温光敏雄性不育小麦穗部发育时期均一化cDNA文库的构建、鉴定和EST分析以及减数分裂时期细胞骨架荧光标记体系探索
|
摘要
小麦温光敏雄性不育材料为我国特有资源,拥有完全独立的自主知识产权,近十年来发展极为迅速,已经成为国内外小麦杂种优势利用的主要育种途径。本实验以温光敏雄性不育系BS20为材料,进行均一化cDNA文库构建、大规模cDNA测序、EST序列生物信息学分析,以及小麦雄蕊发育过程中微管、微丝细胞骨架激光共聚焦荧光标记体系探索等方面的研究,旨在为更好的揭示温光敏雄性不育分子机理搭建平台。
本实验所获得的主要研究结果如下:
1.温光敏雄性不育小麦穗部发育时期均一化cDNA文库构建、鉴定和EST分析
(1)按照雄蕊不同发育时期,即小花分化期,药隔形成期,小孢子母细胞时期,四分体时期,单核花粉期和成熟花粉期,构建了6个独立的温光敏雄性不育小麦穗部和花药组织cDNA文库。除了小孢子母细胞时期构建的文库之外,其余5个文库的重组率均为100%。每个独立文库的库容量在1.6×10~6-2.0×10~7之间,平均插入片段长度大于0.85 kb。
(2)通过与基因组饱和杂交的方法混合6个独立cDNA文库构建穗部发育时期均一化cDNA文库。文库库容量为4.0×10~5,平均插入片段长度为1.3 kb。
(3)从均一化cDNA文库中随机挑选3264个克隆进行5′单向测序,剔除长度小于100 bp序列之后,共获得了3223条去载体序列的EST序列,其平均测序长度为926bp。通过聚类和拼接,结果产生2175条非冗余EST聚类,其中包括423条contig和1752条singleton,我们将它们合称为温光敏雄性不育小麦穗部发育相关UniGene(TPGMSSUniGene)。
(4)通过综合利用不同序列比对工具的优势,检索多个数据库对TPGMSSUni-Gene进行功能注释。结果显示有60%的UniGene有功能注释。其中有264条(占总数的12%)在CSRD数据库中存在高度同源序列。它们主要是编码初生代谢相关基因、信号转导相关基因和转录调节相关基因。
(5)2715个UniGene进行简单重复序列扫描。结果有108个二至六核苷酸为重复基序的SSR位点被找到,其中三核苷酸重复基序的SSR数量最多。
2.小麦减数分裂时期细胞骨架荧光标记体系的探索
(1)多聚赖氨酸直接粘片法:虽然通过改进固定液配方、针对微管微丝采用不同的固定时间和酶解时间,但还是发现这种方法存在细胞脱落情况严重,组织细胞粘片效果不佳和组织碎片荧光背景干扰等问题,不适合进行小麦雄蕊发育过程中微管、微丝细胞骨架荧光标记。
(2)低熔点石蜡切片法:微管、微丝荧光标记效果较好,无背景杂质荧光信号干扰,克服了细胞脱落的情况,可以观察不同发育时期的小孢子细胞骨架结构。通过优化改进实际操作步骤等方面,最终总结出适合小麦雄蕊发育过程中微管、微丝细胞骨架观察的低熔点石蜡切片荧光标记体系。
Thermo-photoperiod-sensitive genie male sterile(TPGMS) wheat line is the unique resource in China,which has independent intellectual property fights.Through the rapid development over the past decade,the TPGMS has become the main means to potentially use heterosis in agricultural crops.In this study,we used the TPGMS wheat,line BS20,to engage a lot of research,including the construction of normalized cDNA library, large-scale EST sequencing,bioinformatics analysis of this EST sequences and exploring the fluorescent labeling system of eytoskeleton of wheat during the meiotic stages.The goal of this study is to establish a platform for functional genomics studies to reveal the molecular mechanism of TPGMS wheat.
The main results are shown as following:
1.Construction,characterization and EST analysis of normalized cDNA library of TPGMS wheat from spike developmental stages
(1) Six individual cDNA libraries of the spikelet of TPGMS Wheat were constructed in the periods of floret differentiation stage,anther development stage,microspore mother cell stage,tetrad stage,mono-nucleate microspores stage and mature pollen stage.Except for one library constructed with microspore mother cell stage,others owned 100%recombinant ratio.The number of recombinant of individual libraries ranges from 1.6×10~6 to 2.0×10~7.The insert fragment size was large than 0.85 kb.
(2) Based on the strategy of saturation hybridization with genomie DNA,we set up a normalized complementary DNA(cDNA) library.The normalized library consisted of 4.0×10~5 clones and had an average insert length of 1.3 kb.
(3) A total of 3,264 clones randomly selected from normalized spike development cDNA library were sequenced,which generated 3,223 vector-trimmed EST sequences with an average sequencing read length of 926 bp.Clustering and assembly analysis resulted in 2,175 unique ESTs from 423 contigs and 1,752 singletons.This final dataset was denoted as the TPGMS wheat Spike development-related UniGene (TPGMSSUniGene) set.
(4) Taking advantage of various tools and database,gene function classification of the 2,175 unique ESTs showed that 60%of the ESTs were predicted to have putative gene function,264(12%) displayed significant homology to genes previously reported to be involved in cold-response related processes.Among these,sequences encoding activities related to primary metabolism,signal transduction,and transcriptional regulation were observed.
(5) The 2,715 unigenes set was searched for potential simple sequence repeats(SSRs).In total,108 di-to pentanucleotide SSRs that fulfilled the criteria of the search were identified,and tri-nucleotide repeats were the most.
2.Exploring the fluorescent labeling system of cytoskeleton of wheat during the meiotic stages
(1) Poly-lysine gluing section method:we improved the composition of stationary liquid, the time of stationary and enzymolysis,but we observed the tissues lose easily during the process of experiment.The anther wall debris disturbed the backdrop of fluorescence.The method is not suitable for labeling cytoskeleton of wheat during the meiotic stages.
(2) Steedman's wax sectioning method:This method was used successfully to labell both the microfilament and microtubule in this experiment,the backdrop of fluorescence was very clean,and only a few cells lost during the process of experiment.By optimizing the actual steps,the cytoskeleton structure of different developmental stages of microspore was observed.We concluded that this optimized method is more suitable for fluorescent labeling during microspore development than other method.
本实验所获得的主要研究结果如下:
1.温光敏雄性不育小麦穗部发育时期均一化cDNA文库构建、鉴定和EST分析
(1)按照雄蕊不同发育时期,即小花分化期,药隔形成期,小孢子母细胞时期,四分体时期,单核花粉期和成熟花粉期,构建了6个独立的温光敏雄性不育小麦穗部和花药组织cDNA文库。除了小孢子母细胞时期构建的文库之外,其余5个文库的重组率均为100%。每个独立文库的库容量在1.6×10~6-2.0×10~7之间,平均插入片段长度大于0.85 kb。
(2)通过与基因组饱和杂交的方法混合6个独立cDNA文库构建穗部发育时期均一化cDNA文库。文库库容量为4.0×10~5,平均插入片段长度为1.3 kb。
(3)从均一化cDNA文库中随机挑选3264个克隆进行5′单向测序,剔除长度小于100 bp序列之后,共获得了3223条去载体序列的EST序列,其平均测序长度为926bp。通过聚类和拼接,结果产生2175条非冗余EST聚类,其中包括423条contig和1752条singleton,我们将它们合称为温光敏雄性不育小麦穗部发育相关UniGene(TPGMSSUniGene)。
(4)通过综合利用不同序列比对工具的优势,检索多个数据库对TPGMSSUni-Gene进行功能注释。结果显示有60%的UniGene有功能注释。其中有264条(占总数的12%)在CSRD数据库中存在高度同源序列。它们主要是编码初生代谢相关基因、信号转导相关基因和转录调节相关基因。
(5)2715个UniGene进行简单重复序列扫描。结果有108个二至六核苷酸为重复基序的SSR位点被找到,其中三核苷酸重复基序的SSR数量最多。
2.小麦减数分裂时期细胞骨架荧光标记体系的探索
(1)多聚赖氨酸直接粘片法:虽然通过改进固定液配方、针对微管微丝采用不同的固定时间和酶解时间,但还是发现这种方法存在细胞脱落情况严重,组织细胞粘片效果不佳和组织碎片荧光背景干扰等问题,不适合进行小麦雄蕊发育过程中微管、微丝细胞骨架荧光标记。
(2)低熔点石蜡切片法:微管、微丝荧光标记效果较好,无背景杂质荧光信号干扰,克服了细胞脱落的情况,可以观察不同发育时期的小孢子细胞骨架结构。通过优化改进实际操作步骤等方面,最终总结出适合小麦雄蕊发育过程中微管、微丝细胞骨架观察的低熔点石蜡切片荧光标记体系。
Thermo-photoperiod-sensitive genie male sterile(TPGMS) wheat line is the unique resource in China,which has independent intellectual property fights.Through the rapid development over the past decade,the TPGMS has become the main means to potentially use heterosis in agricultural crops.In this study,we used the TPGMS wheat,line BS20,to engage a lot of research,including the construction of normalized cDNA library, large-scale EST sequencing,bioinformatics analysis of this EST sequences and exploring the fluorescent labeling system of eytoskeleton of wheat during the meiotic stages.The goal of this study is to establish a platform for functional genomics studies to reveal the molecular mechanism of TPGMS wheat.
The main results are shown as following:
1.Construction,characterization and EST analysis of normalized cDNA library of TPGMS wheat from spike developmental stages
(1) Six individual cDNA libraries of the spikelet of TPGMS Wheat were constructed in the periods of floret differentiation stage,anther development stage,microspore mother cell stage,tetrad stage,mono-nucleate microspores stage and mature pollen stage.Except for one library constructed with microspore mother cell stage,others owned 100%recombinant ratio.The number of recombinant of individual libraries ranges from 1.6×10~6 to 2.0×10~7.The insert fragment size was large than 0.85 kb.
(2) Based on the strategy of saturation hybridization with genomie DNA,we set up a normalized complementary DNA(cDNA) library.The normalized library consisted of 4.0×10~5 clones and had an average insert length of 1.3 kb.
(3) A total of 3,264 clones randomly selected from normalized spike development cDNA library were sequenced,which generated 3,223 vector-trimmed EST sequences with an average sequencing read length of 926 bp.Clustering and assembly analysis resulted in 2,175 unique ESTs from 423 contigs and 1,752 singletons.This final dataset was denoted as the TPGMS wheat Spike development-related UniGene (TPGMSSUniGene) set.
(4) Taking advantage of various tools and database,gene function classification of the 2,175 unique ESTs showed that 60%of the ESTs were predicted to have putative gene function,264(12%) displayed significant homology to genes previously reported to be involved in cold-response related processes.Among these,sequences encoding activities related to primary metabolism,signal transduction,and transcriptional regulation were observed.
(5) The 2,715 unigenes set was searched for potential simple sequence repeats(SSRs).In total,108 di-to pentanucleotide SSRs that fulfilled the criteria of the search were identified,and tri-nucleotide repeats were the most.
2.Exploring the fluorescent labeling system of cytoskeleton of wheat during the meiotic stages
(1) Poly-lysine gluing section method:we improved the composition of stationary liquid, the time of stationary and enzymolysis,but we observed the tissues lose easily during the process of experiment.The anther wall debris disturbed the backdrop of fluorescence.The method is not suitable for labeling cytoskeleton of wheat during the meiotic stages.
(2) Steedman's wax sectioning method:This method was used successfully to labell both the microfilament and microtubule in this experiment,the backdrop of fluorescence was very clean,and only a few cells lost during the process of experiment.By optimizing the actual steps,the cytoskeleton structure of different developmental stages of microspore was observed.We concluded that this optimized method is more suitable for fluorescent labeling during microspore development than other method.
引文
1.曹双河,郭小丽,刘冬成.小麦光温敏核雄性不育基因的初步定位.遗传学报,2004,31(3):293-298
2.曹双河,张相岐,张爱民.光(温)敏雄性不育的调控机理和分子遗传学研究进展.植物学通报,2005,22:19-26
3.陈其军,安瑞,周海梦.使用与Getaway技术兼容的T载体获得入门克隆.生物化学与生物物理进展,2004,31(10):951-954
4.陈志玲.植物细胞周期中微管骨架与微丝骨架的组装及相互作用。中国农业大学博士学位论文,2000
5.储昭晖,彭开蔓,张利达.水稻全生育期均一化cDNA文库的构建及鉴定.科学通报,2002,47(1):1656-1662.
6.丛斌,许萍,杨茂成,张丕方.小麦根尖细胞分化过程中微丝骨架分布格局.复旦大学学报(自然科学版),1996,35(6):661-664
7.何蓓如,董普辉,宋喜悦.小麦温度敏感不育系A3314温敏特性研究.麦类作物学报,2003,3:1-6
8.何觉民,戴君惕,邹应斌.生态雄遗传性不育理论与两系杂交作物Ⅱ:选育生态遗传雄性不育系的一般方法.湖南农学院学报,1994,20(5):412-418
9.何觉民,盛承师,戴君惕等.两系杂交小麦制种方法.中国专利,1996,ZL91106645
10.何觉民.两系杂交小麦研究:态雄性不育的发现、培育及其利用价值.湖南农业科学,1992,5:1-3
11.何群,尤瑞麟.应用Steedman's wax切片法观察植物细胞微管骨架.植物学通报,2004,21(5):547-555
12.贺新强,崔克明.植物细胞次生壁形成的研究进展.植物学通报,2002,19(5):513-522
13.侯春燕,王冬梅,李小娟,韩胜芳,刘娟.细胞骨架解聚药物对小麦与叶锈菌互作诱发的细胞过敏性反应的影响.植物病理学报,2002,32(2):147-152
14.李仕贵,周开达,朱立煌.水稻显性温敏雄性不育基因的定位和遗传分析.科学通报,1999,44:955-958
15.廖伏明,袁隆平.光温敏不育水稻不育性表达不稳定的遗传机制与原因综述.杂交水稻,2003,18(2):1-6
16.凌治萍,俞彰.细胞超微结构与电镜技术(第2版).复旦大学出版社,上海,2004
17.刘刚,王冬梅.细胞信号转导中Ca~((2+))和微管骨架的关系.植物生理学通讯,2006,42(2):331-336
18.刘继红,蔡学泳,何其华.细胞骨架的激光共焦研究技术.中国医学装备,2005,2(6):49-51
19.刘珠琴,张绍铃,徐国华.微丝骨架的构成及其对花粉管极性生长的调控作用.西北植物学报,2005,25(11):2356-2362
20.龙松华,张宁,邱德文,黎定军,黄炜.Gateway技术构建交链孢菌JH505 cDNA 文库.微生物学报,2005.Vo1.45 No.6.
21.罗红兵,觉民,戴君惕.小麦生态雄性不育系ES-10和ES-8的制种特性研究.湖南农业大学学报,1998,4:88-89
22.马廷臣,师凤华,李卓夫.光温敏型雄性不育小麦研究新进展.黑龙江农业科学,2002,(2):32-3
23.梅文倩,宋文强,潘怡.利用Getaway克隆技术大规模克隆拟南芥转录因子.分子植物2004,2(3):358-364.
24.孟祥红,王建波,利容千.光周期对光敏胞质不育小麦花药发育过程中Ca2+分布的影响.植物学报,2000,42(1):15-22
25.庞启华,黄光永,李生荣.两系杂交小麦绵阳32的选育及配套技术.中国种业,2003,(12):50-51
26.秦志列,张风廷,赵昌平.不同花粉密度条件下光温敏雄性不育小麦BS366异交结实分析.麦类作物学报,2007
27.邱芳,金德敏,伏健民.温敏核不育水稻5460S细菌人工染色体文库的构建和鉴定.中国科学(C),1999,29:518-524
28.荣德福,少华,拥军.两极光温敏感型小麦雄性不育系337S的选育.北京农业科学,2001(5):13-17
29.宋国琦,胡银岗,林凡云,董普辉,何蓓.YS型小麦温敏不育系育性转换基因的cDNA-AFLP分析.西北植物学报,2006(4)
30.孙宗修.杂交水稻育种-三系、两系到一系.中国农业科技出版社,1994
31.谭昌华.重庆温光性核不育小麦的育性研究初报.西南农业学报,1992,5:3-8
32.王丰青,何觉民,周斌,何仪.光温敏雄性不育小麦的研究进展.湛江海洋大 学学报,2004,2(6):81-85
33.王凤茹,张笑归.植物细胞骨架及其在花粉萌发中的作用.河北农业科学,2002,6(2):18-23
34.王昕,种康.植物小G蛋白功能的研究进展.植物学通报,2005,22(1):1-10
35.肖玉梅,陈玉玲,黄荣峰,陈珈,王学臣.拟南芥保卫细胞微丝骨架的解聚可能参与了细胞外钙调素诱导的气孔关闭.中国科学(C辑)生命科学,2004,34(2):129-135
36.徐是雄,李春贵,朱激.洋水仙花粉和花粉原生质体中肌动蛋白微丝的共焦显微镜观察.植物学报,1993a,35(1):12-19
37.徐是雄,李春贵,朱激.洋水仙花粉原生质体中微管骨架的免疫荧光及共焦显微镜观察.植物学报,1993b,35(7):513-518
38.徐是雄,朱激,胡适宜.百合生殖细胞分裂过程中微管分布的显微荧光观察.植物学报,1990,32(11):821-836
39.徐是雄.百合花粉原生质体中肌动蛋白微丝的荧光共焦镜观察.植物学报,1992,34(2):907-911
40.徐霞,任海云.微丝骨架动态参与花粉萌发的研究.植物资源与环境学报,2005,14(3):7-11
41.阎隆飞,石德权.高等植物中的收蛋白.生物化学与生物物理学报,1963,3(4):491-496
42.阎隆飞.花粉中的肌动蛋白和肌球蛋白及其在花粉管伸长中的作用.1985,Vo1.30(12):945-945
43.颜龙安.杂交水稻繁制学.中国农业出版社,1999
44.晏慧君,黄兴奇,程在全.cDNA文库构建策略及其分析研究进展.云南农业大学学报,2006,21(1):1-6.
45.杨存义,何蓓如,孟荣华.小麦光温敏”两系”材料研究和利用现状.麦类作物,1997,17:25-27
46.姚雅琴,李蓓,张英利,牛娜,张改生.细胞骨架与小麦雄性不育关系研究初报.西北农林科技大学学报(自然科学版),2005,33(12):39-42
47.余国东.温光型核不育系小麦C49S的育性转换和适应性分析.中国农学通报,1998,14:6-7
48.袁明,傅缨,王风,黄炳权,徐是雄.蓝猪耳受精过程中中央细胞和初生胚乳 细胞中微丝骨架的组装和细胞核的迁移.中国科学(C辑),2002,32(1):13-24
49.翟中和.细胞生物学.高等教育出版社,2000,318-335
50.张爱民,孙其信.杂交小麦产量优势及产量优势的关系.作物学报,1993,19(1):94-96
51.张霖,牛瑞芳.cDNA文库构建方法的进展.生命的化学,2002,22(6):577-579
52.张妙彬,王小菁.植物细胞的形态建成.西北植物学报,2004,24(1):154-160
53.张明,王岳光,刘广田.小麦品质性状杂种优势及其配合力研究.麦类作物学报,2006,26(3):63-66
54.赵昌平,王新.杂种小麦的研究现状与光温敏二系法.北京农业科学,1999,17(2):3-5
55.赵昌平,张立平,李云伏.小麦光温敏核雄性不育相关基因的DDRT-PCR分析.中国生物化学与分子生物学报,2007,23(1):56-62
56.赵昌平.光温敏两系杂交小麦研究进展.第一届国际杂交小麦研究会大会宣读论文北京,1998
57.赵莎,姚家玲.光敏核不育水稻花粉发育过程中的细胞学研究和PCD检测.华中农业大学学报,2007,26(3):283-288
58.周丽华,黄光文.激光扫描共聚焦显微镜在植物学中的应用.激光生物学报,2005,14(1):76-79
59.周美兰,尧楚,邹应斌,何觉民.光温敏核不育小麦ES-14花粉败育的细胞学研究.作物研究,1996,4:20-23
60.Ajay K G,Ju-Kon K,Thomas G O,Anil P R,Yang D C,Leon V K,et al.Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses.Proc Natl Acad Sci,2002,99(25):15898-15903
61.Alberts B,Bray D,Johnson A,Lewis J,Raft M,Roberts K,Watson J D.Molecular Biology of the Cell(Third Edition).Garland Publishing,Inc,New York and London,1994,787-1034
62.Ali M N,Koichiro T,Shuichi K,Yuki M,Masanori S,Masumi I,et al.EGassembler online bioinformatics service for large-scale processing,clustering and assembling ESTs and genomic DNA fragments.Nucleic Acids Res,2006,34:459-462
63.Ali M N,Koichiro T,Shuichi K,Yuki M,Masanori S,Masumi I,et al.EGassembler online bioinformatics service for large-scale processing,clustering and assembling ESTs and genomic DNA fragments. Nucleic Acids Res, 2006, 34:459-462
64. Ana C, Stefan G, Juan Miguel G G, Javier T, Manuel T, Montserrat R. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics, 2005,21 (18):3674-3676
65. Apweiler R, Attwood T K, Bairoch A, Bateman A, Birney E, Biswas M, et al. The InterProScan database, an integrated documentation resource for protein families, domains and functional sites. Nucleic Acids Res, 2001, 29(1):37-40
66. Baluska F, Parker J S, Barlow P W, Specific patterns of cortical and endoplasmic microtubules associated with cell growth and tissue differentiation in roots of maize (Zea mays L.). Cell Sci, 1992,103:191-200
67. Bonaldo M F, Lennon G, Soares M B. Normalization and subtraction: Two approaches to facilitate gene discorery. Genome Res, 1996, 6:791-806
68. Borisy G G, Olmsted J B, Marcum J M, Allen C. Microtubule assembly in vitro. Fed Proc, 1974, 33(2):167-174
69. Brent E, Phil G. Base-calling of automated sequencer traces using phred II. Error probabilities. Genome Res, 1998, 8:186-194
70. Brown R C, Lemmon B E, Mullinax J B. Immunofluorescent staining of microtubules in plant tissues: improved embedding and sectioning techniques using polyethylene glycol (PEG) and Steedman's wax. Bot Acta, 1989,102:54-61
71. Brown R C, Lemmon B E. Methods in plant immunolight microscopy. Method Cell Biol, 1995,49:85-107
72. Caminci P, Shibata Y, Hayastu N, et al. Normalization and subtraction of Cap-trapper selected cDNAs to prepare full- length cDNA libraries for rapid discovery of new genes. Genome Res, 2000,10:1617-1630
73. Cardie L, Ramsay L, Milbourne D, Macaulay M, Marshall D, Waugh R. Computational and experimental characterization of physically clustered simple sequence repeats in plants. Genetics, 2000,156 (2):847-854
74. Chen R Z, Zhao X, Shao Z, Wei Z, Wang Y Y, Zhu L L, et al. Rice UDP-glucose pyrophosphorylasel is essential for pollen callose deposition and its cosuppression results in a new type of thermosensitive genic male sterility. Plant Cell, 2007, 19:847-861
75. Chinnusamy V, Zhu J H, Zhu J K. Gene regulation during cold acclimation in plants. Physiol Plant, 2006,126(1):52-61
76. Chu Z H, Peng K M, Zhang L D, Zhou B, Wei J, Wang S P. Construction and characterization of a normalized whole-life-cycle cDNA library of rice. Chinese Sci Bull, 2003,48:229-235
77. Condeels J S. The identification of F-actin in the pollen tube and protoplama of amanyllis belladon Exptl. Cell Res, 1974, 88: 435-439
78. Dong N V, Subudhi P K, Luong P N. Molecular mapping of a rice thermosensitive genic male sterile (TGMS) gene by AFLP, RFLP and SSR techniques. Thero Appl Genet, 2000, 100:727-734
79. Evgeni M Z, Rolf A. InterProScan-an integration platform for the signature-recognition methods in InterPro. Bioinformatics, 2001, 17(9):847-848
80. Fowler S, Thomashow M F. Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. Plant Cell, 2002,14(8): 1675-1690
81. Franke W W, Herth W, Vanderwoude W J, Morred J D. Tubular and filamentous structures in pollen tubes: possible involement as guide elements in protoplasmic streaming and vectoral migration of secretory vesicles. Planta, 1972, 105:314-317
82. Funada R, Abe H, Furusawa O, Imaizumi H, Fukuzawa K, Ohtani J. The orienlation and localization of cortical microtubules in differentiating conifer tracheids during cell expansion. Plant cell Physiol, 1997, 38:210-212
83. Guo R X, Sun D F, Tan Z B, et al. Two recessive genes controlling thermopho-toperiod-ensitive male sterility in wheat. Theor Appl Genet, 2006,112:1271-1276
84. Hannah M A, Heyer A G, Hincha D K. A global survey of gene regulation during cold acclimation in arabidopsis thaliana. Plant Genet, 2005, 1(2):e26
85. He Q, You R L, Mwange K. Changes of the microtububle arrays during mitosis in prothallus cells of Dryopteris crassirhizoma. Acta Botanica Sinica, 2003, 45(2): 193-199
86. Huang R F, Wang X C, Lou C H. Cytoskeletal inhibitors suppress the Stomatal opening of Vicia faba L. induced by fusicoccin and IAA. Plant Sic, 2000, 156:65-71
87. Huang R F, Wang X C. Roles of cytoplasmic microtubules inthe regulation of Stomatal movement. Acta Bot Sin, 1997,39:253-58
88. Jia J H, Zhang D S, Li C Y, Wang B. Molecular mapping of the reverse thermosensitive genic male sterile gene (rtms1) in rice. Theor Appl Genet, 2001, 103:607-612
89. Jiang S Y, Cai M, Ramachandran S. Oryza sativa MYOSIN XI B controls pollen development by photoperiod-sensitive protein localizations. Dev Biol, 2007, 304:579-592
90. Joos U, Van Aken J, Kristen U. Microtubule are involved in maintainming the cellulat polarity in pollen tubes of Nicotiana sylvestris. Protoplasma, 1995, 187: 182-191
91. Keiichi M, Kanako K, Etsuo S, Naoto K, Tadasu S I, Yuji K, et al. Tissue expression map of a large number of expressed sequence tags and its application to in silico screening of stress response genes in common wheat. Mol Gen Genomics, 2006, 276:304-312
92. Keith W, Earley, Jeremy R et al. Gateway-compatible vectors for plant functional genomics and proteomics. The Plant Journal, 2006,45:616-629
93. Ko M S H. An "equalized cDNA library" by the reassociation of short double-stranded cDNA. Nucl Acids Res, 1990, 18(19):5705-5711
94. Lazo G R, Chao S, Hummel H, Edwards H, Crossman C C, Lui N, et al. Development of an expressed eequence tag (EST) resource for wheat (Triticum aestivum L.): EST generation, unigene analysis, probe selection and bioinformatics for a 16,000-locus bindelineated map. Genetics, 2004,168:585-593
95. Ledbetter M C, Porter K R. A "microtubule" in plant cell fine structure. Cell Biol, 1963, 19:239-250
96. Li Y F, Zhang F T, Sun H, et al. The Fertility Alteration of Photo-Thermo Sensitive Genic Male Sterile Line BS20 in Wheat (Triticum aestivum L.). Euphytica, 2006, 151:207-213
97. Lu Q, Wang F L, Li X H, et al. Separation of the two-locus inheritance of photoperiod- ensitive genic male sterility in rice. Euphyitca, 2001,119:343-348
98. McKusick, V A. Genomics: structural and functional studies of genomes. Genomics, 1997,45:244-249
99. Mei M H, Di X K, Xu C G. Mapping and genetic analysis of the genes for photoperiod sensitive male sterility in rice using the original mutant Nongken 58S. Crop Sci, 1999,39:711-715
100.Mohamed B, Jean D, Barbara B, Mario H, Fathey S. The CBF gene family in hexaploid wheat and its relationship to the phylogenetic complexity of cereal CBFs. Mol Genet Genomics, 2007, 277:533- 554
101.Morgan J L, Seeds N W. Tubulin constancy during morphological differentiation of mouse neuroblastoma cells. Cell Biol, 1975, 67(1):136-145
102.Murai, K. Factors responsible for levels of male sterility in photoperiod-sensitive cytoplasmic male sterile (PCMS) wheat lines. Euphytica, 2001,117:111-116
103.Nathalie P, Charles P, Lee P, John AC, Marie JM, Janice C, et al. Generation, annotation, analysis and database integration of 16,500 white spruce EST clusters. BMC Genomics, 2005, 6: 140-144
104.Nick P, Heuing A, Ehmann B. Plant chaperonins: a role in microtubule dependent wall formation. Protoplasma, 2000,211:234-244
105.Ohara O, Nagase T, Mitsui G, et al .Characterization of size-fractionated cDNA libraries generated by the in vitro recombination-assisted method. DNA Reseach, 2002, 9:47-57.
106.Ohara O, Temple G. Directional cDNA library construction assisted by the in vitro recombination reaction. Nucleic Acids research, 2001, 29:1-8
107.Okayama H, Berg P. High-Efficiency Cloning of Full-Length cDNA. Mol Cell Biol 2, 1982, 161-170.
108.Orvar BL, Sangwan V, Omann F, Dhlndsa R. Early steps in cold sensing by plant cells: the role of actin cytoskeleton and membrane fluidity. Plant J, 2000, 23:785-794.
109.Poovaiah B W. Calcium messenger system: Role of protein phosphory-lation and inositol bisphospholipids. Physiol Plant, 1987, 69:569-573
110.Ronning C M, Stegalkina S S, Ascenzi R A, Bougri O, Hart A L, Utterbach T R, et al. Comparative analyses of potato expressed sequence tag libraries. Plant Physiol, 2003, 131:19-429
111 .Roosens N H C J, Thu T T, Iskandar HM, Jacobs M. Isolation of the ornithine- δ-aminotransferase cDNA and effect of salt stress on its expression in Arabidopsis thaliana. Plant Physiol, 1998, 117:263-271
112.Sambrook J, Russell D W. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001
113.Sarhan F, Danyluk J. Engineering cold-tolerant crops-throwing the master switch. Trends Plant Sci, 1998, 3(8):289-291
114.Sasaki Y F, Ayusawa D, Oishi M. Construction of a normalized cDNA library by introduction of a semisolid mRNA-DNA hybridization system. Nucleic Acids Research, 1994, 22(6):987-992
115.Satoh N, Nagasawa N, Malcomber S, Sakai H, Jackson D. A trehalose metabolic enzyme controls inflorescence architecture in maize. Nature, 2006, 441(4725):227-230
116.Seki M, Narusaka M, Ishida J, Nanjo T, Fujita M, et al. Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. Plant J, 2002, 31(3):279-292
117.Soares M B, Bonaldo M F, Jelene P, Su L, Lawton L, Efstretiadis A. Construction and characterization of a normalized Cdna library. Proc Natl Acad Sci, 1994, 1:9228-9232
118.Stephen F A, Warren G, Webb M, Eugene W M, David J L. Basic local alignment search tool. Mol Biol, 1990,215:403-410
119.Stockinger E J, Gilmour S J, Thomashow M F. Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc Natl Acad Sci, 1997,94(3): 1035-1040
120.Subudhi P K, Bortatati R P, Virmani S S. Molecular mapping of a thermo sensitive genic male sterility in rice using bulked sergeant analysis. Genome, 1997, 40:188-194
121.Tian H Q, Kuang A, Mudgtave, M E, Russell, S D. Calcium distribution in fertile and sterile anthers of a photoperiod-sensitive genic male-sterile rice. Planta 204, 1998, 3:190-192
122.Viswanathan C, Zhu J K. Molecular genetic analysis of cold-regulated gene transcription. Biol Sci , 2002, 357 (1423):877-886
123.Vitha S, Baluska F, Jasik J, Volkmann D, Barlow P W, 2000. Steedman's wax for F-actin visualization. In: Staiger C J, Baluska E, Volkmann D, Barlow P W eds. Actin: A Dynamic Framework for Multiple Plant Cell Functions. Dordrecht / Boston / London: Kluwer Academic Publishers. 619-636
124.Wang B, Xu W W, Wang J Z, et al. Tagging and mapping the thermo-sensitive genic male- terile gene in rice with molecular marker. Theor Appl Genet, 1995, 91:1111-1114.
125.Wang Y G, Jia J H, Li C Y, et al. Study of rice thermo-sensitive genic male sterile gene tms5. Plant Genomics in China II. 2001
126.Wang Y G, Xing Q H, Deng Q Y, Liang F S, Yuan L P, Weng M L, Wang B. Fine mapping of the rice thermo-sensitive genic male-sterile gene tms5. TheorAppl Genet, 2003,107(5):917-921
127.Weissman S M. Molecular genetic techniques for mapping the human genome. Mol Biol Med, 1987,4:133-143
128.Xin Z G, Browse J. Eskimol mutants of Arabidopsis are constitutively freezing-tolerant. Plant Biol, 1998,95:7799-7804
129.Xing Q H, Ru Z G, Li J, et al. Cloning a second form of adenine phosphoribosyl transferace gene TaAPT2) from wheat and analysis of its association with thermosensitive genic male sterility (TGMS). Plant Sci, 2005,169:37-45
130.Xu S X, Liu X D, Feng J H, Lu Y G. Comparative studies on the changes of microtubule distribution and reorganization during the meiotic stages of development in normal(IR36) and a temperature/ photoperiod sensitive male sterile line (Peiai 64S) of rice. Acta Botanica Sinica, 2001, 43(3): 221-226
131.Xu S X, Liu X D, Zhu H L, Lu Y G. Further studies on microtubule organizational changes during megagametogenesis in rice embryo sac. Acta Botanica Sinica, 2001, 43(9): 910-917
132.Xu S X, Wang L J, Qiu Z P, Ye Y J, Yu X H. Actin visualization in living immature pollen 44(6): of rice using a GFP-mouse talin fusion protein. Acta Botanica Sinica, 2002, 642-648
133.Xu S X, Ye X L, Wang L J, Qiu Z P, Ye Y J. F-actin Visualization in Generative and sperm cells of living pollen of rice using a GFP-mouse talin fusion protein. Acta Botanica Sinica, 2003,45(8): 949-958
134.Xu S X, Ye X L. Changes in the pattern of organization of microtubules during microspore in rice. Acta Botanica Sinica, 1998,40(7): 585-590
135.Yamaguchi Y, Ikeda R, Hirasawa H. Linkage analysis of thermo sensitive genic male sterility gene tms2 in rice. Breeding Science, 1997,4794:371-373
136.Ye X L, Yeung E, Xu S X, Liang C Y. Microtubule structure and male sterility in a gene- ytoplasmic male sterile line of rice Zhen Shan 97A. Acta Botanica Sinica, 2003,45(2): 183-192
137.Ye X L, Yeung E, Zee S Y, Tung S H. Confocal microscopic observations on microtubular cytoskeleton changes during megasporogenesis and megagame- togenesis in Phaius tankervilliae (Aiton) Bl. Acta Botanica Sinica, 1996, 38: 677-685
138.Zhang Q F, Shen B Z, Dai X K, et al. Using bulked extremes and recessive class to map gene for photoperiod-sensitive genic male sterility in rice. Proc Natl Acad Sci, 1994,91:675-8679
139.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 MO17 from multiple tissues and developmental stages. Mol Biol, 2005, 39(2): 177-184
140.Zhang Z X, Zhang F D,Tang W H, et al. Construction and Characterization of Normalized cDNA Library of Maize Inbred MO17 from Multiple Tissues and Developmental Stages. Molecular Biology, 2005, 39(2): 198-206.
141.Zhu J H, Dong C H, Zhu J K. Interplay between cold-responsive gene regulation, metabolism and RNA processing during plant cold acclimation. Curr Opin Plant Biol, 2007,10:290-295
142.Zhu J H, Dong C H, Zhu J K. Interplay between cold-responsive gene regulation, metabolism and RNAprocessing during plant cold acclimation. Curr Opin Plant Biol, 2007,10:290-295
143.Zhulidov P A, Bogdanova E A, Shcheglov A S, et al. Simple cDNA normalization using kamchatka crab duplex-specific nuclease. Nucleic Acids Research, 2004, 32(3), e37
2.曹双河,张相岐,张爱民.光(温)敏雄性不育的调控机理和分子遗传学研究进展.植物学通报,2005,22:19-26
3.陈其军,安瑞,周海梦.使用与Getaway技术兼容的T载体获得入门克隆.生物化学与生物物理进展,2004,31(10):951-954
4.陈志玲.植物细胞周期中微管骨架与微丝骨架的组装及相互作用。中国农业大学博士学位论文,2000
5.储昭晖,彭开蔓,张利达.水稻全生育期均一化cDNA文库的构建及鉴定.科学通报,2002,47(1):1656-1662.
6.丛斌,许萍,杨茂成,张丕方.小麦根尖细胞分化过程中微丝骨架分布格局.复旦大学学报(自然科学版),1996,35(6):661-664
7.何蓓如,董普辉,宋喜悦.小麦温度敏感不育系A3314温敏特性研究.麦类作物学报,2003,3:1-6
8.何觉民,戴君惕,邹应斌.生态雄遗传性不育理论与两系杂交作物Ⅱ:选育生态遗传雄性不育系的一般方法.湖南农学院学报,1994,20(5):412-418
9.何觉民,盛承师,戴君惕等.两系杂交小麦制种方法.中国专利,1996,ZL91106645
10.何觉民.两系杂交小麦研究:态雄性不育的发现、培育及其利用价值.湖南农业科学,1992,5:1-3
11.何群,尤瑞麟.应用Steedman's wax切片法观察植物细胞微管骨架.植物学通报,2004,21(5):547-555
12.贺新强,崔克明.植物细胞次生壁形成的研究进展.植物学通报,2002,19(5):513-522
13.侯春燕,王冬梅,李小娟,韩胜芳,刘娟.细胞骨架解聚药物对小麦与叶锈菌互作诱发的细胞过敏性反应的影响.植物病理学报,2002,32(2):147-152
14.李仕贵,周开达,朱立煌.水稻显性温敏雄性不育基因的定位和遗传分析.科学通报,1999,44:955-958
15.廖伏明,袁隆平.光温敏不育水稻不育性表达不稳定的遗传机制与原因综述.杂交水稻,2003,18(2):1-6
16.凌治萍,俞彰.细胞超微结构与电镜技术(第2版).复旦大学出版社,上海,2004
17.刘刚,王冬梅.细胞信号转导中Ca~((2+))和微管骨架的关系.植物生理学通讯,2006,42(2):331-336
18.刘继红,蔡学泳,何其华.细胞骨架的激光共焦研究技术.中国医学装备,2005,2(6):49-51
19.刘珠琴,张绍铃,徐国华.微丝骨架的构成及其对花粉管极性生长的调控作用.西北植物学报,2005,25(11):2356-2362
20.龙松华,张宁,邱德文,黎定军,黄炜.Gateway技术构建交链孢菌JH505 cDNA 文库.微生物学报,2005.Vo1.45 No.6.
21.罗红兵,觉民,戴君惕.小麦生态雄性不育系ES-10和ES-8的制种特性研究.湖南农业大学学报,1998,4:88-89
22.马廷臣,师凤华,李卓夫.光温敏型雄性不育小麦研究新进展.黑龙江农业科学,2002,(2):32-3
23.梅文倩,宋文强,潘怡.利用Getaway克隆技术大规模克隆拟南芥转录因子.分子植物2004,2(3):358-364.
24.孟祥红,王建波,利容千.光周期对光敏胞质不育小麦花药发育过程中Ca2+分布的影响.植物学报,2000,42(1):15-22
25.庞启华,黄光永,李生荣.两系杂交小麦绵阳32的选育及配套技术.中国种业,2003,(12):50-51
26.秦志列,张风廷,赵昌平.不同花粉密度条件下光温敏雄性不育小麦BS366异交结实分析.麦类作物学报,2007
27.邱芳,金德敏,伏健民.温敏核不育水稻5460S细菌人工染色体文库的构建和鉴定.中国科学(C),1999,29:518-524
28.荣德福,少华,拥军.两极光温敏感型小麦雄性不育系337S的选育.北京农业科学,2001(5):13-17
29.宋国琦,胡银岗,林凡云,董普辉,何蓓.YS型小麦温敏不育系育性转换基因的cDNA-AFLP分析.西北植物学报,2006(4)
30.孙宗修.杂交水稻育种-三系、两系到一系.中国农业科技出版社,1994
31.谭昌华.重庆温光性核不育小麦的育性研究初报.西南农业学报,1992,5:3-8
32.王丰青,何觉民,周斌,何仪.光温敏雄性不育小麦的研究进展.湛江海洋大 学学报,2004,2(6):81-85
33.王凤茹,张笑归.植物细胞骨架及其在花粉萌发中的作用.河北农业科学,2002,6(2):18-23
34.王昕,种康.植物小G蛋白功能的研究进展.植物学通报,2005,22(1):1-10
35.肖玉梅,陈玉玲,黄荣峰,陈珈,王学臣.拟南芥保卫细胞微丝骨架的解聚可能参与了细胞外钙调素诱导的气孔关闭.中国科学(C辑)生命科学,2004,34(2):129-135
36.徐是雄,李春贵,朱激.洋水仙花粉和花粉原生质体中肌动蛋白微丝的共焦显微镜观察.植物学报,1993a,35(1):12-19
37.徐是雄,李春贵,朱激.洋水仙花粉原生质体中微管骨架的免疫荧光及共焦显微镜观察.植物学报,1993b,35(7):513-518
38.徐是雄,朱激,胡适宜.百合生殖细胞分裂过程中微管分布的显微荧光观察.植物学报,1990,32(11):821-836
39.徐是雄.百合花粉原生质体中肌动蛋白微丝的荧光共焦镜观察.植物学报,1992,34(2):907-911
40.徐霞,任海云.微丝骨架动态参与花粉萌发的研究.植物资源与环境学报,2005,14(3):7-11
41.阎隆飞,石德权.高等植物中的收蛋白.生物化学与生物物理学报,1963,3(4):491-496
42.阎隆飞.花粉中的肌动蛋白和肌球蛋白及其在花粉管伸长中的作用.1985,Vo1.30(12):945-945
43.颜龙安.杂交水稻繁制学.中国农业出版社,1999
44.晏慧君,黄兴奇,程在全.cDNA文库构建策略及其分析研究进展.云南农业大学学报,2006,21(1):1-6.
45.杨存义,何蓓如,孟荣华.小麦光温敏”两系”材料研究和利用现状.麦类作物,1997,17:25-27
46.姚雅琴,李蓓,张英利,牛娜,张改生.细胞骨架与小麦雄性不育关系研究初报.西北农林科技大学学报(自然科学版),2005,33(12):39-42
47.余国东.温光型核不育系小麦C49S的育性转换和适应性分析.中国农学通报,1998,14:6-7
48.袁明,傅缨,王风,黄炳权,徐是雄.蓝猪耳受精过程中中央细胞和初生胚乳 细胞中微丝骨架的组装和细胞核的迁移.中国科学(C辑),2002,32(1):13-24
49.翟中和.细胞生物学.高等教育出版社,2000,318-335
50.张爱民,孙其信.杂交小麦产量优势及产量优势的关系.作物学报,1993,19(1):94-96
51.张霖,牛瑞芳.cDNA文库构建方法的进展.生命的化学,2002,22(6):577-579
52.张妙彬,王小菁.植物细胞的形态建成.西北植物学报,2004,24(1):154-160
53.张明,王岳光,刘广田.小麦品质性状杂种优势及其配合力研究.麦类作物学报,2006,26(3):63-66
54.赵昌平,王新.杂种小麦的研究现状与光温敏二系法.北京农业科学,1999,17(2):3-5
55.赵昌平,张立平,李云伏.小麦光温敏核雄性不育相关基因的DDRT-PCR分析.中国生物化学与分子生物学报,2007,23(1):56-62
56.赵昌平.光温敏两系杂交小麦研究进展.第一届国际杂交小麦研究会大会宣读论文北京,1998
57.赵莎,姚家玲.光敏核不育水稻花粉发育过程中的细胞学研究和PCD检测.华中农业大学学报,2007,26(3):283-288
58.周丽华,黄光文.激光扫描共聚焦显微镜在植物学中的应用.激光生物学报,2005,14(1):76-79
59.周美兰,尧楚,邹应斌,何觉民.光温敏核不育小麦ES-14花粉败育的细胞学研究.作物研究,1996,4:20-23
60.Ajay K G,Ju-Kon K,Thomas G O,Anil P R,Yang D C,Leon V K,et al.Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses.Proc Natl Acad Sci,2002,99(25):15898-15903
61.Alberts B,Bray D,Johnson A,Lewis J,Raft M,Roberts K,Watson J D.Molecular Biology of the Cell(Third Edition).Garland Publishing,Inc,New York and London,1994,787-1034
62.Ali M N,Koichiro T,Shuichi K,Yuki M,Masanori S,Masumi I,et al.EGassembler online bioinformatics service for large-scale processing,clustering and assembling ESTs and genomic DNA fragments.Nucleic Acids Res,2006,34:459-462
63.Ali M N,Koichiro T,Shuichi K,Yuki M,Masanori S,Masumi I,et al.EGassembler online bioinformatics service for large-scale processing,clustering and assembling ESTs and genomic DNA fragments. Nucleic Acids Res, 2006, 34:459-462
64. Ana C, Stefan G, Juan Miguel G G, Javier T, Manuel T, Montserrat R. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics, 2005,21 (18):3674-3676
65. Apweiler R, Attwood T K, Bairoch A, Bateman A, Birney E, Biswas M, et al. The InterProScan database, an integrated documentation resource for protein families, domains and functional sites. Nucleic Acids Res, 2001, 29(1):37-40
66. Baluska F, Parker J S, Barlow P W, Specific patterns of cortical and endoplasmic microtubules associated with cell growth and tissue differentiation in roots of maize (Zea mays L.). Cell Sci, 1992,103:191-200
67. Bonaldo M F, Lennon G, Soares M B. Normalization and subtraction: Two approaches to facilitate gene discorery. Genome Res, 1996, 6:791-806
68. Borisy G G, Olmsted J B, Marcum J M, Allen C. Microtubule assembly in vitro. Fed Proc, 1974, 33(2):167-174
69. Brent E, Phil G. Base-calling of automated sequencer traces using phred II. Error probabilities. Genome Res, 1998, 8:186-194
70. Brown R C, Lemmon B E, Mullinax J B. Immunofluorescent staining of microtubules in plant tissues: improved embedding and sectioning techniques using polyethylene glycol (PEG) and Steedman's wax. Bot Acta, 1989,102:54-61
71. Brown R C, Lemmon B E. Methods in plant immunolight microscopy. Method Cell Biol, 1995,49:85-107
72. Caminci P, Shibata Y, Hayastu N, et al. Normalization and subtraction of Cap-trapper selected cDNAs to prepare full- length cDNA libraries for rapid discovery of new genes. Genome Res, 2000,10:1617-1630
73. Cardie L, Ramsay L, Milbourne D, Macaulay M, Marshall D, Waugh R. Computational and experimental characterization of physically clustered simple sequence repeats in plants. Genetics, 2000,156 (2):847-854
74. Chen R Z, Zhao X, Shao Z, Wei Z, Wang Y Y, Zhu L L, et al. Rice UDP-glucose pyrophosphorylasel is essential for pollen callose deposition and its cosuppression results in a new type of thermosensitive genic male sterility. Plant Cell, 2007, 19:847-861
75. Chinnusamy V, Zhu J H, Zhu J K. Gene regulation during cold acclimation in plants. Physiol Plant, 2006,126(1):52-61
76. Chu Z H, Peng K M, Zhang L D, Zhou B, Wei J, Wang S P. Construction and characterization of a normalized whole-life-cycle cDNA library of rice. Chinese Sci Bull, 2003,48:229-235
77. Condeels J S. The identification of F-actin in the pollen tube and protoplama of amanyllis belladon Exptl. Cell Res, 1974, 88: 435-439
78. Dong N V, Subudhi P K, Luong P N. Molecular mapping of a rice thermosensitive genic male sterile (TGMS) gene by AFLP, RFLP and SSR techniques. Thero Appl Genet, 2000, 100:727-734
79. Evgeni M Z, Rolf A. InterProScan-an integration platform for the signature-recognition methods in InterPro. Bioinformatics, 2001, 17(9):847-848
80. Fowler S, Thomashow M F. Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. Plant Cell, 2002,14(8): 1675-1690
81. Franke W W, Herth W, Vanderwoude W J, Morred J D. Tubular and filamentous structures in pollen tubes: possible involement as guide elements in protoplasmic streaming and vectoral migration of secretory vesicles. Planta, 1972, 105:314-317
82. Funada R, Abe H, Furusawa O, Imaizumi H, Fukuzawa K, Ohtani J. The orienlation and localization of cortical microtubules in differentiating conifer tracheids during cell expansion. Plant cell Physiol, 1997, 38:210-212
83. Guo R X, Sun D F, Tan Z B, et al. Two recessive genes controlling thermopho-toperiod-ensitive male sterility in wheat. Theor Appl Genet, 2006,112:1271-1276
84. Hannah M A, Heyer A G, Hincha D K. A global survey of gene regulation during cold acclimation in arabidopsis thaliana. Plant Genet, 2005, 1(2):e26
85. He Q, You R L, Mwange K. Changes of the microtububle arrays during mitosis in prothallus cells of Dryopteris crassirhizoma. Acta Botanica Sinica, 2003, 45(2): 193-199
86. Huang R F, Wang X C, Lou C H. Cytoskeletal inhibitors suppress the Stomatal opening of Vicia faba L. induced by fusicoccin and IAA. Plant Sic, 2000, 156:65-71
87. Huang R F, Wang X C. Roles of cytoplasmic microtubules inthe regulation of Stomatal movement. Acta Bot Sin, 1997,39:253-58
88. Jia J H, Zhang D S, Li C Y, Wang B. Molecular mapping of the reverse thermosensitive genic male sterile gene (rtms1) in rice. Theor Appl Genet, 2001, 103:607-612
89. Jiang S Y, Cai M, Ramachandran S. Oryza sativa MYOSIN XI B controls pollen development by photoperiod-sensitive protein localizations. Dev Biol, 2007, 304:579-592
90. Joos U, Van Aken J, Kristen U. Microtubule are involved in maintainming the cellulat polarity in pollen tubes of Nicotiana sylvestris. Protoplasma, 1995, 187: 182-191
91. Keiichi M, Kanako K, Etsuo S, Naoto K, Tadasu S I, Yuji K, et al. Tissue expression map of a large number of expressed sequence tags and its application to in silico screening of stress response genes in common wheat. Mol Gen Genomics, 2006, 276:304-312
92. Keith W, Earley, Jeremy R et al. Gateway-compatible vectors for plant functional genomics and proteomics. The Plant Journal, 2006,45:616-629
93. Ko M S H. An "equalized cDNA library" by the reassociation of short double-stranded cDNA. Nucl Acids Res, 1990, 18(19):5705-5711
94. Lazo G R, Chao S, Hummel H, Edwards H, Crossman C C, Lui N, et al. Development of an expressed eequence tag (EST) resource for wheat (Triticum aestivum L.): EST generation, unigene analysis, probe selection and bioinformatics for a 16,000-locus bindelineated map. Genetics, 2004,168:585-593
95. Ledbetter M C, Porter K R. A "microtubule" in plant cell fine structure. Cell Biol, 1963, 19:239-250
96. Li Y F, Zhang F T, Sun H, et al. The Fertility Alteration of Photo-Thermo Sensitive Genic Male Sterile Line BS20 in Wheat (Triticum aestivum L.). Euphytica, 2006, 151:207-213
97. Lu Q, Wang F L, Li X H, et al. Separation of the two-locus inheritance of photoperiod- ensitive genic male sterility in rice. Euphyitca, 2001,119:343-348
98. McKusick, V A. Genomics: structural and functional studies of genomes. Genomics, 1997,45:244-249
99. Mei M H, Di X K, Xu C G. Mapping and genetic analysis of the genes for photoperiod sensitive male sterility in rice using the original mutant Nongken 58S. Crop Sci, 1999,39:711-715
100.Mohamed B, Jean D, Barbara B, Mario H, Fathey S. The CBF gene family in hexaploid wheat and its relationship to the phylogenetic complexity of cereal CBFs. Mol Genet Genomics, 2007, 277:533- 554
101.Morgan J L, Seeds N W. Tubulin constancy during morphological differentiation of mouse neuroblastoma cells. Cell Biol, 1975, 67(1):136-145
102.Murai, K. Factors responsible for levels of male sterility in photoperiod-sensitive cytoplasmic male sterile (PCMS) wheat lines. Euphytica, 2001,117:111-116
103.Nathalie P, Charles P, Lee P, John AC, Marie JM, Janice C, et al. Generation, annotation, analysis and database integration of 16,500 white spruce EST clusters. BMC Genomics, 2005, 6: 140-144
104.Nick P, Heuing A, Ehmann B. Plant chaperonins: a role in microtubule dependent wall formation. Protoplasma, 2000,211:234-244
105.Ohara O, Nagase T, Mitsui G, et al .Characterization of size-fractionated cDNA libraries generated by the in vitro recombination-assisted method. DNA Reseach, 2002, 9:47-57.
106.Ohara O, Temple G. Directional cDNA library construction assisted by the in vitro recombination reaction. Nucleic Acids research, 2001, 29:1-8
107.Okayama H, Berg P. High-Efficiency Cloning of Full-Length cDNA. Mol Cell Biol 2, 1982, 161-170.
108.Orvar BL, Sangwan V, Omann F, Dhlndsa R. Early steps in cold sensing by plant cells: the role of actin cytoskeleton and membrane fluidity. Plant J, 2000, 23:785-794.
109.Poovaiah B W. Calcium messenger system: Role of protein phosphory-lation and inositol bisphospholipids. Physiol Plant, 1987, 69:569-573
110.Ronning C M, Stegalkina S S, Ascenzi R A, Bougri O, Hart A L, Utterbach T R, et al. Comparative analyses of potato expressed sequence tag libraries. Plant Physiol, 2003, 131:19-429
111 .Roosens N H C J, Thu T T, Iskandar HM, Jacobs M. Isolation of the ornithine- δ-aminotransferase cDNA and effect of salt stress on its expression in Arabidopsis thaliana. Plant Physiol, 1998, 117:263-271
112.Sambrook J, Russell D W. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001
113.Sarhan F, Danyluk J. Engineering cold-tolerant crops-throwing the master switch. Trends Plant Sci, 1998, 3(8):289-291
114.Sasaki Y F, Ayusawa D, Oishi M. Construction of a normalized cDNA library by introduction of a semisolid mRNA-DNA hybridization system. Nucleic Acids Research, 1994, 22(6):987-992
115.Satoh N, Nagasawa N, Malcomber S, Sakai H, Jackson D. A trehalose metabolic enzyme controls inflorescence architecture in maize. Nature, 2006, 441(4725):227-230
116.Seki M, Narusaka M, Ishida J, Nanjo T, Fujita M, et al. Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. Plant J, 2002, 31(3):279-292
117.Soares M B, Bonaldo M F, Jelene P, Su L, Lawton L, Efstretiadis A. Construction and characterization of a normalized Cdna library. Proc Natl Acad Sci, 1994, 1:9228-9232
118.Stephen F A, Warren G, Webb M, Eugene W M, David J L. Basic local alignment search tool. Mol Biol, 1990,215:403-410
119.Stockinger E J, Gilmour S J, Thomashow M F. Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc Natl Acad Sci, 1997,94(3): 1035-1040
120.Subudhi P K, Bortatati R P, Virmani S S. Molecular mapping of a thermo sensitive genic male sterility in rice using bulked sergeant analysis. Genome, 1997, 40:188-194
121.Tian H Q, Kuang A, Mudgtave, M E, Russell, S D. Calcium distribution in fertile and sterile anthers of a photoperiod-sensitive genic male-sterile rice. Planta 204, 1998, 3:190-192
122.Viswanathan C, Zhu J K. Molecular genetic analysis of cold-regulated gene transcription. Biol Sci , 2002, 357 (1423):877-886
123.Vitha S, Baluska F, Jasik J, Volkmann D, Barlow P W, 2000. Steedman's wax for F-actin visualization. In: Staiger C J, Baluska E, Volkmann D, Barlow P W eds. Actin: A Dynamic Framework for Multiple Plant Cell Functions. Dordrecht / Boston / London: Kluwer Academic Publishers. 619-636
124.Wang B, Xu W W, Wang J Z, et al. Tagging and mapping the thermo-sensitive genic male- terile gene in rice with molecular marker. Theor Appl Genet, 1995, 91:1111-1114.
125.Wang Y G, Jia J H, Li C Y, et al. Study of rice thermo-sensitive genic male sterile gene tms5. Plant Genomics in China II. 2001
126.Wang Y G, Xing Q H, Deng Q Y, Liang F S, Yuan L P, Weng M L, Wang B. Fine mapping of the rice thermo-sensitive genic male-sterile gene tms5. TheorAppl Genet, 2003,107(5):917-921
127.Weissman S M. Molecular genetic techniques for mapping the human genome. Mol Biol Med, 1987,4:133-143
128.Xin Z G, Browse J. Eskimol mutants of Arabidopsis are constitutively freezing-tolerant. Plant Biol, 1998,95:7799-7804
129.Xing Q H, Ru Z G, Li J, et al. Cloning a second form of adenine phosphoribosyl transferace gene TaAPT2) from wheat and analysis of its association with thermosensitive genic male sterility (TGMS). Plant Sci, 2005,169:37-45
130.Xu S X, Liu X D, Feng J H, Lu Y G. Comparative studies on the changes of microtubule distribution and reorganization during the meiotic stages of development in normal(IR36) and a temperature/ photoperiod sensitive male sterile line (Peiai 64S) of rice. Acta Botanica Sinica, 2001, 43(3): 221-226
131.Xu S X, Liu X D, Zhu H L, Lu Y G. Further studies on microtubule organizational changes during megagametogenesis in rice embryo sac. Acta Botanica Sinica, 2001, 43(9): 910-917
132.Xu S X, Wang L J, Qiu Z P, Ye Y J, Yu X H. Actin visualization in living immature pollen 44(6): of rice using a GFP-mouse talin fusion protein. Acta Botanica Sinica, 2002, 642-648
133.Xu S X, Ye X L, Wang L J, Qiu Z P, Ye Y J. F-actin Visualization in Generative and sperm cells of living pollen of rice using a GFP-mouse talin fusion protein. Acta Botanica Sinica, 2003,45(8): 949-958
134.Xu S X, Ye X L. Changes in the pattern of organization of microtubules during microspore in rice. Acta Botanica Sinica, 1998,40(7): 585-590
135.Yamaguchi Y, Ikeda R, Hirasawa H. Linkage analysis of thermo sensitive genic male sterility gene tms2 in rice. Breeding Science, 1997,4794:371-373
136.Ye X L, Yeung E, Xu S X, Liang C Y. Microtubule structure and male sterility in a gene- ytoplasmic male sterile line of rice Zhen Shan 97A. Acta Botanica Sinica, 2003,45(2): 183-192
137.Ye X L, Yeung E, Zee S Y, Tung S H. Confocal microscopic observations on microtubular cytoskeleton changes during megasporogenesis and megagame- togenesis in Phaius tankervilliae (Aiton) Bl. Acta Botanica Sinica, 1996, 38: 677-685
138.Zhang Q F, Shen B Z, Dai X K, et al. Using bulked extremes and recessive class to map gene for photoperiod-sensitive genic male sterility in rice. Proc Natl Acad Sci, 1994,91:675-8679
139.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 MO17 from multiple tissues and developmental stages. Mol Biol, 2005, 39(2): 177-184
140.Zhang Z X, Zhang F D,Tang W H, et al. Construction and Characterization of Normalized cDNA Library of Maize Inbred MO17 from Multiple Tissues and Developmental Stages. Molecular Biology, 2005, 39(2): 198-206.
141.Zhu J H, Dong C H, Zhu J K. Interplay between cold-responsive gene regulation, metabolism and RNA processing during plant cold acclimation. Curr Opin Plant Biol, 2007,10:290-295
142.Zhu J H, Dong C H, Zhu J K. Interplay between cold-responsive gene regulation, metabolism and RNAprocessing during plant cold acclimation. Curr Opin Plant Biol, 2007,10:290-295
143.Zhulidov P A, Bogdanova E A, Shcheglov A S, et al. Simple cDNA normalization using kamchatka crab duplex-specific nuclease. Nucleic Acids Research, 2004, 32(3), e37