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1 作物学报 ACTA AGRONOMICA SINICA 206, 42(5): ISSN ; CODEN TSHPA9 DOI: /SP.J 棉花不同 GbU6 启动子截短克隆及功能鉴定雷建峰刘晓东,* 李月徐新霞 2 阿尔祖古丽塔什蒲艳张巨松 /, ; 2, : U6 CRISPR/Cas9 sgrna, CRISPR/Cas9 GbU6-5P ( 66 bp), Transfer PCR 6 U6, bp, 6 GUS 6 GbU6-5Ps::GUS-pCAMBIA300 GbU6-5P::GUS-pCAMBIA300 GUS, 7 GbU6-5P GUS,, U6 GbU6-P GbU6-7P 3 GbU6 U6, U6 U6 CRISPR/Cas9, sgrna, : ; GbU6 ; ; ; Cloning and Functional Analysis of Different Truncated GbU6 Promoters in Cotton LEI Jian-Feng, LI Yue, XU Xin-Xia 2, AERZUGULI Tashi, PU Yan, ZHANG Ju-Song, and LIU Xiao-Dong,* College of Agronomy, Xinjiang Agricultural University, Laboratory of Agricultural Biotechnology of Xinjiang Agricultural University, Urumqi , China; Center of Bazhou Agricultural Technology Promotion, Korla 84000, China 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 genome editing vector. According to the sequence of GbU6-5P promoter cloned, six different truncated U6 promoters were successfully cloned using Transfer PCR method and their length were 672, 468, 358, 280, 202, and 05 bp, respectively. Together with GbU6-5P::GUSpCAMBIA300, GUS fusion expression vectors driven by corresponding truncated promoter were constructed and transformed into cotton pollen by the vacuum in filtration transformation method. Results of GUS histochemical staining showed that the cloned seven truncated GbU6-5P promoters could drive GUS expression in cotton pollen and all corresponding cotton pollen 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 with transcription 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 only ( ) (XJGRI205084) This study was supported by the National Natural Science Foundation of China ( ) and the Science and Technology Innovation Projects for Graduate Students in Xinjiang Uygur Autonomous Region (XJGRI205084). (Corresponding authors):, xiaodongliu75@aliyun.com;, xjndzjs@63.com : @63.com Received( ): ; Accepted( ): ; Published online( ): URL:

2 conform to the requirement of constructing CRISPR/Cas9 genome editing vector, but also improve the transcription of sgrna, which may enhance the efficiency of genome editing finally. Keywords: Cotton; GbU6 promoter; Truncated cloning; Vacuum infiltration; Cotton pollen,,,, ΙΙ CRISPR/Cas9 (ZFN) TALE (TALEN), [-3] sgrna Cas9 2, sgrna crrna tracerrna(trans-activating crrna), CRISPR/Cas9, sgrna U6 U3,, : G, A sgrna DNA, [4-6] U6 sgrna Hyun [7] AtU6 sgrna, FT SPBPlike-4 CRISPR/Cas9, T Thomas [8] U6, 9 Jiang [9] U6 sgrna, OsSWEET4 OsSWEET U6, [0], U6, [6,] CRISPR/Cas9 U6 0 U6 5 U6, GbU6-5P ( 66 bp) [2],, CRISPR/Cas9 U6 200~400 bp [3-4], 00 bp [5] Cas9 ( 4000 bp), Cas9 35S 000 bp [4-5], 2 sgrna Cas9, sgrna U6, U6 CRISPR/Cas9 5 U6, GbU6 GUS [6-7],, U6 材料与方法. AtU6-26P::sgRNA [8] pbi0 GV30 6 Fermentas ; T4 DNA Blunt Zero Trans-T Taq DNA RNase A KD Plus DNA kb Plus DNA Ladder ; Phusion DNA ( ) ; ;.2 GbU6-5P GbU6-5P AtU6-26P::sgRNA 6 GbU6-5P Transfer PCR ( ) 204 GbU6-5P (787 bp) GbU6-5P, PCR GbU6-5P, AtU6-26P::sgRNA PCR Transfer PCR [9], GbU6-5P Transfer PCR Dpn I (4 U), 37 3 h, 0 µl GbU6-5P BamH I Hind III, T-GbU6-5Ps

3 5 : GbU6 677 表本研究中使用的引物序列 Table Primer sequences used in this study Primer name Forward sequence (5' 3') Reverse sequence (5' 3') T-GbU6-5P2 CCGCCAGTGTGCTGGAATTGCCCT CTAGCTCTAAAACAGGTCTTCTCGAA TAACAGACTATCAACAAGCCAAGC GACCCACTTATTTGACGCTTCTTTCGC T-GbU6-5P3 CCGCCAGTGTGCTGGAATTGCCCTTA CTAGCTCTAAAACAGGTCTTCTCGAAG AGAATGCTGTTGAATTCCCATC ACCCACTTATTTGACGCTTCTTTCGC T-GbU6-5P4 CCGCCAGTGTGCTGGAATTGCCCTTAAC CTAGCTCTAAAACAGGTCTTCTCGAAGA TAATATACATTGGTTCCAAGG CCCACTTATTTGACGCTTCTTTCGC T-GbU6-5P5 CCGCCAGTGTGCTGGAATTGCCCTTAAA CTAGCTCTAAAACAGGTCTTCTCGAAGA CCCGATTGTAATGTGAAGTC CCCACTTATTTGACGCTTCTTTCGC T-GbU6-5P6 CCGCCAGTGTGCTGGAATTGCCCTTAAGGA CTAGCTCTAAAACAGGTCTTCTCGAAGACCC TAAGTGATAAACACTGAGC ACTTATTTGACGCTTCTTTCGC T-GbU6-5P7 CCGCCAGTGTGCTGGAATTGCCCTTAA CTAGCTCTAAAACAGGTCTTCTCGAAGACCC AGGACGTGGTAGCATACTTC ACTTATTTGACGCTTCTTTCGC GUS TAAGTGGGTCTTCATGTTACGTCCTGTAGAAAC TAAACAGGTCTTCGAATTCCCGATCTAGTAACA T-GbU6-P4 CCGCCAGTGTGCTGGAATTGCCCTTAAGCC GGGTTTCTACAGGACGTAACATGAAGACCC AAAATTATAATGACTCGAAC GATAATAAGTTTCTTCCTTTGCC T-GbU6-P5 CCGCCAGTGTGCTGGAATTGCCCTTAATTTTCC GGGTTTCTACAGGACGTAACATGAAGACCCGAT CATTGAAGGGACAAC AATAAGTTTCTTCCTTTGCC T-GbU6-3P4 CCGCCAGTGTGCTGGAATTGCCCTTAATTTTCC GGGTTTCTACAGGACGTAACATGAAGACCCAGC CATTGAAGGGACAAC CAAGTGATCCTTCTTTTAC T-GbU6-3P5 CCGCCAGTGTGCTGGAATTGCCCTTAACCTGTC GGGTTTCTACAGGACGTAACATGAAGACCC AGATAGACGCCATT AGCCAAGTGATCCTTCTTTTAC T-GbU6-7P4 CCGCCAGTGTGCTGGAATTGCCCTTAACTTAA GGGTTTCTACAGGACGTAACATGAAGACCC CGAGAGTTGAGTTGGT ATTACCTGCTAGCTGCTTCTT T-GbU6-7P5 CCGCCAGTGTGCTGGAATTGCCCTTAATCCC GGGTTTCTACAGGACGTAACATGAAGACCC ACTCTACAGCCATAAG ATTACCTGCTAGCTGCTTCTT T-GbU6-9P4 CCGCCAGTGTGCTGGAATTGCCCTTAAAGT GGGTTTCTACAGGACGTAACATGAAGACCC AAAAGGTTGTTCCCAACC AGCCTAAGCTGGCTGCTGT T-GbU6-9P5 CCGCCAGTGTGCTGGAATTGCCCTTAA GGGTTTCTACAGGACGTAACATGAAGA GTTGAGGGAGTTGTGACAC CCCAGCCTAAGCTGGCTGCTGT Underlined sequences are the part of receptor plasmid partial nucleotide sequence..3 GbU6-5P::GUS GUS T-GbU6-5Ps Bbs I,,,, PCR, GbU6-5Ps::GUS Hind III BamH I pcambia300 6 GbU6-5Ps:: GUS,,,,, GbU6-5Ps::GUS-P300 GbU6-5Ps GbU6-5Ps::GUS GV30,.4 GbU6-5Ps::GUS-P300 CaMV35S::GUS pcambia LB ( 50 µg ml Kan 25 µg ml Rif) 28, 80 min, OD 600 = 0.6~.2, 6 ml LB ( 50 µg ml Kan 25 µg ml Rif), OD, 28, 80 min, OD 600 =.6, g 5 min,, [7] [0.% H 3 BO 3 0.3% Ca(NO 3 ) 2 0.2% MgSO 4 7H 2 O 0.% KNO 3 45% ],,

4 图不同截短 GbU6-5P 启动子驱动报告基因 GUS 的融合表达载体构建 Fig. Construction of GUS reporter gene fusion expression vector driven by different truncated GbU6-5P promoters, 0.05 MPa 30 min 3, 2.5 GbU6-5Ps::GUS 00 g min,,, 4~5, 00 g min,, GUS (0.5 mol L, ph 7.0; 0.5 mol L EDTA, ph 8.0; 0% Triton X-00; 20 mmol L X-Gluc), 37, 80 min 3~4 h, 00 g min, GUS 4~5, 00 g min, GUS GUS 620 nm ( ).6 2 GbU6-5P6 GbU6-5P7,, (%) = ( / ) 00, 3,,,.7 4 U6 PCR, 4 U6, GbU6-P GbU6-3P GbU6-7P GbU6-9P Transfer PCR 4 U6 2 ( ) GbU6-5P2::GUS, GbU6-P GbU6-3P GbU6-7P GbU6-9P U6 GUS, pcambia300 GbU6-P4::GUS GbU6-P5::GUS GbU6-3P4:: GUS GbU6-3P5::GUS GbU6-7P4::GUS GbU6-7P5:: GUS GbU6-9P4::GUS GbU6-9P5::GUS GV30, 2 结果与分析 2. GbU6-5P GbU6-5P, Transfer PCR, 6 GbU6-5P ( 2), GbU6-5P2: 672 bp GbU6-5P3: 468 bp GbU6-5P4: 358 bp GbU6-5P5: 280 bp GbU6-5P6: 202 bp GbU6-5P7: 05 bp BamH I Hind III ( ), GbU6-5P

T-GbU6-5P3, 3: T-GbU6-5P4, 4: T-GbU6-5P5, 5: T-GbU6-5P6, 6:T-GbU6-5P7. 2.

5 5 : GbU6 679 图 2 6 个不同截短 GbU6-5P 启动子的 Transfer PCR 扩增产物电泳检测 Fig. 2 Transfer PCR amplification of six truncated GbU6-5P promoters M: kb DNA marker; :T-GbU6-5P2, 2: T-GbU6-5P3, 3: T-GbU6-5P4, 4: T-GbU6-5P5, 5: T-GbU6-5P6, 6:T-GbU6-5P GbU6-5P::GUS 6 GbU6-5Ps GUS, : EcoR I GbU6-5P2::GUS, 3272 bp 2608 bp ; EcoR V GbU6-5P3:: GUS GbU6-5P4::GUS GbU6-5P5::GUS, GbU6-5P3::GUS bp, GbU6-5P4::GUS bp, GbU6-5P5::GUS bp ; GbU6-5P6::GUS GbU6-5P7::GUS Pst I Mfe I, GbU6-5P6::GUS 3404 bp 249 bp, GbU6-5P7::GUS 3307 bp 249 bp ( 3), 6 GbU6-5P::GUS pcambia300,,, GbU6-5P GUS GbU6-5P:: GUS-P300 ( ) 图 3 GbU6-5Ps::GUS 表达载体的酶切鉴定 Fig. 3 Identification of different truncated GbU6-5Ps::GUS expression vectors by restriction enzyme digestion M: kb DNA marker: A: GbU6-5P2::GUS; B: GbU6-5P3::GUS; C: GbU6-5P4::GUS; D: GbU6-5P5::GUS; E: GbU6-5P6::GUS; F: GbU6-5P7::GUS. 2.3 GbU6-5P GUS, GbU6-5P 6 GbU6-5P GUS,, GbU6-5P6::GUS GbU6-5P7::GUS,, GUS, ( 4) 2.4 GUS GbU6-5P, GUS, 620 nm, pcambia300 GUS 3, CaMV35S GUS, GbU6-5P7 GbU6-5P6,, ( 5), 2.5, GbU6-5P6 GbU6-5P7, ( 6) GbU6-5P6 23.7%, 2.%, 95.9%; GbU6-5P7 25.%, 4.5%, 73.% U6 4 U6

6 680 作物学报 Fig. 4 Fig. 5 第 42 卷图 4 不同截短 GbU6-5P 启动子驱动 GUS 在棉花花粉中的瞬时表达 GUS expression driven by different truncated GbU6-5P promoters in cotton pollen 图 5 不同截短 GbU6-5P 启动子对应的花粉 GUS 染色后染液光吸收值 Light absorbance value at 620 nm of staining solution after histochemical stain corresponding to different truncated GbU6-5P promoters 不同小写字母表示处理间在 0.05 的水平上差异显著 Different letters indicate significant difference among different treatments at the 0.05 probability level. 别命名为 GbU6-P4 (440 bp), GbU6-P5 (92 bp); GbU6-3P4 (460 bp), GbU6-3P5 (2 bp); GbU6-7P4 (45 bp), GbU6-7P5 (90 bp); GbU6-9P4 (470 bp), GbU6-9P5 (200 bp), GUS 融合表达载体酶切鉴定结 7) 在 620 nm 处光吸收值的结果也显示, GbU6-P5 果图略通过农杆菌真空渗透转化棉花花粉, 经 3 显著高于 GbU6-P4; 在 GbU6-7P4 几乎没有光吸收值的情况下, GbU6-7P5 的光吸收值却很高(图 8) 讨论 GUS 组织化学染色发现, 对于 GbU6-P4::GUS ΙΙ 型 CRISPR/Cas9 基因组编辑系统自 203 年诞生 GbU6-P5::GUS GbU6-7P5::GUS 可以检测到蓝色以来, 已在水稻[9,20-22] 小麦[20,23] 玉米[24] 番茄[25] 花粉, 而其他克隆的不同截短的 GbU6 启动子并没大豆[8]等作物中成功应用 U6 启动子作为利用 CRISPR/ 有检测到蓝色花粉(图略) 结果还显示较短的 Cas9 基因编辑技术进行棉花分子育种的重要组成部 GbU6-P5::GUS GbU6-7P5::GUS 比对应长片段的分, 需要它能在生殖细胞中具有高效转录活性, 产 GbU6-P4::GUS GbU6-7P4::GUS 染色显著增强(图生可遗传的新种质研究表明, 在真核生物中, USE

7 5 : GbU6 68 图 6 真空与非真空处理转化效率比较 Fig. 6 Comparison of transformation efficiency between vacuum and non-vacuum treatments 0.05 Different letters indicate significant difference among different treatments at the 0.05 probability level. 图 7 其他截短 GbU6 启动子花粉 GUS 染色 Fig. 7 GUS staining analysis of other truncated GbU6 promoter in cotton pollen 图 8 其他截短 GbU6 启动子 GUS 染液光吸收值 Fig. 8 Light absorbance value of GUS staining solution for other truncated GbU6 promoter 0.05 Different letters indicate significant difference among different treatments at the 0.05 probability level. TATA U6 2 [6,26] 5 GbU6, 5 GbU6 60 bp USE 30 bp TATA, 5 GbU6 GbU6-5P (66 bp) CRISPR/Cas9, U6, U6 GbU6-5P 6, USE TATA,, ( 4 5) 2 U6, U6 U6 ( 7), U6 ( ) U6,, USE TATA, U6, Nekrasov [5] 79 bp U6, GbU6-5P7 05 bp, U6 U6 60 bp USE 30 bp TATA GbU6-3P GbU6-9P, GbU6-3P GbU6-9P, U6,,,,,, GUS, U6,,

8 [7,27],,,,, GbU6-5P::GUS,,,, [6-7],,, GbU6-5P6 GbU6-5P7 95.9% 73.%,, GbU6, U6 sgrna, CRISPR/Cas9 4 结论 3 GbU6 ; GbU6, U6, GbU6-5P7 GbU6-P5 GbU6-7P5 CRISPR/Cas9 References [] Liu L, Fan X D. CRISPR-Cas system: a powerful tool for genome engineering. Plant Mol Biol, 204, 85: [2] Fichtner F, Urrea Castellanos R, Ülker B. Precision genetic modifications: a new era in molecular biology and crop improvement. Planta, 204, 239: [3] Cong L, Ran F A, Cox D, Lin S, Barretto R, Habib N, Hsu P D, Wu X, Jiang W, Marraffini L A, Zhang F. Multipl exgenome engineering using CRISPR/Cas systems. Science, 203, 339: [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, 203, 9: 39 [5] Belhaj K, Chaparro-Garcia A, Kamoun S, Patron N J, Nekrasov V. Editing plant genomes with CRISPR/Cas9. Curr Opin Biotechnol, 205, 33: [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: [7] Hyun Y, Kim J, Cho S W, Choi Y, Kim J S, Coupland G. Site-directed mutagenesis in Arabidopsis thaliana using dividing tissue-targeted RGEN of the CRISPR/Cas system to generate heritable null alleles. Planta, 205, 24: [8] Jacobs T B, LaFayette P R, Schmitz R J, Parrott W A. Targeted genome modifications in soybean with CRISPR/Cas9. BMC Biotechnol, 205, 5: 6 [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, 203, 4: e88 [0] Wang M B, Helliwell C A, Wu L M, Waterhouse P M, Peacock W J, Dennis E S. Hairpin RNAs derived from RNA polymerase II and polymerase III promoter-directed transgenes are processed differently in plants. RNA Biol, 2008, 4: [] Domitrovich A M, Kunkel G R. Multiple, dispersed human U6 small nuclear RNA genes with varied transcriptional efficiencies. Nucl Acids Res, 2003, 3: [2],,,,,,,. U6., 205, 48: 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 Agric Sin, 205, 48: (in Chinese with English abstract) [3] 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, 204, 79: [4] 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, 204, : [5] Nekrasov V, Staskawicz B, Weigel D, Jones J D, Kamoun S. Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guide dendonuclease. Nat Biotechnol, 203, 3: [6],.., 2004, 6: Li X, Wang X D. Study on transformation of cotton pollen using Agrobacterium tumefaciens. Cotton Sci, 2004, 6: (in Chinese with English abstract) [7],,,.., 2008, 20: Zhang Y H, Huang L P, Zhou X Y, Wang D M. The preliminary study on transformation of cotton pollen using Agrobacteriummediated vacuum infiltration. Cotton Sci, 2008, 20: (in Chinese with English abstract) [8] 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, 203, 23: [9] Erijman A, Shifman J M, Peleg Y. A single-tube assembly of DNA using the transfer-pcr (TPCR) platform. Methods Mol Biol, 204, 6: 89 0 [20] Shan Q, Wang Y, Li J, Gao C. Genome editing in rice and wheat

9 5 : GbU6 683 using the CRISPR/Cas system. Nat Prot, 204, 9: [2] Endo M, Mikami M, Toki S. Multi-gene knockout utilizing off-target mutations of the CRISPR/Cas9 system in rice. Plant Cell Physiol, 204, 56: 4 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, 203, 23: [23] Santosh K U, Jitesh K, Anshu A, Rakesh T. RNA-guided genome editing for target gene mutations in wheat. Genes Genomes Genetics (Bethesda), 203, 3: [24] Liang Z, Zhang K, Chen K, Gao C. Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas System. J Genet Genomics, 204, 4: [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, 204, 66: [26] Marshallsay C, Kiss T, Filipowicz W. Amplification of plant U3 and U6 snrna gene sequences using primers specific for an upstream promoter element and conserved intragenic regions. Nucl Acids Res, 990, 8: [27] Tomson M, Manjula S. Optimization of Agrobacteriummediated transient gene expression and endogenous gene silencing in Piper colubrinum Link. by vacuum infiltration. Plant Cell Tissue Organ Cult, 20, 05: 3 9

Syngene GBOXHR Retsch MM400 KQ DE Eppendorf Table 1 The sources of Astragali Radix and Hedysarum Radix No. 1 AMM2011_NMGTY Astr

Syngene GBOXHR Retsch MM400 KQ DE Eppendorf Table 1 The sources of Astragali Radix and Hedysarum Radix No. 1 AMM2011_NMGTY Astr PCR 龙平, 崔占虎, 李虔全, 徐建平, 张春红, 周立社 * *, 李旻辉 ( 包头医学院, 内蒙古包头 014060) DNA 30 28 DNA trnl-trnf PCR PCR 136 bp 323 bp 10% PCR PCR Astragalus membranaceus Fisch. Bge. var. mongho- licus Bge. Hsiao A. membranaceus