轉(zhuǎn)基因煙草的方法

1、轉(zhuǎn)基因煙草的方法1.接種單菌落于加有KAN,RIF和STR的3ML液體YEB培養(yǎng)基中，28度培養(yǎng)2天，轉(zhuǎn)接與20ML YEB液體培養(yǎng)基中，28度繼續(xù)培養(yǎng)至OD600=0.6-0.8。2.菌液經(jīng)4000RPM離心10分鐘，倒掉上清液，用MS液體培養(yǎng)基重懸菌體，調(diào)OD600=0.6-0.83.取無菌煙草葉片，去除邊緣，主葉脈，剪成大小約1CM*1CM的小塊，浸泡在菌體懸浮液中，2-3分鐘，期間不斷輕輕搖晃。4.取出葉片，用滅菌濾紙吸取表面菌液，將葉片至于共培養(yǎng)培養(yǎng)基上（MS+6-BA 1MG/L,NAA 0.1MG/L）28度黑暗共培養(yǎng)2-3天。5.將共培養(yǎng)后的葉片在無菌水和加入羧芐青霉素的無菌水

2、中清洗一遍，滅菌濾紙吸取多余水分，然后轉(zhuǎn)到分化選擇培養(yǎng)基上（MS+6-BA 1MG/L,NAA 0.1MG/L，潮霉素10-60MG/L,CARB 500MG/L）25度光照培養(yǎng)，每兩周更換一次培養(yǎng)基，直至分化出愈傷組織，進而分化出不定芽。6.將長至2CM以上的不定芽切下并轉(zhuǎn)移到生根培養(yǎng)基上（1/2MS+NAA 0.1MG/L，潮霉素20-100MG/L,CARB 500MG/L）誘導(dǎo)生根。7.將一次生根的再生苗切取根尖，接種到新的生根培養(yǎng)基上，誘導(dǎo)生根，不能生根的芽丟掉。王哲,孫敬克,王世翔,肖婧婧，煙草潮霉素抗性濃度的篩選與研究，18（4）,2009LOC_Os10g33000 seque

3、nce informationGenomic sequence length: 2932 nucleotidesCDS length: 2103 nucleotidesProtein length: 700 amino acidsPutative Function: retrotransposon protein, putative, unclassifiedGenomic Sequence13110.t02599ATGGTGGAAGAAAGAGAAAGCTTCGAGAGTCAGTGGGCGCCCTCCGATGTGACAGAAGATAATCTGAAGGAGATGGTGGCGCACGGCGTTC

4、TTCCAGCGAAGGAGATCATCGGGTGGCGACCGGCATTTGGCGAAGCATTCCCGACTCCCGATACGCATGAAATCGTGGTATTTGCTCACTTCTTCTATGGCGGATTTTCTCTTCCAACCTCAAGATTTTTCCGGGGCATTCTGAACTTCTACGGGATTAGCTTGCATCATCTGAATCCAAACTCCATAGTGCATATTGCCAATTTTATCCACGCCTGCGAAGCTTTTCTGGGCATTAGGCCTCATTTTGCCTTGTTCCGCCGCATCTTCTTCCTCAAGCCCCAGCCAAATAAGAGCAAAC

5、CATGCGTAGTTGGGGGTGCTGGTTTCCAACTCAGGGGAACACTAAGCCAAAAATACTTCTCCATGCCCTTCAAGACCTCAAATAAAGGCTGGCACGCAAACTGGTTCTATGTTCAGAATCCTGAGCCCGCACTCCTGAGCACGCAAACTGTCTTCCTCCGGTTTACCAGGACACATGGAACTCATTGCCTATGGGAGATGAAGCTGCTCAGGCAGTAGAACTGATGGAGAGGATGATAAAGCTGAAAGAACAGGGTCTTCAAGGAGAGCAGATCACCCAGCACTTCATCAAGTGTCGGTT

6、AGCTCCAATCAAAGAAAGATCCCGCACAGCTTTTGAGTTCGACGGCAAGCATGATCCAAATCGGGAAGATCCAGATTCTCTTGACTTTAAGGTCATGAAAGAAAGAATGTACAAGATCTTCTCAAACGCTATTGTCGTCAGCTATTCACATCTGCTGCCCGTTGTGCCATTCAACGCATTCAACCCTCCTCCACCGGTATGCACTCAAACATTTCAACCTTCCTCAAAATATGATCTTGTCTTCCTGTTGAATCAATTAATAATGAAAGATATTACACCCAGGAATTTGCTTTGATGAAA

7、TCGGATCCCCCAATCGCTCAACGCCGATCTCCCCGCCACCAAACTGGGCAAGGGAGCGGAGGACCAAGAATTCGGTCTGAAACTCAACCAAGTGCTTCTGATTCAGTCGGCCAGTCAGACTCTCGAAAGAGGAAGCTGGTTCTGAGTGACGACGAAGCCGATGATACCACCAGGAGGCCTGGAGACCGAGAGACAACCAAGAAACTATCAAAACAGGCTACTCCAAAGAAGAAAACATCCAGTCGCCCTGCGCCAAAGATCAGGAAGTCTTCTAGGTACAACTCATATAACGATCTCTCT

8、TGCATTGATGCACATCCATTGCAGCAATAATACTAATAATCAAATTTGCAGGAAACCATCTGATATAGATCCGTCAGGGAAAGACTCTGACCCGACTAGCGTGGACGCAAATTCTTCCAAAGAAATCGGGACAACTGCCGAGGACCGTCCAAGTGACAATCAGCCGGCAACCGATAATGTAGAATCCGGTGATCAACCCCCGATTGGAAACCAGTCGGCCGAAGCCGAAGCCGGGGTTAATCAGGAGCCCCCGACTAGAAACCAGTCGGACGCAAGGCCAAGCCAAGAGATTCCAGAGGT

9、TGGAGCTCAAGCAGATAGCCTTCGCAGTCAAGACGCCAGCAATAACCAGAGATCTAGATCTCCTTTAAAGGCATCCGAATCCACTCATCCTCATCCAGAAATCATTATGGGTAAAACACTTTGTCTGAAACCATGTCAACCCAAATAACTCTGACCAATTGTCTAACCTCTGTTAGTAGATACATTAGGGCCAATGATCAGTGATGAAGAAGAAATTCCCCGCATACAAGCAGCTGAGGATTCACGCCCTCCGATTCTGGTCAAGTGGTGGGATGACAACATGCAGCCTCAAGGGACCGT

10、GATAAATAAGCAAAAAGAGGACGAAGAGTTATGCCTACTGAAGAAGGCACTTGGTCAGGCAACCCACATTGTAAATGTAAGCCCTCTGGTACCTGATCTCAACCATCTTGTTTGCCATTTAATTCAGGCGCTAATTAAACTGTCTTGCAGAGGATTCATCTTAGGAACGAAGCTAAAACGGAAACCTTAGAGAGGCTAGTCCCTCATCTCGGGACCCTCGAAGCCACCAGGAGTCTGCTGCACGAGACAAAAGAGCTTGCCAGGAAGAATGAGCATGATCTGAGGAATCGAATTGCTGAG

11、CTCCAGGAATCAAATTTTGAACTGAGTGGCTCATCAAAAGGTACGTACAAACCCTACTTAACTGACTCATATTGTTATCTTTGCAATCTTGATTAACCGTAGTCAATGTAGTGCAAACTGCCAAGATATCTCAGTTGGAGAAGCAGATTCAAATTCTGGAAAACGACAAGGCAGAGCTGGCAAGACAAAGAGACCTGGCTTTAAAGGAAGTTGAAGGTATCAACAATCAATATCTCAAAAGCCATTATCCTCCATATGCTGACTTATTAATGTGAATCTGTTGATGCAGACCGCAAGATT

12、AAATCTCAAGCTCAATTCGACGTCCTGGTTAGCAAGATCAAAAAACTTGAAGGAGCCCGGGATGAGGTTGATAACGCTGCTGCCCCAATTGTTCAAGCGATGTTCTTAAATAACAACGGCCCGAGTGCACTTGATGCTTCCGAAATTTTCGACAAGCTGAGAGTTGCTCCGGATATATATTTCAAGAACATCAAAGAAGCTGGAAGTATGGGAGTAAGTATGGCGTTGGCGATGACTAAATCCCTTTATCCGAGAGTTGATATTGACGTCATTGATGGCTTTGCAGACGGGACGAGCGAA

13、GAAGCTGCTCTTGACCTCATCAGTGATGCGCAGAAAGCGGCTGACAAAATTGCTGCTGATGTAGTTGAGAGATTTCAGGACACCGATCTTCGACCGACTGGTCCTGGTAATTCTGATGATGAAAAGACTGAGACTGATTAACDS13110.m02899ATGGTGGAAGAAAGAGAAAGCTTCGAGAGTCAGTGGGCGCCCTCCGATGTGACAGAAGATAATCTGAAGGAGATGGTGGCGCACGGCGTTCTTCCAGCGAAGGAGATCATCGGGTGGCGACCGGCATTTGGCGAAGCATTCCCG

14、ACTCCCGATACGCATGAAATCGTGGACACATGGAACTCATTGCCTATGGGAGATGAAGCTGCTCAGGCAGTAGAACTGATGGAGAGGATGATAAAGCTGAAAGAACAGGGTCTTCAAGGAGAGCAGATCACCCAGCACTTCATCAAGTGTCGGTTAGCTCCAATCAAAGAAAGATCCCGCACAGCTTTTGAGTTCGACGGCAAGCATGATCCAAATCGGGAAGATCCAGATTCTCTTGACTTTAAGGTCATGAAAGAAAGAATGTACAAGATCTTCTCAAACGCTATTGTCGTCAGCTAT

15、TCACATCTGCTGCCCGTTGTGCCATTCAACGCATTCAACCCTCCTCCACCGGAATTTGCTTTGATGAAATCGGATCCCCCAATCGCTCAACGCCGATCTCCCCGCCACCAAACTGGGCAAGGGAGCGGAGGACCAAGAATTCGGTCTGAAACTCAACCAAGTGCTTCTGATTCAGTCGGCCAGTCAGACTCTCGAAAGAGGAAGCTGGTTCTGAGTGACGACGAAGCCGATGATACCACCAGGAGGCCTGGAGACCGAGAGACAACCAAGAAACTATCAAAACAGGCTACTCCAAAGAAG

16、AAAACATCCAGTCGCCCTGCGCCAAAGATCAGGAAGTCTTCTAGGAAACCATCTGATATAGATCCGTCAGGGAAAGACTCTGACCCGACTAGCGTGGACGCAAATTCTTCCAAAGAAATCGGGACAACTGCCGAGGACCGTCCAAGTGACAATCAGCCGGCAACCGATAATGTAGAATCCGGTGATCAACCCCCGATTGGAAACCAGTCGGCCGAAGCCGAAGCCGGGGTTAATCAGGAGCCCCCGACTAGAAACCAGTCGGACGCAAGGCCAAGCCAAGAGATTCCAGAGGTTGGAGCT

17、CAAGCAGATAGCCTTCGCAGTCAAGACGCCAGCAATAACCAGAGATCTAGATCTCCTTTAAAGGCATCCGAATCCACTCATCCTCATCCAGAAATCATTATGGTAGATACATTAGGGCCAATGATCAGTGATGAAGAAGAAATTCCCCGCATACAAGCAGCTGAGGATTCACGCCCTCCGATTCTGGTCAAGTGGTGGGATGACAACATGCAGCCTCAAGGGACCGTGATAAATAAGCAAAAAGAGGACGAAGAGTTATGCCTACTGAAGAAGGCACTTGGTCAGGCAACCCACATTGTA

18、AATAGGATTCATCTTAGGAACGAAGCTAAAACGGAAACCTTAGAGAGGCTAGTCCCTCATCTCGGGACCCTCGAAGCCACCAGGAGTCTGCTGCACGAGACAAAAGAGCTTGCCAGGAAGAATGAGCATGATCTGAGGAATCGAATTGCTGAGCTCCAGGAATCAAATTTTGAACTGAGTGGCTCATCAAAAGTCAATGTAGTGCAAACTGCCAAGATATCTCAGTTGGAGAAGCAGATTCAAATTCTGGAAAACGACAAGGCAGAGCTGGCAAGACAAAGAGACCTGGCTTTAAAGGAA

19、GTTGAAGACCGCAAGATTAAATCTCAAGCTCAATTCGACGTCCTGGTTAGCAAGATCAAAAAACTTGAAGGAGCCCGGGATGAGGTTGATAACGCTGCTGCCCCAATTGTTCAAGCGATGTTCTTAAATAACAACGGCCCGAGTGCACTTGATGCTTCCGAAATTTTCGACAAGCTGAGAGTTGCTCCGGATATATATTTCAAGAACATCAAAGAAGCTGGAAGTATGGGAGTAAGTATGGCGTTGGCGATGACTAAATCCCTTTATCCGAGAGTTGATATTGACGTCATTGATGGCTTT

20、GCAGACGGGACGAGCGAAGAAGCTGCTCTTGACCTCATCAGTGATGCGCAGAAAGCGGCTGACAAAATTGCTGCTGATGTAGTTGAGAGATTTCAGGACACCGATCTTCGACCGACTGGTCCTGGTAATTCTGATGATGAAAAGACTGAGACTGATTAAProtein13110.m02899MVEERESFESQWAPSDVTEDNLKEMVAHGVLPAKEIIGWRPAFGEAFPTPDTHEIVDTWNSLPMGDEAAQAVELMERMIKLKEQGLQGEQITQHFIKCRLAPIKERSRTAFEFDGKHDPNRE

21、DPDSLDFKVMKERMYKIFSNAIVVSYSHLLPVVPFNAFNPPPPEFALMKSDPPIAQRRSPRHQTGQGSGGPRIRSETQPSASDSVGQSDSRKRKLVLSDDEADDTTRRPGDRETTKKLSKQATPKKKTSSRPAPKIRKSSRKPSDIDPSGKDSDPTSVDANSSKEIGTTAEDRPSDNQPATDNVESGDQPPIGNQSAEAEAGVNQEPPTRNQSDARPSQEIPEVGAQADSLRSQDASNNQRSRSPLKASESTHPHPEIIMVDTLGPMISDEEEIPRIQAAEDSRPPILVKWWDDNMQPQG

22、TVINKQKEDEELCLLKKALGQATHIVNRIHLRNEAKTETLERLVPHLGTLEATRSLLHETKELARKNEHDLRNRIAELQESNFELSGSSKVNVVQTAKISQLEKQIQILENDKAELARQRDLALKEVEDRKIKSQAQFDVLVSKIKKLEGARDEVDNAAAPIVQAMFLNNNGPSALDASEIFDKLRVAPDIYFKNIKEAGSMGVSMALAMTKSLYPRVDIDVIDGFADGTSEEAALDLISDAQKAADKIAADVVERFQDTDLRPTGPGNSDDEKTETD*104341 ttgcttgttt gtt

23、tatatgc agttgatctt gcatcattgc tcttactggt aggacgcttt 104401 ctacgacagg ttggtacatc aagcttgaga tgttgagaag ctataatagt aaactcttca 104461 aaatgttgga gctggcacta gcagatgaaa cagtgcaagc gatggactaa tttaatcctt 104521 cttctttttg ccgagtaact tagaataaat ctaaatgtaa ggtgccaatt gcgataagga 104581 aaatatttta tttctatgaa

24、aattttgatt gtttgatcat tttgctattg atgtcatttt 104641 cagaattagg tgggaaaaga tgaactgatt ttgtacagtt cagtgtgctg acaaagctga 104701 agaaaggaga aacaggggat gccaattccc gctgtttggc actgtatata tgctcagtta 104761 aatgtccgat aaacaataag ggtgtgtttg aggagattgg gaagatacgc aaaacgaggt 104821 gagccattag ctcatgatta attgagta

25、tt aactatttta aatttcaaaa atggattaat 104881 atgatttttt aaagcaactt tcttatagaa aatttttaca aaaaacacac cgtttaatag 104941 tttgggaagc gtgcgtgcgg aaaacgaggt gctttctccc tcaatgtcct ccaaacgaac 105001 gctgcctaag tgaagaaaaa gtacataagc aaagaggaat tacactgtgt cgaaacaaga 105061 tttcggcaat aataaaaggg ggtggctatc aagct

26、aggaa agtggatggg tttgaagaca 105121 aggaatttta tacaggttca ggccttctta ttcaagaagt aataccctac tcctgtccgg 105181 ggatgattcc gccgagtgtg ttattgattg tataattggg aaaaaagaat cgtgccccaa 105241 gaggcacacg gaccctcctt atacagggga agggatccgt aatacaaagt acaacccata 105301 tcctaacgga atatgggatt acagataaaa cacaatcgta ac

27、cgactagg atcccgggta 105361 ttttcttgac atacaagttt agaatctaac ccgagtcctc gtaaaaatat tttatctata 105421 tttggtaccg tatatccagc tggaatataa ccgccatgta gatatggggt aaccataatc 105481 tccacagtag cccccgagac cttcacagtt gaccaaaata gtcttctcga aacaaaaaca 105541 ataatccgag tgctttaatt caacttgctc cggcttgccg agtgctaaaa gactgtaaag 105601 ggcgaaaata aaacatggtg catcgagtag atgcacctaa ttaggtgtag cccctgacta 105661 tgtgattagt tgaaaaacta aacatatagt caagaaccga gtggttttat agtgaagcac 105721 gcatggtact tagtaagata