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1 作物学报 ACTA AGRONOMICA SINICA 2009, 35(5): ISSN ; CODEN TSHPA9 DOI: /SP.J 采用一种新型 RNAi 载体培育转基因高直链淀粉马铃薯郭志鸿王亚军张金文 2 张玉宝王金牛谢忠奎,* 陈正华 3, ; 2, ; 3, 000 : PCR nos axi ubi.u4, Sbe Sbe2 SIII, ubi.u4 SIII nos axi nos 3 UTR RNAi pcusni, 3 2, 6, 4, 53.80%~85.33%; RT-PCR, 80% Sbe Sbe2 mrna, 3 UTR RNAi pcusni Sbe Sbe2, RNAi pcusni,, pcusni BamH I Xba I SIII,, : RNAi; nos ; ; Development of Transgenic High-Amylose Potato Using a Novel RNAi Vector GUO Zhi-Hong, WANG Ya-Jun, ZHANG Jin-Wen 2, ZHANG Yu-Bao,WANG Jin-Niu, XIE Zhong-Kui,*, and CHEN Zheng-Hua 3 Cold and Arid Region s Environmental and Engineering Institute, Chinese Academy of Sciences, Lanzhou , China; 2 College of Agronomy, Gansu Agricultural University, Lanzhou , China; 3 Postdoctoral Scientific Research Station of Gansu Yasheng Industrial (Group). Co. Ltd., Beijing Branch, Beijing 000, China Abstract: Amylose from potato starch is of great advantage for applications in many fields because of its higher degree of polymerization and lower gelling temperature compared with cereal starch. However, there is no natural mutant of high-amylose potato. RNAi technique is efficient and specific for plant gene silence but traditional RNAi vector is laborious to prepare. To design an easily-prepared RNAi vector and to develop transgenic high-amylose potato, PCR technique was employed to amplify the nos terminator, the tobacco axi and the tobacco ubi.u4 promoter, and to sub-clone a fused fragment SIII which is partially homologous to potato Sbe and to Sbe2. Then, a newly designed vector pcusni containing ubi.u4 promotor antisense SIII antisense nos terminator axi gene intron sense nos terminator was generated and transformed into potato varieties Longshu 3, Gannongshu 2, and Atlantic by Agrobacterium-mediated transformation. Sixteen transgenic potato plants were obtained, and the amylose content in 4 of them increased significantly, which ranged from 53.80% to 85.33% of total starch. But with the increase of amylose content, starch content decreased in transgenic potato plants. Results of semi-quantitative RT-PCR indicated that the accumulation of mrnas for Sbe and Sbe2 was not detectable in transgenic plants with amylose content higher than 80%, indicating that the novel RNAi vector pcusni was highly efficient for the simultaneous silence of Sbe and Sbe2 in potato. The generation of the RNAi vector pcusni makes it much easier to prepare RNAi vectors targeting to other plant genes. The only thing is to insert a fragment of the target gene or the target genes in restriction sites between BamH I and Xba I in vector pcusni to replace SIII and there is no need to construct an inverted repeat of target gene. Keywords: RNAi; nos terminator; Inverted repeat; High-amylose potato (KSCX2-YW-N-46-05) * (Corresponding author):, wxhcas@lzb.ac.cn : guozhhong@yahoo.com.cn Received( ): ; Accepted( :

2 80 35 RNA (RNA interference, RNAi) RNA (double-stranded RNA, dsrna), [-2], [3-5] RNAi,,,, RNAi RNAi, RNA (small interference RNA, sirna), RNA RNA RNA (complement RNA, crna), dsrna, Dicer sirna, RNA mrna 5 [6-9] RNAi, 3 ( ) RNAi, mrna, 3 ( ) RNAi, RNAi,,,,,,, (starch branching enzyme, SBE) α-,4, α-,6, [0] SBE, SBEI SBEII,, [-2], Sbe Sbe2, [3-4] nos, Sbe Sbe2 SIII ubi.u4 nos,, Sbe Sbe2, 材料与方法. NC pbluescript SK(+) pbi2 prnaiiii [5] DH5α LBA4404, T4 DNA DNA TaKaRa, RNA Concert Plant RNA Reagent Invitrogen, DNA Marker RT-PCR Taq MasterMix TIANGEN, (Nylon Membrane, positive geladen) Southern blot Roche, Sigma,.2 GenBank pbi2 (GI ) nos nosu (5 -gcgg ccgcgatcgttcaaacatttggcaata-3 ) nosl (5 -tctagattcccgatctagtaacatagatgaca-3 ), 258 bp, nosu 5 Not I, nosl 5 Xba I ( ) pbi2, nosu nosl, PCR nos axi (GI: ) tinu (5 -gcggccgcgtg GTCCATTTCAATTTTGTT-3 ) tinl (5 -gagctcctgc AAATGTTACAACAAGGCT-3 ), ( Not I Sac I ), 82 bp CTAB DNA, tinu tinl, DNA, PCR axi [6] ubi.u4 ubi (5 -aagcttggaggctaactacgttagagc-3 ) ubi2 (5 -ggatcctctgtatatacagaaaaggtt-3 ), Hind III BamH I

3 5 : RNAi 8, DNA ubi.u4 ; [5] SIII sal (5 -ggatccgaacgaggacgatcatcatacca- 3 ) sbu (5 -tctagatgatgttgacagtaagccagtc- 3 ), BamH I Xba I 4 pbluescript SK+ Xba I Not I Not I Sac I BamH I Xba I Hind III BamH I,, pusni.3 RNAi pusni Hind III/Sac I pbi2 pbiusin, pbiusin Hind III/EcoR I pcambia- 300, pcusni.4 [7] pcusni LBA4404, 3 2, [8] 2 cm,.5 CTAB [9] DNA DNA, sbu, sal PCR,, Souhern, PCR DNA Sac I 0.8%, 20 SSC, 80 2 h ubi.u4,, DIG High Prime DNA Labeling and Detection Starter Kit I.6, [20], λ =500 nm λ 2 =600 nm, λ 3 =700 nm λ 4 =590 nm 3,.7 RT-PCR RNA, RT-PCR, [5] 2 结果与分析 2. nosu nosl, pbi2 PCR, 250 bp (, ), nos ; tinu tinl, DNA PCR, 200 bp (, 2), axi ; sal sbu, pcusni PCR, 700 bp (, 3), SIII ; ubi ubi2, DNA PCR, 800 bp (, 4), ubi.u4, 4,, nos axi SIII ubi.u4, ubi.u4 SIII nos axi pusni 图基因 PCR 扩增 Fig. PCR amplification of genes M DNA marker III; nos ; 2 axi ; 3 SIII; 4 ubi.u4 M: DNA marker III; : nos terminator; 2: axi intron; 3: SIII; 4: ubi.u4 promoter. 2.2 pcusni pcusni nosu nosl tinu tinl PCR, 250 bp ( 2, ) 200 bp ( 2, 2), nos axi ; EcoR I/Sac I, 250 bp ( 3, 4), tinu nosl PCR,

4 bp ( 2, 3), axi nos ; tinl nosu, 450 bp ( 2, 4), axi nos ; sbu sal PCR, 70 bp ( 2, 5), SIII ; BamH I/ Sac I, 200 bp ( 3, 3), SIII nos axi Hind III/ BamH I ubi ubi2 PCR, 800 bp ( 3, 2; 2, 6), ubi.u4 Hind III/ EcoR I, bp ( 3, ), ubi.u4 SIII nos axi nos Sbe Sbe2 3 UTR RNAi pcusni, , 44 PCR, bp, ubi.u4, 3 ( 5), SIII Southern, ~2 ( 6) 6, 3 9 (3A~3A8 3A5), 2 4 (3A9~3A2), 3 (3A3~ 3A4 A6) 2.4, 6 4, 53.80%, 85.33%, ; 87.5% 80% 4, 3 2 (3A3 3A6), 2 (3A), (3A4) 80% (SY) 图 2 pcusni PCR 检测 Fig. 2 PCR detection of pcusni M DNA marker III; nosu+nosl ; 2 tinu+tinl ; 3 tinu+nosl ; 4 tinl+nosu ; 5 sbu+sal ; 6 ubi+ubi2 M: DNA marker III; : amplified with nosu+nosl; 2: amplified with tinu+tinl; 3: amplified with tinu+nosl; 4: amplified with tinl+nosu; 5: amplified with sbu+sal; 6: amplified with ubi+ubi2. 图 3 pcusni 酶切分析 Fig. 3 Restriction analysis of pcusni M: DNA marker III; Hind III/ EcoR I ; 2 Hind III/BamH I ; 3 BamH I/ Sac I ; 4 EcoR I/Sac I M: DNA marker III; : digested by Hind III/ EcoR I; 2: digested by Hind III/BamH I; 3: digested by BamH I/ Sac I; 4: digested by EcoR I/Sac I. 图 4 pcusni 表达框架示意图 Fig. 4 Schematic diagram of pcusni expressing frame RB ; ubi P ubi.u4 ; anti-siii SIII; anost nos ; Tin axi ; nost nos ; CaMV 35S CaMV35S ; HPTII ; poly A CaMV 35S ; LB RB: right border; ubi P: tobacco ubi.u4 promoter; anti-siii: SIII in antisense oritention; anost: nos terminator in antisense oritention; Tin: tobacco axi intron; CaMV35S: CaMV35S promoter; NOS P: nos promoter; HPT II: hygromycine phosphotransferase gene; poly A: CaMV 35S terminator; LB: left border.

5 5 : RNAi 83 图 5 Hyg 抗性再生植株的 PCR 检测 Fig. 5 PCR detection of regenerated plants resistant to Hyg M DNA marker V; ~6 ; 7 3 M: DNA marker V; 6: regenerated plants; 7: Longshu 3. 图 6 转基因植株 Southern 杂交 Fig. 6 Southern blotting of transgenic plants : 3A3; 2: 3A5; 3: 3A0; 4: 3A; 5: 3A2; 6: 3A3; 7: 3A5; 8: Longshu 3. 25%,,, 2.5 Sbe Sbe Sbe2 mrna, 800 bp, Actin, PCR ; 3 Sbe Sbe2 mrna ( 7, ), 80% 80% ( 7, 2~ 6), SBE mrna, Sbe Sbe2 ( 7, 7~0), Sbe Sbe2 Table 表转基因植株直链淀粉和淀粉糖含量 The amylose and starch content in transgenic plants ( ) Variety (line) 3 Longshu 3 Amylose content (%) Starch content (%) 23.65± ±.96 3A 74.30± ±2. 3A ± ±2.36 3A ± ±2.68 3A ±2.69.0±.89 3A ± ±3.02 3A6 8.30± ±2.22 3A7 79.8±.70.32±.59 3A ± ±2.38 3A ± ±.98 2 Gannongshu ± ±2.26 3A ± ±2.27 3A ± ±3.37 3A 85.24± ±.85 3A ± ±2.0 Atlantic 22.6± ±.75 3A3 68.2± ±2.26 3A ± ±.29 3A6 2.99± ±2.9 Amylose content is the percentage to total starch. 图 7 半定量 RT-PCR 检测 Sbe 和 Sbe2 mrna 的积累 Fig. 7 Semi-quantitative RT-PCR detection of mrna derived from Sbe and Sbe2 M: DNA marker III; : Longshu 3; 2: 3A3; 3: 3A6; 4: 3A; 5: 3A4; 6: 3A0; 7: 3A5; 8: 3A6; 9: 3A5; 0: 3A2. 3 讨论 3 UTR RNAi dsrna 5 5 ( RNAi dsrna 5 ) ubi.u4 SII nos axi nos, nos, SIII nos axi nos mrna, nos dsrna, RNAi,

6 84 35 sirna, sirna, RNA RNA SIII RNA, SIII, SIII mrna 3 UTR RNAi,, 3 UTR, 3 UTR (5 ),, 3 UTR, nos,, axi, ubi.u4, nos RNAi pcusni, RNAi, BamH I Xba I, nos axi, nos 800 bp,,, 6 pcusni, 4 Sbe Sbe2,, 87.5%, 80% 4, 25%, nos 3 UTR RNAi Sbe Sbe2, RNAi Sbe Sbe2, ( ~2 ), 2,,, RNA, Sbe Sbe2, Sbe, Sbe [4], Sbe Sbe2,, ;, [4] 4 结论 3 UTR RNAi pcusni RNAi, Sbe References [] Waterhouse P M, Helliwell C A. Exploring plant genomes by RNA-induced gene silencing. Nat Rev Genet, 2002, 4: [2] Louisa M. RNAi for plant functional genomics. Comparative Funct Genom, 2004, 5: [3] Ogita S, Uefuji H, Yamguchi Y, Koizumi N, Samo H. Producing decaffeinated coffee plants. Nature, 2003, 423: 823 [4] Kusaba M, Miyahara K, Lida S, Fukuoka H, Takario T, Sassa H, Nishimura M, Nishio T. Low glutenin content : A dominant mutation that suppresses the glutenin multigene family via RNA silencing in rice. Plant Cell, 2003, 5: [5] Wang M, Abbott D, Waterhouse P M. A single copy of a virus derived transgene encoding hairpin RNA gives immunity to barley yellow dwarf virus. Mol Plant Pathol, 2000, : [6] Dalmay T, Hamilton A J, Rudd S, Angell S, Baulcombe D C. An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a tansgene but not by a virus. Cell, 2000, 0: [7] Hamilton A J, Baulcombe D C. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science, 999, 286: [8] Han Y, Grierson D. Relationship between small antisense RNAs and aberrant RNAs associated with sense transgene mediated gene silencing in tomato. Plant J, 2002, 29: [9] Sijen T, Fleenor J, Simmer F, Thijssen K L, Parrish S, Timmons L, PlasterK R H A, Fire A. On the role of RNA amplification in dsrna-triggered gene silencing. Cell, 200, 07: [0] Fernie A R, Willmitzer L, Trethewey N R. Sucrose to starch:

7 5 : RNAi 85 Transition in molecular plant physiology. Trends Plant Sci, 2002, 7: 35 4 [] Safford R, Jobling S A, Sidebottom C, Westcott R J, Cooke D, Tober K J, Strongitharm B H, Russell A L, Gidley M J. Consequences of antisense RNA inhibition of starch branching enzyme activity on properties of starch. Carbohydrate Polymers, 998, 35: [2] Jobling S A, Schwall G P, Westcott R J, Sidebottom C M, Debet M, Gidley M J, Jeffcoat R, Safford R. A minor form of starch branching enzyme in potato (Solanum tuberosum L.) tubers has a major effect on starch structure: Cloning and characterization of multiple forms of SBE A. Plant J, 999, 8: 63 7 [3] Schwall G P, Safford R, Westcott R J, Jeffcoat R, Tayal A, Shi Y, Gidley M J, Jobling S A. Production of very-high-amylose potato starch by inhibition of SBE A and B. Nat Biotechnol, 2000, 8: [4] Hofvander P, Andersson M, Larsson C T, Larsson H. Field performance and starch characteristics of high-amylose potatoes obtained by antisense gene targeting of two branching enzymes. Plant Biotechnol J, 2004, 2: [5] Guo Z-H( ), Zhang J-W( ), Wang D( ), Chen Z-H( ). Using RNAi technology to produce high-amylose potato plants. Sci Agric Sin ( ), 2008, 4(2): (in Chinese with English abstract) [6] Kang T J, Kwon T H, Kim T G, Loc N H, Yang M S. Comparing constitutive promoters using CAT activity in transgenic tobacco plants. Mol Cells, 2003, 6: 7 22 [7] Wang G-L( ), Fang H-J( ). Plant Gene Engineering ( ), 2nd edn. Beijing: Science Press, pp (in Chinese) [8] Paul H, Marja S R, Evert J, Richard G F V. Transformation of a large number of varieties: Genotype-dependent variation in efficiency and somaclonal variability. Euphytica, 2002, 24: 3 22 [9] Murray M G, Thompson W F. Rapid isolation of high molecular weight DNA. Nucl Acids Res, 980, 8: [20] Chen Y-Q( ). Mothods and Technologies of Biochemistry Experiment ( ). Beijing: Science Press, pp 75 78(in Chinese)

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇