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作物学报 ›› 2009, Vol. 35 ›› Issue (4): 602-607.doi: 10.3724/SP.J.1006.2009.00602

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

中棉所36均一化全长cDNA文库的构建与鉴定

吴东，刘俊杰，喻树迅，范术丽，宋美珍   

  1. 中国农业科学院棉花研究所/农业部棉花遗传改良重点实验室，河南安阳455000
  • 收稿日期:2008-09-01 修回日期:2008-12-13 出版日期:2009-04-12 网络出版日期:2009-01-16
  • 通讯作者: 喻树迅 E-mail:yu@cricaas.com.cn Tel:0372-2562201
  • 基金资助:

    本研究由国家高技术研究发展计划（863计划）项目（2006AA10A109）资助。

Establishment and Identification of a Normalized Full-length cDNA Library of CCRI36

WU Dong,LIU Jun-Jie**,YU Shu-Xun*,FAN Shu-Li,SONG Mei-Zhen   

  1. Cotton Research Institute of Chinese Academy of Agricultural Sciences/Key Laboratory of Cotton Genetic Improvement, Ministry of Agriculture,Anyang 455000,China
  • Received:2008-09-01 Revised:2008-12-13 Published:2009-04-12 Published online:2009-01-16
  • Contact: YU Shu-Xun E-mail:yu@cricaas.com.cn Tel:0372-2562201

PDF

1999

被引次数

29

摘要：

以短季棉中棉所36(CCRI36)顶端分生组织和花蕾为材料, 以DSN(duplex-specific nuclease)均一化技术与SMART(switching mechanism at 5¢ end of RNA transcript)建库技术相结合, 构建CCRI 36花发育期均一化全长cDNA文库。经检测原始文库滴度为1.7×106 cfu mL-1。随机挑取100个克隆, 利用PCR方法测得文库重组率达100%, 插入片段平均长度为1.2 kb。以两个高丰度表达基因Histon3和UBQ7为探针, 进行虚拟Northern blot检测显示, 其丰度在均一化cDNA中均明显降低, 说明均一化效果显著, 为节约筛库成本和EST有效测序奠定了基础；同时, 利用常规PCR扩增技术从cDNA文库中筛选阳性信号, 获得了花发育相关蛋白基因。初步证实CCRI36花发育期均一化全长cDNA文库构建成功, 为深入研究棉花花发育机理及发掘与早熟相关的功能基因奠定了基础。

关键词: 花发育, 均一化, cDNA文库

Abstract:

Short season cotton (SSC) breeding plays an important role in solving the competition for more growing area between cotton and food crops. To explore the premature mechanism of SSC, to clone genes that have a close relation with premature flowering, and to speed process of premature cultivar breeding, we established a normalized full-length cDNA library using the flower and bud of CCRI 36 by DSN (duplex-specific nuclease)-normalization method combined with SMART (switching mechanism at 5' end of RNA transcript) technique. The titer of un-amplified cDNA library was about 1.7×106 cfu mL-1. The average size of cDNA inserts was 1 200 bp with a recombination rate of 100%. The abundance of transcripts Histon3 and UBQ7 decreased obviously in normalized cDNA library comparing with that in non-normalized samples detected by Virtual Northern Blot. Meanwhile, protein genes associated with flower development were obtained on the basis of the positive signal of cDNA library by PCR. These results indicated that the normalized full-length cDNA library has been established successfully, which is convenient for further study on the molecular mechanism and gene cloning of flower development.

Key words: Flower development, Normalization, cDNA Library

[1]Yu S-X(喻树迅), Song M-Z(宋美珍), Fan S-L(范术丽), Yuan R-H(原日红). Studies on biochemical assistant breeding technology of earliness without premature senescence of the shot-season upland cotton. Sci Agric Sin (中国农业科学), 2005, 38(4): 664-670

[2] Berier G. Structural and metobolic changes in the shoot apex in transition to flowering. Canadian J Bot, 1997, 49: 803-819

[3] Zeevaart J A D. DAN multiplication as a requirement for expression of floral stimulus in pharbitis nil. Plant Physiol, 1962, 37: 296-304

[4] Buban T, Hesemana C U. Cytochemical in investigations of shoot of apple trees: I. DNA and RNA, histon content of meristematic cell nuclei in terminal bud of spur with and without fruits. Acta Bot Hung, 1997, 25: 53-63

[5] Ceban A I. The content and accumulation of mucleic acids in the over wintering eyes of the vine during different stages of seasonal development. Fiziologiya Rastenii, 1986, 15: 329-335(in Russian)

[6] Li L(李丽), Zhang Y-R(张艳茹), Chang L-M(常立民), Song J-Y(宋金跃). The changes of nucleic acid content during floral bud differentiation in Guoguang apple. Northern Hort (北方园艺), 1998, (z1): 53-54(in Chinese)

[7] Liljegren S J, Gustafson-Brown C, Pinyopich A, Ditta G S, Yanofsky M F. Interactions among APEATALA1, LEAFY and TERMINAL FLOWER 1 specify meristem fate. Plant Cell, 1999, 11: 1007-1018

[8] Kotake T, Takada S, Nakahigashi K, Ohto M, Goto K. Atabidopsis TERMINAL FLOWER 2 gene encodes a heterochromatin protein 1 homolog and represses both FLOWERING LOCUS to regulate flowering time and several floral homeotic genes. Plant Cell Physiol, 2003, 44: 555-564

[9] Bradley D, Vincent C, Carpenter R, Coen E. Pathways for inflorescence and floral induction in Antirrhinum. Development, 1996, 122: 1535-1544
[10] Kelly A J, Bonnlander M B, Meeks-Wanger D R. NFL, the tobacco homolog of FLORICAULA and LEAFY, is transcriptionally expressed in both vegetative and floral meristems. Plant Cell, 1995, 7: 225-234
[11] Schwarz-Sommer Z, Huijser P, Nacken W, Saedler H, Sommer H. Genetic control of flower development by homeotic genes in Antirrhinum majus. Science, 1990, 250, 931-936
[12] Weige D, Meyerowitz E M. The ABCs of floral homeotic genes. Cell, 1994, 78: 203-209
[13] Wiemann S, Mehrle A, Bechtel S, Wellenreuther R, Pepperkok R, Poustka A. cDNAs for functional genomics and proteomics: The German consortium. Comptes Rendus Biol, 2003, 326: 1003-1009

[14] Pear J R, Kawagoe Y, Schreckengost W E, Delmer D P, Stalker D M. Higher plants contain homologs of the bacterial celA genes

encoding the catalytic subunit of cellulose synthase. Proc Natl Acad Sci USA, 1996, 93: 12637-12642

[15] Li X B, Cai L, Cheng N H, Liu J W. Molecular characterization of the cotton GhTUB1 gene that is preferentially expressed infiber. Plant Physiol, 2002, 130: 666-674

[16] Haigler C H, Zhang D, Wilkerson C G. Biotechnological improvement of cotton fibre maturity. Physiol Plant, 2005, 124: 285-294

[17] Udall J, Swanson J, Haller K, Rapp R, Sparks M, Hatfield J, Yu Y, Wu Y, Dowd C, Arpat A, Sickler B, Wilkins T, Guo J, Chen X, Scheffler J, Taliercio E, Turley R, McFadden H, Payton P, Klueva N, Allen R, Zhang D, Haigler C, Wilkerson C, Suo J, Schulze S, Pierce M, Essenberg M, Kim H, Llewellyn D, Dennis E, Kudrna D, Wing R, Paterson A, Soderlund C, Wendel J. A global assembly of cotton ESTs. Genome Res, 2006, 16: 441-450

[18] Ji S J, Lu Y C, Feng J X, Wei G, Li J, Shi Y H, Fu Q, Liu D, Luo J C, Zhu Y X. Isolation and analyses of genes preferentially expressed during early cotton fiber development by subtractive PCR and cDNA array. Nucl Acids Res, 2003, 31: 2534-2543

[19] Zhu Y Y, Machleder E M, Chenchik A, Li R, Siebert P D. Reverse transcriptase template switching, a SMART approach for full-length cDNA library construction. Biotechniques, 2001, 30: 892-897

[20] Zhang Z X, Zhang F D, Tang W H, Pi Y J, Zheng Y L. Construction and characterization of normalized cDNA Library of maize inbred M017 from multiple tissues and developmental stages. Mol Biol, 2005, 39: 198-206

[21] Shagin D A, Rebrikov D V, Kozhemyako V B, Altshuler I M, Shcheglov A S, Zhulidov P A, Bogdanova E A, Staroverov D B, Rasskazov V A, Lukyanov S. A novel method for SNP detection using a new duplex-specific nuclease from crab hepatopancreas. Genome Res, 2002, 12: 1935-1942

[22] Zhulidov P A, Bogdanova E A, Shcheglov A S, Vagner L L, Khaspekov G L, Kozhemyako V B, Matz M V, Meleshke-Vitch E, Moroz L L, Lukyanov S A. Simple cDNA normalization using kamchatka crab duplex-specific nuclease. Nucl Acids Res, 2004, 32: e37

[23] Lu S-D(卢圣栋). Current Protocols for Molecular Biology (现代分子生物学实验技术), 2nd edn. Beijing: Higher Education Press, 1999, pp 338-339(in Chinese)
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