S, Liu J L, et al. A versatile zero background T-vector system for gene cloning and functional genomics1 [C][W][OA]. Plant Physiology, 2009,150(3):1111-1121.
[45] 姜玲, 秦长平, 伏卉. Gateway TM系统快速构建番木瓜环斑病毒CP基因反向重复序列表达载体 . 农业生物技术学报, 2008,16(3):526-529.
[46] Schwab R, Ossowski S, Riester M, et al. Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell, 2006,18:1121-1133.
doi: 10.1105/tpc.105.039834 pmid: 16531494
[47] 邢珍娟, 王振营, 何康来, 等. 转Bt基因玉米幼苗残体中CrylAb杀虫蛋白田间降解动态. 中国农业科学, 2008,41(2):412-416.
[48] 王镭, 才华, 柏锡, 等. 转OsCDPK7基因水稻的培育与耐盐性分析. 遗传, 2008,30(8):1051-1052.
[49] 吕品, 柴晓杰, 王丕武, 等. 大豆胰蛋白酶抑制剂KSTI3基因的克隆及其植物表达载体的构建. 吉林农业大学学报, 2007,29(3):275-278.
[50] 娄玲玲, 郑唐春, 曲冠证, 等. 可用于植物转化的RNAi载体构建方法. 安徽农业大学学报, 2012,39(1):120-123.
[51] Manamohan M, Sharath Chandra G, Asokan R, et al. One-step DNA fragment assembly for expressing intron-containing hairpin RNA in plants for gene silencing. Analytical Biochemistry, 2013,433(2):189-191.
doi: 10.1016/j.ab.2012.09.034
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