欢迎访问作物学报,今天是

作物学报 ›› 2016, Vol. 42 ›› Issue (05): 675-683.doi: 10.3724/SP.J.1006.2016.00675

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

棉花不同GbU6启动子截短克隆及功能鉴定

雷建峰1,李月1,徐新霞2,阿尔祖古丽•塔什1,蒲艳1,张巨松1,刘晓东1,*   

  1. College of Agronomy, Xinjiang Agricultural University, Laboratory of Agricultural Biotechnology of Xinjiang Agricultural University, Urumqi 830052, China;
    Center of Bazhou Agricultural Technology Promotion, Korla 841000, China
  • 收稿日期:2015-10-25 修回日期:2016-01-11 出版日期:2016-05-12 网络出版日期:2016-02-18
  • 通讯作者: 刘晓东, E-mail: xiaodongliu75@aliyun.com;张巨松, E-mail: xjndzjs@163.com
  • 基金资助:

    本研究由国家自然科学基金项目(31560534)和新疆维吾尔自治区研究生科研创新项目(XJGRI2015084)资助。

Cloning and Functional Analysis of Different TruncatedGbU6Promoters in Cotton

LEI Jian-Feng1,LI Yue1,XU Xin-Xia2,AERZUGULI•Tashi1,PU Yan1,ZHANG Ju-Song1,LIU Xiao-Dong1,*   

  1. 刘晓东, E-mail: xiaodongliu75@aliyun.com;张巨松, E-mail: xjndzjs@163.com
  • Received:2015-10-25 Revised:2016-01-11 Published:2016-05-12 Published online:2016-02-18
  • Contact: Liu Xiaodong, E-mail: xiaodongliu75@aliyun.com;Zhang Jusong, E-mail: xjndzjs@163.com
  • Supported by:

    This study was supported by the National Natural Science Foundation of China(31560534) and the Xinjiang Uygur Autonomous Region Graduate Science and Technology Innovation Projects for Graduate Students (XJGRI2015084).

摘要:

U6启动子是CRISPR/Cas9基因组编辑载体系统中驱动sgRNA转录的重要元件, 而使用较短序列的启动子也是构建CRISPR/Cas9基因组编辑载体的基本要求之一。将已经克隆的海岛棉GbU6-5P启动子(长度为1166bp), 采用Transfer PCR方法成功地截出6个长度不同的U6启动子, 其长度分别为672、468、358、280、202和105bp, 并分别构建了6个启动子驱动的GUS融合植物表达载体。将构建好的6个GbU6-5Ps::GUS-pCAMBIA1300与初始克隆的GbU6-5P::GUS-pCAMBIA1300植物表达载体一起利用农杆菌真空渗透转化法分别转染棉花花粉。GUS组织化学染色显示, 克隆到的7个不同截短大小的GbU6-5Ps启动子均能驱动GUS基因在棉花花粉中转录, 棉花花粉被染成蓝色但颜色深浅存在显著差异。结果显示启动子长度越短, 其转录活性越高。而且另外两种棉花U6启动子GbU6-1P和GbU6-7P也表现出类似的结果。本研究克隆了3个短小的、在棉花花粉细胞中具有转录功能的GbU6启动子。结果显示更短的U6启动子具有更高的转录活性, 而且这一特点在不同U6启动子上具有共性。这预示着使用更短U6启动子不仅符合构建CRISPR/Cas9基因组编辑载体的要求, 而且会提高sgRNA的转录水平, 进而可能提高基因组编辑效率。

关键词: 棉花, GbU6启动子, 截短克隆, 真空渗透, 棉花花粉

Abstract:

U6 promoter is an important element for the transcription of sgRNA in the CRISPR/Cas9 genome editing system. Using a short promoter is also one of the basic requirements for the construction of CRISPR/Cas9 genomeediting vector. According to the sequence of GbU6-5Ppromoter cloned, six different truncated U6promoterswere successfully cloned using Transfer PCR method and their length were 672, 468, 358, 280, 202 and 105 bp, respectively.Together with GbU6-5P::GUS-pCAMBIA1300, GUS fusion expression vectors driven bycorresponding truncated promoter were constructed and transformed into cotton pollen by the vacuum infiltrationtransformation method.Results of GUS histochemical staining showed that the cloned seven truncatedGbU6-5Ppromoters could drive GUS expression in cotton pollen and all corresponding cottonpollen could be stained blue, but there exist different shades among them. Results showed that the shorter promoter, the stronger transcription activity, and so did the two other GbU6 promoter. In this study three short GbU6 promoters withtranscription activity in cotton pollen were cloned. GUS staining results showed that the shorter U6 promoter had higher transcriptional activity, which was the common characteristics in different U6 promoter.The above results indicated that using shorter U6 promoter not onlyconform to the requirement of constructing CRISPR/Cas9 genome editing vector,but also improve the transcription of sgRNA, which mayenhance the efficiency of genome editing finally.

Key words: Cotton, GbU6 promoter, Truncated cloning, Vacuum infiltration, Cotton pollen

[1]Liu L, Fan X D. CRISPR-Cas system: a powerful tool for genome engineering. Plant MolBiol, 2014, 85: 209–218
[2]Fichtner F,Urrea Castellanos R,Ülker B.Precision genetic modifications: A new era in molecular biology and crop improvement. Planta, 2014, 239:921–939
[3]Cong L,Ran FA,Cox D,Lin S,Barretto R,Habib N,Hsu PD,Wu X,Jiang W,Marraffini LA,Zhang F.MultiplexgenomeengineeringusingCRISPR/Cassystems.Science,2013, 339:819–823
[4]Belhaj K, Chaparro-Garcia A, Kamoun S,Nekrasov V. Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods, 2013,9: 39
[5]Belhaj K, Chaparro-Garcia A, Kamoun S, Patron N J, Nekrasov V. Editing plant genomes with CRISPR/Cas9. Curr Opin Biotechnol, 2015, 33: 76–84
[6]Li X, Jiang D H, Yong K L, Zhang D B. Varied transcriptional efficiencies of multiple Arabidopsis U6 small nuclear RNA genes. J Integr Plant Biol, 2007, 49: 222–229
[7]Hyun Y, Kim J, Cho S W,Choi Y, Kim JS, Coupland G. Site-directed mutagenesis in Arabidopsis thaliana using dividing tissue-targeted RGEN of the CRISPR/Cas system to generate heritable null alleles. Planta, 2015, 241: 271–284
[8]Jacobs T B,LaFayette P R,Schmitz R J,Parrott W A.Targeted genome modifications in soybean with CRISPR/Cas9.BMC Biotechnol, 2015, 15: 16
[9]Jiang W,Zhou H,Bi H,Fromm M, Yang B, Weeks D P.Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucl Acids Res, 2013, 41:e188
[10]Wang M B, Helliwell CA, Wu L M, Waterhouse PM, Peacock WJ, Dennis ES.Hairpin RNAs derived from RNA polymerase II and polymerase III promoter-directed transgenes are processed differently in plants. RNA Biol,2008, 14: 903–913
[11]DomitrovichAM,Kunkel GR. Multiple, dispersed human U6 small nuclear RNA genes with varied transcriptional efficiencies. Nucl Acids Res, 2003, 31:2344–2352
[12]雷建峰,伍娟,陈晓俊,於添平, 倪志勇,李月, 张巨松,刘晓东.棉花花粉中高效转录U6启动子的克隆及功能分析.中国农业科学,2015, 48: 3794–3802
Lei J F, Wu J, Chen X J, Yu T P, Ni Z Y, Li Y, Zhang J S, Liu X D. Cloning and functional analysis of cotton U6 promoter with high transcription activity in cotton pollen. Sci AgricSin, 2015, 48: 3794–3802(in Chinese with English abstract)
[13]Fauser F, Schiml S, Puchta H. Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J,2014,79: 348–359
[14]Feng Z Y, Mao Y F, Xu N F, Zhang B T, Wei P L, Yang D L, Wang Z, Zhang Z J, Zheng R, Yang L, Zeng L, Liu X D, Zhu J K. Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc Natl Acad Sci USA, 2014, 111: 4632–4637
[15]Nekrasov V, Staskawicz B, Weigel D, Jones JD, Kamoun S.TargetedmutagenesisinthemodelplantNicotianabenthamianausingCas9RNA-guidedendonuclease.Nat Biotechnol, 2013,31:691–693
[16]李晓,王学德.根癌农杆菌转化棉花花粉的研究.棉花学报,2004,16:323–327
Li X, Wang X D.Study on transformation of cotton pollen using Agrobacterium tumefaciens. Cotton Sci, 2004, 16:323–327(in Chinese with English abstract)
[17]张燕红,黄乐平,周小云,王冬梅. 农杆菌真空渗透法转化棉花花粉的初步研究. 棉花学报, 2008, 20:354–358
Zhang Y H, Huang L P, Zhou X Y, Wang D M. The preliminary study on transformation of cotton pollen using agrobacterium-mediated vacuum infiltration.Cotton Sci,2008, 20:354–358 (in Chinese with English abstract)
[18]Feng Z Y, Zhang B, Ding W, Liu X D, Yang D L, Wei P, Cao F, Zhu S, Zhang F, Mao Y, Zhu J K. Efficient genome editing in plants using a CRISPR/Cas system. Cell Res, 2013, 23:1229–1232
[19]Erijman A, Shifman JM, Peleg Y. A single-tube assembly of DNA using the transfer-PCR (TPCR) platform.Methods Mol Biol, 2014,1116: 89–101
[20]Shan Q, Wang Y, Li J, Gao C.Genome editing in rice and wheat using the CRISPR/Cas system.Nat Prot, 2014, 9: 2395–2410
[21]Endo M, Mikami M, Toki S. Multi-gene knockout utilizing off-target mutations of the CRISPR/Cas9 system in rice. Plant Cell Physiol, 2014, 56: 41–47
[22]Miao J, Guo D, Zhang J, Huang Q, Qin G, Zhang X, Wan J, Gu H, Qu L J.Targeted mutagenesis in rice using CRISPR-Cas system. Cell Res, 2013, 23: 1233–1236
[23]Santosh K U, Jitesh K, Anshu A, Rakesh T. RNA-guided genome editing for target gene mutations in wheat.G3 (Bethesda), 2013, 3: 2233–2238
[24]Liang Z, Zhang K, Chen K,Gao C. Targeted mutagenesis in Zea maysusing TALENs and the CRISPR/CasSystem. J Genet Genom, 2014, 41: 63–68
[25]Christopher B, Vladimir N, Zachary B. Lippman, Joyce V E. Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 System.Plant Physiol, 2014, 166: 1292–1297
[26]MarshallsayC, KissT, FilipowiczW. Amplification of plant U3 and U6 snRNA gene sequences using primers specific for an upstream promoter element and conserved intragenic regions.Nucl Acids Res, 1990, 18:3459–3466
[27]Tomson M,Manjula S. Optimization of Agrobacterium-mediated transient gene expression and endogenous gene silencing in Piper colubrinum Link.by vacuum infiltration. Plant CellTissue Organ Culture, 2011, 105: 113–119

[1] 周静远, 孔祥强, 张艳军, 李雪源, 张冬梅, 董合忠. 基于种子萌发出苗过程中弯钩建成和下胚轴生长的棉花出苗壮苗机制与技术[J]. 作物学报, 2022, 48(5): 1051-1058.
[2] 孙思敏, 韩贝, 陈林, 孙伟男, 张献龙, 杨细燕. 棉花苗期根系分型及根系性状的关联分析[J]. 作物学报, 2022, 48(5): 1081-1090.
[3] 闫晓宇, 郭文君, 秦都林, 王双磊, 聂军军, 赵娜, 祁杰, 宋宪亮, 毛丽丽, 孙学振. 滨海盐碱地棉花秸秆还田和深松对棉花干物质积累、养分吸收及产量的影响[J]. 作物学报, 2022, 48(5): 1235-1247.
[4] 郑曙峰, 刘小玲, 王维, 徐道青, 阚画春, 陈敏, 李淑英. 论两熟制棉花绿色化轻简化机械化栽培[J]. 作物学报, 2022, 48(3): 541-552.
[5] 张艳波, 王袁, 冯甘雨, 段慧蓉, 刘海英. 棉籽油分和3种主要脂肪酸含量QTL分析[J]. 作物学报, 2022, 48(2): 380-395.
[6] 张特, 王蜜蜂, 赵强. 滴施缩节胺与氮肥对棉花生长发育及产量的影响[J]. 作物学报, 2022, 48(2): 396-409.
[7] 赵文青, 徐文正, 杨锍琰, 刘玉, 周治国, 王友华. 棉花叶片响应高温的差异与夜间淀粉降解密切相关[J]. 作物学报, 2021, 47(9): 1680-1689.
[8] 岳丹丹, 韩贝, Abid Ullah, 张献龙, 杨细燕. 干旱条件下棉花根际真菌多样性分析[J]. 作物学报, 2021, 47(9): 1806-1815.
[9] 曾紫君, 曾钰, 闫磊, 程锦, 姜存仓. 低硼及高硼胁迫对棉花幼苗生长与脯氨酸代谢的影响[J]. 作物学报, 2021, 47(8): 1616-1623.
[10] 马欢欢, 方启迪, 丁元昊, 池华斌, 张献龙, 闵玲. 棉花GhMADS7基因正调控棉花花瓣发育[J]. 作物学报, 2021, 47(5): 814-826.
[11] 许乃银, 赵素琴, 张芳, 付小琼, 杨晓妮, 乔银桃, 孙世贤. 基于GYT双标图对西北内陆棉区国审棉花品种的分类评价[J]. 作物学报, 2021, 47(4): 660-671.
[12] 周冠彤, 雷建峰, 代培红, 刘超, 李月, 刘晓东. 棉花CRISPR/Cas9基因编辑有效sgRNA高效筛选体系的研究[J]. 作物学报, 2021, 47(3): 427-437.
[13] 卢合全, 唐薇, 罗振, 孔祥强, 李振怀, 徐士振, 辛承松. 商品有机肥替代部分化肥对连作棉田土壤养分、棉花生长发育及产量的影响[J]. 作物学报, 2021, 47(12): 2511-2521.
[14] 王晔, 刘钊, 肖爽, 李芳军, 吴霞, 王保民, 田晓莉. 转PSAG12-IPT基因对棉花叶片衰老及产量和纤维品质的影响[J]. 作物学报, 2021, 47(11): 2111-2120.
[15] 杨琴莉, 杨多凤, 丁林云, 赵汀, 张军, 梅欢, 黄楚珺, 高阳, 叶莉, 高梦涛, 严孙艺, 张天真, 胡艳. 棉花花器官突变体的鉴定及候选基因的克隆[J]. 作物学报, 2021, 47(10): 1854-1862.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!