温敏型大白菜雄性不育系育性转化机理的研究
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摘要
大白菜温敏雄性不育系在杂种优势利用中占有十分重要的地位,但温度调控育性转化的机理尚不清楚。本研究采用cDNA-AFLP技术和蛋白质双向凝胶电泳技术从转录和表达两个水平对大白菜温度敏感雄性不育系进行了温度调控育性转化机理的探究,分析了来自同一遗传背景,在不育和可育条件下幼蕾的基因转录(幼蕾≤1.0㎜,包括顶端分生组织)和表达(约1.5㎜幼蕾)的差异。探究了大白菜温敏雄性不育系幼蕾发育过程中,温度调控育性转化的机理。获得的主要研究结果如下:
     1.建立了一套适合大白菜转录水平差异分析的cDNA-AFLP技术。该方法是将提取的大白菜幼蕾RNA,经cDNA一链合成试剂盒反转录合成cDNA一链,再采用DNA polymeraseⅠ和RNase H以适当的比例和反应时间合成cDNA二链,将合成的cDNA二链作为模板进行AFLP分析。利用该技术合成的cDNA二链质量较高,差异显示分析表明该技术具有稳定性好、假阳性低、所需RNA少等优点。
     2.利用建立的cDNA-AFLP技术,分析了温敏型大白菜细胞质雄性不育系TsCMS7311低温处理期间及处理后幼蕾(≤1.0㎜,包括顶端分生组织)中的基因转录水平的差异。用256对引物扩增出连续性差异片段212条,其表现归为2类4种差异带型,其中以诱导带型产生为主,有175条差异带,表现为“无→有→无”和“无→有”2种带型;诱导沉默带型37条,包括“有→无→有”和“有→无”2种带型。
     3.对差异片段进行回收、克隆、测序,共得到100条EST序列,其大小为100-700bp。经BLASTx序列比对分析发现, 34条EST序列与已知蛋白相似性大于62%,表明温度调控育性的转化与新陈代谢、能量、防御、细胞构建、蛋白质合成及运输、转录翻译、细胞通信及信号转导等相关蛋白有关;有49条EST序列与已知表达序列标签有很高相似性,一致性均达到了90%以上,且47条同源EST来源于芸薹属; 17条EST序列在两个库中没有找到与之同源的序列,或相似性很低,无法进行分析,推测可能是一些新的基因片段,并将其中的3条长于150bp的EST登录GeneBank,获得的登录号分别为: EST80:登录号FE905220, dbEST_Id 54764252; EST182:登录号FE905221, dbEST_Id 54764253; EST162:登录号FE905222, dbEST_Id 54764254。
     4.对EST-58, EST-114和EST-188三个ESTs进行了电子克隆,并通过RT-PCR验证,获得了2个大白菜新基因——大白菜木葡聚糖内糖基转移酶EXT基因(登录号:EU579461)和大白菜酪氨酸磷酸酯酶样蛋白PAS基因(登录号:EU650783),一个伸长的EST188,在GeneBank登录(登录号为:FE905223;dbEST_Id:54764255)。对EST-58的电子延伸得到了长1196bp的新生序列,RT-PCR验证结果表明与电子克隆的结果一致性达98%,该序列具有完整的阅读框架,编码292 AA,同源比对分析得知该序列是新的大白菜木葡聚糖内糖基转移酶EXT基因。对EST-114的电子延伸得到了长1036bp的新生序列,RT-PCR验证结果表明与电子克隆的结果一致性达98%,该序列具有完整的阅读框架,编码229 AA,同源比对分析得知该序列是新的大白菜酪氨酸磷酸酯酶样蛋白PAS基因。对EST-188的电子延伸得到了长877bp的新生序列,RT-PCR验证结果表明与电子克隆的结果一致性达96%,但该序列阅读框架不完整,作为EST递交了GeneBank。
     5.建立了一套有效的大白菜特异β-酯酶的回收、纯化方法。即采用制备型电泳与切胶回收相结合的技术对目的β-酯酶进行富集,自制双向电泳系统进一步分离纯化。制备型凝胶两边染色,切割回收中间未染色的目的条带区。通过电洗脱及冷冻干燥的方法得到目的酯酶的粗干粉,然后再经过自制双向凝胶电泳系统进一步分离纯化。该系统双向凝胶电泳的第一向是等电聚焦电泳,采用国产竖直板电泳槽进行,既经济又高效。第二向是SDS电泳,即等电聚焦电泳完毕进行考马斯亮兰蛋白染色,切下目的条带,直接进行SDS电泳再分离,减少了非目的条带对分析的干扰,使分析结果更可靠。
     6.获得了特异β-酯酶多肽的部分氨基酸序列。通过双向凝胶电泳的分离纯化,共获得了与育性转化相关的6种多肽,对其中分子量为57.1KD和62KD的两个多肽进行Q-TOF质谱测序,分别得到了3段短肽序列和2段短肽序列。并且分子量为62KD的目的条带测出的2段短肽序列与分子量为57.1KD中的两段短肽序列完全相同,预示着两条肽链氨基酸序列相似性很强。
     7.将获得的3段短肽序列同源比对,分析发现它们与p27SJ蛋白有很高的序列一致性(60-100%)。
The thermo-sensitive male sterile of Chinese cabbage (Brassica campestris L. ssp. Pekinensis) play an extremely important role in heterosis utilization, but the mechanism of temperature regulating fertility is still not clear. The technologies of cDNA-AFLP and the two-dimensional GEL electrophoresis of protein were used to research the mechanism of temperature regulating fertility transform at two levels of transcription and expression respectively. The differences of gene transcript in little buds (little bud≤1.0㎜, including apical meristem) and gene express in little buds (little bud≤1.5㎜) were continuously analyzed. The main results are as follows:
     1. A cDNA-AFLP (cDNA Amplified Fragment Length Polymorphism) technology was established, which was suitable for analyzing the gene differentially-expression of Chinese cabbage. The RNA extracted from small buds (≤1.0㎜, including apical meristem ) was used to synthesize 1st Strand cDNA with PrimeScriptTM 1st Strand cDNA Synthesis Kit. The 2st Strand cDNA was synthesized with the suitable proportion of DNA polymeraseⅠand RNase H and the appropriate reaction time, and then to carry on the AFLP analysis. In a word, the technology had lots merits. Such as stable zymograms, high quality 2st Strand cDNA, low false positive, and few RNA template.
     2. The plants of TsCMS7311 with flower buds were treated under low temperature first, then the RNA was extracted from small buds (≤1.0㎜, including apical meristem ) of them in 10 continues days , and were analyzed with cDNA-AFLP. The results showed that 212 differentially-expressed fragments were obtained with 256 primer pairs, and which was divided into two species and four zymogram types as the day going. In which, one mainly species was inducting produce zymogram types, including type“absent→present→absent”and type“absent→present”, totalled 175 fragments. The other species was inducting silence zymogram types, including type“present→absent→present”and type“present→absent”, totalled 175 fragments.
     3. 100 ESTs with 100-700bp length were obtained by recovered, cloned and sequenced. 34 ESTs of these 100 ESTs were highly homologous (>62%) with the known proteins of GeneBank. By Blastx search to NCBI dbEST, it was found that the low temperature inducing fertility-changeover was related to many proteins, such as metabolism, energy, defense, cell construction, protein synthesis and transport, transcription and translation, Cellular communication, signal transduction mechanism correlative protein and so on, which maybe form a network. 49 ESTs of these 100 ESTs were highly homologous (>90%) with the known ESTs of GeneBank, in which 47 ESTs comes from Brassica campestris. The other 17 ESTs had not homologous sequences of GeneBank. As implied that they were new gene fragment. Three ESTs with length longer 150bp were submitted to GeneBank. They were: EST80: GenBank_Accn FE905220, dbEST_Id 54764252; EST182: GenBank_Accn FE905221, dbEST_Id 54764253; EST162: GenBank_Accn FE905222, dbEST_Id 54764254.
     4. The EST-58, EST-114, EST-188 were amplified by In silico cloning and the RT-PCR was used to confirm the authenticity. Two new genes of Chinese cabbage were obtained, which are xyloglucan endotransglycosylase (XET) gene (GenBank_Accn is EU579461) and PAS (PASTICCINO) gene (GenBank_Accn is EU650783). And one new sequence (GenBank_Accn is FE905223; dbEST_Id is 54764255) full length is 877bp, come from EST-188. The new sequence come from EST-58, whose full length is 1196bp, contained a complete ORF, encoding 292 animo acids. The confirm result of RT-PCR is identity to it with 98%. Homology analyse showed that it was the Chinese cabbage gene of XET. The new sequence come from EST-114, whose full length is 1036bp, contained a complete ORF, encoding 229 animo acids. The confirm result of RT-PCR is identity to it with 98%. Homology analyse showed that it was the Chinese cabbage gene of PAS. The new sequence comes from EST-188, whose full length is 877bp. The confirm result of RT-PCR is identity to it with 96%. Whose ORF was not complete. So it was submitted to GeneBank as EST.
     5. Established an effective method ofβ-EST recycling and purification from Chinese cabbage. Namely, riched the goalβ-EST with one-dimensional active GEL electrophoresis and cutting goal bands technology, and further purified with self-made two-dimensional GEL electrophoresis. The one-dimensional active GEL was only dyed two margins in order to denote the goal band area middle to reclaim. The reclaimed bands were made up to rude powder by electroelution and freeze-dry respectively, and then were further separated by two-dimensional GEL electrophoresis. In the two-dimensional GEL electrophoresis, the first dimensional IEF was optimized using vertical slab made in China, which is economy and effective. After IEF,the GEL was dyed and cut the goal bands. These goal bands were separated by the second dimensional SDS GEL further. This method reduced the non-goal bands disturbance, and made the analysis results more reliable.
     6. Obtained the partial amino acid sequences of the specialβ-EST poly-peptides. By the separation and purification of two-dimensional GEL electrophoresis, 6 kinds poly-peptides related to fertility changeover were obtained. The poly-peptides with molecular weight 57.1KD and 62KD were analyzed by Q-TOF mass spectrum. We obtained three sequences of short peptides from 57.1KD poly-peptide and two sequences of short peptides from 62KD poly-peptide, and the two short peptides sequences of 62KD poly-peptide are same as that from the 57.1KD poly-peptide. The result indicates that the sequences of the two polypeptides might have high similarity.
     7. By comparing the sequence with protein database of GeneBank, it was found that the three short peptides obtained were highly homologous (60-100%) with protein p27SJ.
引文
[1] 张战凤. 大白菜胞质雄性不育相关基因的克隆及差异表达研究[D]. 西北农林科技大学,硕士论文,2006.
    [2] Hanson MR. Plant mitochondrial mutation and male sterility[J]. Annu Rev Genet, 1991, 25:461-486
    [3] 张耀文, 王俊生, 田建华等. 油菜 CMS 育性不稳定的研究进展[J]. 西北农业学报,2003, 12(1):8-11
    [4] Kaul M L H. Male Sterility in Higher Plants[J]. Berlin, Spring Verlag,1988,2-5
    [5] 夏广清, 何启伟. 大白菜雄性不育及其机理的研究进展[J]. 山东农业科学, 2004, 6: 73-76
    [6] Tatlioglu T. Genetic control of temperature-sensitivity of cytoplasmic male sterility (cms) in chives[J] .Plant Breeding,1987,99(1):65-76
    [7] 傅廷栋, 杨光圣, 杨小牛. Some investigation on Polima cytoplasmic male sterility in B.napus. Eucarpia Cruciferae Newsletter[J]. 1987, 12(1):46-47
    [8] 张鲁刚, 郝东方. 结球白菜温度敏感细胞质雄性不育系 CMS7311 育性转换的研究(英文) [J]. 植物学报, 2001, 43(11):1123-1128
    [9] 张鲁刚, 郝东方, 柯桂兰. 波里马胞质大白菜雄性不育系 CMS3411-7 温度敏感特性的研究[J]. 园艺学报, 2001, 28(5): 415-420
    [10] 邵双, 郭晓雷, 冯辉. 温敏型大白菜不育系的小孢子败育特性[J]. 2005, 16(1):43-45
    [11] 杨晓云, 曹寿椿. 不结球白菜波里马胞质雄性不育系温度敏感的特征[J].中国蔬菜,1999(4): 15-17
    [12] 李素萍, 陈利平, 陈贵华. 辣(甜)椒雄性不育研究进展[J]. 内蒙古农业科技, 1999(6):28-31
    [13] Bhadual S K, Sawhney V K. Esterase activity and isozymes during the ontogeny of stamens of male fertile Lycopersicon esculentum Mill a male sterile stamenless-2 mutant and the low temperature-reverted mutant[J]. Plant Science,1987,52(1):187-194
    [14] Nieuwhof M , Efect of temperature on the expression of male sterility in Brussels sprouts[J]. Euphytica,1968,17.265-273
    [15] Dickson M.H., A temperature sensitive male sterile gene in broccoli[J]. Hort Sci,1970, 2 :13 -14
    [16] Mustchler M.A., Temperonture-influencal instability in a genic male-sterile common bean [J], HortS cience,1984,19:401-402.
    [17] 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[J]. 植物学通报, 2005,22(1): 19-26
    [18] 杨光圣, 傅廷栋. 环境条件对油菜细胞质雄性不育的影响[J]. 中国油料, 1987, 9(3): 45-51
    [19] 李殿荣, 田建华. 杂交油菜遗传育种的研究与应用[C].中加油菜育种项目年会暨学术讨论会论文集.乌鲁木齐:1994
    [20] Shengwu Hu, Jiying Huang. Temperature and genotype on the expression of CMS in winter rapeseed Shaan 2A. J. of 94 Sino-British workship on Brassica Genetics and Breeding[J]. 1994, 17:26
    [21] 王际凤, 陆作楣. 温敏核不育基因在籼型三系遗传背景下的育性表达[J]. 中国水稻科学, 2006, 20(5):487-492
    [22] 杨晓云, 曹寿椿. 不结球白菜波里马胞质雄性不育系花药发育的细胞形态学研究[J]. 南京农业大学学报, 1997, 20 (3):36-43
    [23] 刘志, 刘少卿, 余筱南. 特棉 S-1 雄性不育发生的细胞学基础及可育花粉对温度的反应[J]. 湖南农业大学学报(自然科学版), 2007, 33(4):403-406
    [24] 黄玉祥, 陈良碧, 周建林等. 不同温度条件下温敏核不育水稻雄性不育的细胞学观察[J]. 湖南师范大学自然科学学报, 2000, 23(1): 65-67
    [25] 梁海鹰, 谢锦云, 陈平等. 光温敏核不育水稻幼穗育性转换期蛋白质组的初步分析[J]. 广东海洋大学学报, 2007, 27(3): 117-122
    [26] 肖辉海, 陈良碧. 温敏雄性核不育水稻几种同功酶的热激反应[J]. 常德师范学院学报(自然科学版), 1999, 11(3):64-67
    [27] 周仲华, 朱四元, 陈金湘. 棉花温敏雄性不育与膜脂过氧化的关系研究[J]. 江西农业大学学报, 2007, 29(4): 518-521
    [28] 周广洽. 温敏核不育水稻的光温生态生理学[M]. 湖南师范大学出版社.3-17
    [29] 肖翊华. 光敏感核不育水稻的光周期及其生理学[M]. 武汉大学出版社.150-158
    [30] 田长恩, 段俊, 梁承邺等. 水稻细胞质雄性不育系及其保持系幼穗发育过程中内源激素的变化[J]. 热带亚热带植物学报, 1998, 6(2):137-143
    [31] 赵玉锦, 童哲, 陈华君等. 内源植物激素与光敏核不育水稻农垦 58S 育性的关系[J]. 植物学报,1996,38(12):936-941
    [32] 李英贤, 张爱民, 黄铁成. 小麦雄性不育与叶片中内源激素含量的关系[J]. 农业生物技术学报,1998,6(2):185-190
    [33] 唐祈林, 荣延昭, 胡长远等. 不同核背景的玉米 CMS 系内源激素关系研究[J]. 四川农业大学学报,2002, 9(3): 202-211
    [34] 解海岩, 蒋培东, 王晓玲等. 棉花细胞质雄性不育花药败育过程中内源激素的变化[J]. 作物学报, 2006, 32(7): 1094-1096
    [35] 关天霞, 党占海, 张建平等. 亚麻温敏型雄性不育系POD活性和内源激素含量比较分析[J].甘肃农业大学学报, 2007, 2(6): 66-70
    [36] 关天霞, 党占海, 张建平等. 亚麻温敏型雄性不育系花蕾发育过程中内源激素的变化[J]. 中国油料作物学报, 2007, 29(3): 248-253
    [37] 李德红, 骆炳山, 屈映兰. 光敏核不育水稻幼穗的乙烯生成与育性转换[J]. 植物生理学报, 1996, 22(3): 320-326
    [38] 骆炳山, 李文斌, 屈映兰等. 湖北光敏感核不育水稻育性转换机理初探[J]. 华中农业大学学报, 1990, (9):7
    [39] 汤继华, 赫忠友, 陈伟程等. 玉米温敏核雄性不育育性转换与内源激素的关系[J]. 作物学报, 2003, 29(3): 336-338
    [40] 邱义兰, 李红, 陈松等. 温敏雄性核不育水稻可育花药与不育花药的钙分布[J]. 生命科学研究,2007,11(2):167-171
    [41] 余凤群,傅廷栋. 甘蓝型油菜几个雄性不育系花药的细胞形态学研究[J].武汉植物学研究,1990,8:209-215
    [42] 杨光圣. 甘蓝型油菜雄性不育的分类和综合利用研究[D]. 武汉,华中农业大学, 博士学位论文 1998
    [43] 赵坚义, Bo kistiansson. 温、湿度对油菜波里马细胞质雄性不育基因表达影响的研究[C].中国油料作物科学技术新进展, 中国作物学会油料作物专业委员会编.
    [44] Fan Z, Stefansson B R. Influence of temperature on sterility of two cytoplasmic male sterility in rape(B. napus L.) [J], Can J Plant Sci,1986,66:221-227
    [45] Burns D R, Scarth R and McVetty P B E. Temperature and genotypic effects on the expression of pol cytoplasmic male sterility in summer rape[J]. Can J Plant Sci,1991,71:655-661
    [46] 李加纳,唐章林,谌利等. 温度对波里马胞质不育系油菜育性变化时期和临界值的影响研究[J].西南农业大学学报,1995,17(5):391-395
    [47] 唐章林, 李加纳, 谌利等.温度对甘蓝型油菜细胞质雄性不育系及其杂种一代育性的影响研究[C].全国作物育种学术讨论会论文集.中国作物学会第六届理事会鱀全国作物育种学术讨论会.北京,1998.北京,中国农业科技出版社,1998, 305-311
    [48] 傅廷栋. 杂交油菜育种与利用[M]. 武汉:湖北科学技术出版社,1994.
    [49] 李殿荣. 杂交油菜秦油二号论文集[C]. 北京: 农业出版社,1993
    [50] 袁美. 甘蓝型油菜生态型细胞质雄性不育两用系温度调控机理的初步研究[D],华中农业大学,博士论文 2002
    [51] 陈良碧, 周广洽, 黄玉祥. 温光条件对水稻安农 S-1/衡农 S-1 的育性及生理的影响[J]. 植物学报,1994,36(增刊):119-123.
    [52] 吴秋云, 何觉民, 罗红兵等. 生态雄性小育小麦 ES-50 育性转换机制的研究[J] . 湖南农业大学学报:自然科学版, 2001, 27( 2) : 89- 91.
    [53] 赵凤梧, 李慧敏, 李爱国等. 冬小麦温敏型雄性小育系 LT-1-3A 选育及育性转换与遗传研究[J]. 核农学报, 2001, 15(2):65- 69.
    [54] 何蓓如, 董普辉, 宋喜悦等. 小麦温度敏感小育系 A3314 温敏特性研究[J]. 麦类作物学报, 2003, 23(1) :1- 6.
    [55] 邹应斌, 周关兰, 何觉民等. 生态型雄性小育小麦育性转换机制[J] . 湖南农业科学, 1992(6): 5- 7.
    [56] 魏荷, 郭建夫, 王丰青等. 两个新籼稻光温敏核不育系的育性转换特性研究[J]. 中国农学通报, 2006, 22(12):157-161
    [57] 张自国, 杨静, 元生朝等.光敏核小育水稻在热带稻区的育性转换特性与利用研究[J]. 作物学报, 1995,21(4):485-490.
    [58] 卢兴桂,哀潜华,姚克敏等.我国主要水稻光温敏核小育系类型的气候适应性[J].中国水稻科学,2001,15(2):81-87.
    [59] 汤继华, 张凤瑞, 付志远等. 玉米温敏核雄性不育系育性转换的生态机制研究[J]. 河南农业大学学报, 2006, 40(5): 455-458
    [60] 张立平, 赵昌平, 单福华等. 小麦光温敏雄性不育系 BS210 育性的主基因+多基因混合遗传分析[J].作物学报, 2007, 33(9):1553-1557
    [61] 宋喜悦, 何蓓如, 马翎健等. 小麦温敏不育系 A3314 温敏不育性的遗传研究[J]. 中国农业科学 2005,38(6):1095-1099
    [62] 袁美, 杨光圣, 傅廷栋等. 甘蓝型油菜生态型细胞质雄性不育两用系的研究 8-8112AB 的温度敏感性及其遗传[J]. 作物学报, 2003, 29(3): 330-335
    [63] Feng Hui. Inheritance of and Utilization Model for Genetic Male Sterility in Chinese cabbage. Acta Horticulturae, 1995, 402 : 133-140
    [64] 张鲁刚, 柯桂兰.大白菜异源胞质雄性不育遗传规律及其恢复性的研究[J]. 西北农业大学报, 1994,3(3): 45-50
    [65] 杜德志, 刘青元, 赵洪潮. 甘蓝型玻里马不育胞质三系杂种的选育[J]. 中国油料作物学报, 1998, 20(1):7-11
    [66] 缪颖, 曹家树, 陈林姣. 芸苔属植物雄性不育的发育生物学研究[J]. 生命科学, 2000, 12(3):105-108
    [67] 赵昌平, 张立平, 李云伏等. 小麦光温敏雄性不育相关基因的 DDRT-PCR 分析及功能预测[J]. 中国生物化学与分子生物学报, 2007, 23(1):56-62
    [68] 庄楚雄, 徐是雄, 卢永根等. 运用 cDNA 缩减杂交法克隆水稻花粉发育相关的cDNA[J]. 科学通报,1999, 44(17):1842-1846
    [69] 陈章权. 温敏核雄性不育水稻育性转换相关基因的克隆及其 RNAi 植物表达载体的构建[D]. 华南热带农业大学,博士论文, 2002.
    [70] 卢利方, 冯仁军, 张银东. 玉米温敏核雄性不育相关基因克隆[J].华南热带农业大学学报, 2006,12(1): 7-11
    [71] 李祥. 利用 HSP70 基因人工创造植物雄性不育系的研究[D].湖南农业大学, 2003.
    [72] 陈建南, 傅鸿仪, 路子显等. 高粱细胞质雄性不育与HSC_(70)mRNA的关系[J]. 科学通报 , 1998,43(23):2525-2530
    [73] 陈建南, 傅鸿仪, 路子显等. 温度、热激蛋白与高梁育性的变化. 遗传学报, 1998, 25(4): 356-361
    [74] CHEN J N, FU H Y, Que qiang,et al. Effect of the Expression of HSP70 gene on the Fertility of Sorghum [J]. Developmental&Reproductive Biology. 1998,7 (2):51-62.
    [75] 龙跃生, 陈良碧. 光敏、温敏雄性核不育水稻不育基因研究进展[J]. 2001, 2: 44-49
    [76] 王斌, 牟同敏, 陈一华. 水稻光温敏不育的遗传学和分子生物学研究[M].两系杂交水稻理论与技术.北京:科学出版社,2001: 39-60
    [77] 姜大刚, 卢森, 周海等. 用 EST 和 SSR 标记定位水稻温敏不育基因 tms5[J]. 科学通报,2006,51(2):148-151
    [78] Wang B, Xu WW, Wang JZ, et al. Ray JD, Nguyen HT. Tagging and mapping the thermo-sensitive genic male-steri1e gene in rice with molecular marker[J]. Theoretical and Applied Genetics, 1995,91: 1111-1114
    [79] Yamaguchi Y, Ikeda R, Hirasawa H. Linkage analysis of thermo-sensitive genic male sterility gene tms2 in rice[J]. Breeding Science, 1997, 47: 371- 373
    [80] Subudhi PK,Bortatati RP,Vi mani SS Molecular mapping of a thermo-sensitive genic male steri1ity in rice using bulked sergeant analysis. Genome, 1997,40: 188-194
    [81] Dong NV, Subudhi PK, Luongl PN. Molecular mapping of a rice thermo sensitive genic male sterile by AFLP,RFLPand SSR techniques[J]. Theoretical and Applied Genetics, 2000, 100: 727-734
    [82] Reddy OUK, Sddiq EA,Ali J. Identifing molecular markers for the gene(s) governing thermo sensitive genic male sterility in rice[J]. International Rice Research notes, 23: l0-11
    [83] Wang Q, Weng ML, Wang B. Construction of genetic linkage map and mapping of thermo-sensitive genic male sterile gene tms5[J]. Plant Genomics in China, 2000
    [84] Koh H, Son YH, Heu MH. Molecular mapping of a new genic male-sterility gene causing chalky endospermin in rice (Oryza satlva L) [J].Euphytice, 1999,106: 57-62
    [85] 李仕贵, 周开达, 朱立煌. 水稻温敏显性核不育基因的遗传分析和分子标记定位[J]. 科学通报. 1999,44: 955-957
    [86] Jia JH, Zhang DS, Li CY, et al. Molecular mapping of the reverse thermo-sensitive genicmale steri1e gene (rtmsl) in rice [J]. Theoretical and Applied Genetics.2001, 103: 607-612
    [87] 邱芳, 金德敏, 伏健民等. 温敏核不育水稻5460细菌人工染色体文库的构建和鉴定[J]. 中国科学(辑),1999, 29: 518-524
    [88] 曾汉来. 温度导致的水稻雄性不育现象研究概述[J]. 水稻文摘, 1993, 12(5):1-6
    [89] 梅国志, 汪向明. 农垦 58S 型光周期敏感雄性不育的遗传分析[J]. 华中农业大学学报. 1990, 9(4): 400-406
    [90] 周庭波. 新论植物雄性不育遗传. 湖南农业科学[J],1997,4:15-17
    [91] 何觉民, 戴君惕, 邹应斌. 生态遗传雄性不育理论与两系杂交植物. Ⅰ.生态遗传雄性不育理论[J].湖南农学院学报,1994,20(1):1-5
    [92] 晋坤贞, 万广辉, 秋建遨. 磁水对番茄酯酶同工酶的影响[J]. 西北植物学报, 1994, 14( 2) : 102-106.
    [93] 宋水山. Ralstonia eutropha CH34 酯酶基因的克隆和序列分析[J]. 微生物学报, 2001,41(4):402-407
    [94] 王煜, 田廷亮等. 油菜种子老化过程中的生理生化变异. 中国油料,1994,16(3):11-14
    [95] Candalios J G. Isoenzymes in development and differentiation.[J]. Ann Rev plant physical,1974,25:225~258
    [96] 邓明华. 辣椒胞质雄性不育株生理生化特性及离体培养研究[D]. 博士论文,2003
    [97] 张少丽, 张鲁刚, 吉雪花等. 与大白菜温度敏感胞质雄性不育系育性转化相关的同工酶分析初探[J]. 中国农学通报, 2005, 21(5):90-95。
    [98] 唐章林, 李加纳, 堪利等. 温度对油菜细胞质雄性不育系酯酶同工酶的影响研究[J]. 西南农业大学学报,1995,17(4):325-328
    [99] 杨斌, 李桐森, 王晓波等. 2 种拟青霉代谢产物对烟蚜乙酞胆碱酯酶和羧酸酯酶的影响[J]. 云南大学学报(自然科学版), 2005,27(2):166-169
    [100] 杨银书, 刘增加, 宫占威等. 百部碱对德国小镰酯酶活性的影响[J]. 中国媒介生物学及控制杂志, 1998,9(5):341-343
    [101] 乔传令, 王靖, 邢建民. 不同地区小菜蛾种群的抗药性及酯酶同工酶的研究[J]. 农药学学报, 2000,2(4): 33-39
    [102] 寇宇, 倪晓平, 乔传令等. 杭州淡色库蚊抗性及酯酶活性测试[J]. 中国媒介生物学及控制杂志, 2002, 13(1):22-23
    [103] Hemingwav J, Callagban A, Amin A M. Mechanisms of organophosphate and carbamate resistance in Culex quinquef asciatus from Saudi Arabia. Med. Vet [J]. Entomol.,1990, 4: 275 一 283
    [104] Villani F, Hemingway J. The detection and interaction of multiple organophosphorus and carbmate insecticide resistance genes in field populations of Culex pipiens from Italy [J]. Pestic.Biochem. Physiol.,1987, 27: 216- 228
    [105] Bonning B C, Hemingway J, Romi R et al. Interaction of insecticide resistance genes in field populations of CuIex pipiens (Diptera: Culicidae) from Italy in response to changing insecticide selection pressure[J]. Bull.Entoml.Res.,1991, 81: 5 一 10
    [106] 滕霞, 孙曼霁. 羧酸酯酶研究进展[J]. 生命科学, 2003,15(1):31-35
    [107] 王淑华, 魏毓棠, 冯辉. 大白菜雄性不育株与可育株花蕾生理生化特性比较分析[J]. 沈阳农业大学学报, 1998, 29(2):132-137
    [108] 胡美华,陈竹君,王炳良. 榨菜胞质雄性不育系及其保持系过氧化物酶和酯酶同工酶的比较研究[J]. 浙江农业学报, 2000, 12(4):201-205
    [109] 董伟,陈玲.甘蓝雄性不育系和保持系花药中可溶性蛋白质的分离及几种同工酶鉴定[J]. 西南农业大学学报, 1993, 15(3):255-258
    [110] 柯桂兰, 赵稚雅, 宋胭脂等. 大白菜异源胞质雄性不育系 CMS3417--7 的选育及应用[J]. 园艺学报, 1992, 19(4): 333-340
    [111] 周长久, 张友良. 萝卜雄性不育性的几种特性研究[J]. 园艺学报, 1994, (1):65-70
    [112] 张鲁刚,柯桂兰. 大白菜胞质雄性不育系和保持系同工酶及可溶性蛋白质分析[J]. 西北植物学报, 1999, 8(4):67-70
    [113] 李惠敏, 秦新民. 沙田柚花粉萌发前后同工酶分析[J]. 广西师范大学学报(自然科学版), 2004, 22(3) :85-90
    [114] 吴乃虎. 基因工程原理, 第二版, 北京:科学出版社,1998: 356-364
    [115] 薛淑群, 刘伟, 匡友谊. 蛋白质组学研究的技术体系及进展[J]. 水产学杂志, 2004,17(1):77-81
    [116] 李军. 补骨脂种子抗菌肤的分离纯化与基因克隆[D]. 西南农业大学, 2005
    [117] 韩曜平, 苏月菊, 饶定齐等. 山溪鲵皮肤β-microsem inoprotein 样蛋白的分离纯化及其 cD NA 克隆[J].南京农业大学学报, 2007, 30 (3):94 一 99
    [118] 程冬梅, 范三红, 刘香莉等. 小麦返白系胆色素原脱氨酶纯化及编码cDNA序列分析[J]. 中国生物化学与分子生物学报, 2006, 22(12): 973-978
    [119] 王大东, 王嘉玺. 蛋白质组与蛋白质组学[J]. 国外医学分子生物学分册. 2000, 22(3):129-133
    [120] O'Farrell P H. High Resolution Two-Dimensional Electrophoresis of Proteins[J].Biol Chem,1975,250: 4007-4021
    [121] 杜鹏, 冯伟华, 郭俊生. 蛋白质组双向电泳技术研究进展[J]. 卫生研究, 2005,34(2):237-240
    [122] 刘立军, 薛光行. 水稻光敏核不育基因相关蛋白产物初步研究[J]. 作物学报, 1995, 21(2):251-253
    [123] 王台, 童哲, 匡廷云等. 光敏核不育水稻农垦58S 41 kD蛋白质的N-端序列分析[J]. 植物学报, 1997, 39 (10):979-982
    [124] 王台, 童哲, 匡延云等. 光敏核不育水稻 61kD 特异蛋白质的纯化和 N-端序列分析[J].植物学报, 1996,3 8( 10):7 72-776
    [125] 李大东, 王斌. 水稻线粒体 at 必基因的克隆及其与细胞质雄性不育的关系[J].遗传,1990, 12(4):1 -4
    [126] 夏快飞. 温敏核不育水稻双低-s 水稻花药蛋白质的研究[D]. 湖南师范大学, 2001
    [127] 谢锦云, 李小兰, 陈平等. 温敏核不育水稻花药蛋白质组初步分析[J]. 中国生物化学与分子生物学报, 2003, 19( 2):215一221
    [128] 曹孟良, 郑用链, 张启发. 光敏核不育水稻农垦58S与正常可育农垦58N蛋白质双向电泳的对比分析[J]. 华中农业大学学报, 1992, 1l: 305-311
    [129] 王志强, 刘文芳, 肖翊华. HPGMR‘农垦 58s 叶片中过氧化物酶活性变化的比较研究[J]. 华中农业大学学报, 1990, 9: 466-468
    [130] 高原, 王国秀, 刘中来. 一种经济、简便的双向电泳方法[J]. 生物化学与生物物理进展, 2003,30(1):156-158
    [131] 郭志雄, 林琳, 吕柳新. 垂直板小型分析型蛋白质双向电泳技术体系的改进[J].福建农林大学学报(自然科学版), 2006, 35(2):182-186
    [132] 王东辉, 韩韬, 龚化勤等. 一种利用普通垂直电泳槽回收PAGE胶蛋白条带的简便方法[J]. 植物生理学通讯, 2004, 40(1):75-77
    [133] Bachem CWB, Hoeven RS van der, Bruijn SM de,et al. Visualization of differential gene expression using a novel method of RNA finger-printing based on AFLP: analysis of gene expression during potato tuber development[J]. Plant Journal, 1996, 9: 745-753
    [134] Kuhn E. From library screening to microarray technology: strategies to determine gene expression profiles and to identify differentially regulated genes in plants[J]. Anna Bot.2001, 87: 139-155
    [135] Bachem CWB, Oomen RJFJ, Visser RGF. Transcript imaging with cDNA-AFLP: A step-by-step protocol. Plant Molecular Biology Reporter[J].1998,16:157-173
    [136] Christian W, B.Bachem, Ronald J F. J, et al. Transcript Imaging with cDNA-AFLP: A Step-by-step Protocol [J]. Plant Molecular Biology Reporter, 1998, 16: 157-173.
    [137] Milioni D, Sado PE, Stacey NJ,et al. Early gene expression associated with the commitment and differentiation of a plant rtacheary element is revealed by cDNA-amplified length polymorphism analysis[J]. Plant Cell. 2002, 14:2813-2824
    [138] Money T, Reader S, Qu L J, et al. AFLP-based Mma fingerpring. Nucleic Acids Rsearch[J], 1996, 24: 2616-2617
    [139] Fukuda T, Kido A, Kajino K, et al. Cloning of Differentially Expressed Genes in Highly and Low Metastatic Rat Osteosarcomas by a Modified cDNA-AFLP Method. Biophysical Research Communications[J], 1999, 261(1): 35-40
    [140] Durrant WE, Rowland O, Piedras P, et al. cDNA-AFLP reveals a striking overlap in race-specific resistance and wound response gene expression profiles[J]. Plant cell, 2000, 12: 963-977.
    [141] Suares M C, Bemal A, Gutierrez J, et al .Developing Expressed Sequence Tags(ESTs) from Polymorphic Transcript Derived Fragments in Cassava(Manihot-Esculenta Crantz) [J]. Genome. 2000, 43: 62—67
    [142] Habu Y, Fukada-Tanaka S, Hisetomi Y, et al. Amplified restriction fragment lengthpolymorphism based mRNA fingerprinting using a single restriction enzyme that recognizes a 4-bp sequence[J]. Biochemical and Biophysical Research Communications, 1997, 234: 516-521
    [143] Dellagi A, Birch PRJ, Heilbrom J, et al. DNA-AFLP analysis of differential Gene-Expression in the Prokaryotic Plant Pathogen Erwinia carotovora[J]. Micribiology, 2000, 146: 165-171
    [144] Kojima T, Habu Y, Lida S, et al. Direct isolation of differentially expressed gene from a specific chromosome region of common whet. Application of the amplified fragment length polymorphism based messenger RNA fingerprinting(amf) method in combination with a Deletion line of wheat[J]. Molecular and Generl Gengeics. 2000, 263(4): 635-641
    [145] Johnes Jt, Rower H. A comparison the efficiency of differential display and cDNA-AFLP as a tools for the isolation of differentially expressed parasite genes[J]. Fundamental and Applied Nemadity, 1998, 21:81-88
    [146] Terrvn N, Kouze P, MOritagu MV. Plant genomics[J]. FEBS Letters.1999, 452:3-6
    [147] 郭秀林, 张正斌, 李景娟等. cDNA-AFLP 技术在植物抗逆性相关基因表达研究中的应用[J]. 西北农林科技大学学报(自然科学版), 2006,34(11):87-92
    [148] Gill R, Sanseau P. Rapid in silico cloning of genes using expressed sequence tags (ESTs) [J]. Biotechnol Annu Rev, 2000, 5:25-44
    [149] 李晓晓, 李蕊, 李雅轩等. 大豆脱氢抗坏雪酸还原酶基因的电子克隆及进化分析[J]. 大豆科学, 2007, 26 (1): 45-50
    [150] 王冬冬, 朱延明, 李勇等. 电子克隆技术及其在植物基因工程中的应用[J]. 东北农业大学学报, 2006,37(3): 403-408
    [151] 张成岗,贺福初. 生物信息学方法与实践[M]. 北京:科学出版社,2002. 249
    [152] Huang X. An improved sequence assembly program[J]. Genomics, 1996, 33(1): 21-31
    [153] 黄骥, 张红生, 曹雅君等. 一个新的水稻C2H2型锌指蛋白cDNA的克降与序列分析[J]. 南京农业大学学报, 2002, 25(2):110- 112
    [154] 孟宪萍, 李晓晓, 李雅轩等. 大豆尿黑酸叶绿基转移酶基因的克隆与进化分析[J]. 华北农学报,2007,22(4):14-18
    [155] 张会敏, 姜民国, 冯友军. 稻瘟菌I型烯醇化酶基因全长cDNA的电子克隆[J].生物信息学, 2006,4(2):57-61
    [156] 李蕊, 孟宪萍, 胡英考等. 截形苜蓿吡哆醇生物合成基因的电子克隆和进化分析. 江苏农业科学, 2007, 1: 81-84
    [157] 张国裕, 康俊根, 张延国等. 青花菜快速碱化因子 RALP 的克隆与序列分析[J]. 园艺学报, 2006, 33(3): 561 一 565
    [158] 曹凤, 王亚红, 金伟波等. 小麦尿卟啉酸合成酶基因克隆及序列分析[J]. 西北植物学报, 2007,27(7):1299- 1304
    [159] 郭志爱, 藏庆伟, 景蕊莲等. 小麦小 G 蛋白 Rab2 基因的克隆及其表达分析[J]. 作物学报, 2007, 33( 2):201 一 207
    [160] 钮旭光, 韩梅. 水稻 1,3,4-三磷酸肌醇 5/6-激酶基因的电子克隆及表达[J]. 安徽农业科学,2006, 34(24) : 6408- 6409, 6411
    [161] Huang Ji, Wang Jian-Fei, Zhang Hong-sheng, et al. In silico cloning of glucose-6-phosphate dehydrogenase cDNA from rice (Oryza sativa L.)[J]. Acta Genetica Sinica. 2002, 29(11): 1012-1016.
    [162] 李任华, 才宏伟, 王象坤. 一条重要水稻酯酶同工酶带的发现[J]. 科学通报, 1994,39(22):2095-2096
    [163] 刘贵华, 蔡明, 盛晓燕等. 甘蓝型油菜雄性不育的遗传研究. Ⅱ.甘蓝型油菜异核型雄性不育材料的酯酶同工酶分析[J]. 中国油料, 1994,16(4): 1-3
    [164] 于澄宇, 胡胜武, 郭蔼光等. 甘蓝型油菜 5 个同质 CMS 系及其保持系、杂交种的同工酶比较[J]. 西北农业学报, 2003, 12(1):5- 7
    [165] 张战凤, 张鲁刚, 王绮等. 大白菜胞质雄性不育系育性相关线粒体 DNA 片断的克隆及序列分析[J]. 西北农业学报, 2006,15(3): 12-115
    [166] Sarkissian, N.D., Darbinyan, A., Otte, J.,et al. p27SJ, a novel protein in St John's Wort, that suppresses expression of HIV-1 genome[J]. Gene Therapy. 2006, 13: 288-295.
    [167] Lewis, A.P., D. Crowther. DING proteins are from Pseudomonas[J]. FEMS Microbiology Letters. 2005, 2: 215-222.
    [168] Scott K, Wu L. Functional properties of a recombinant bacterial DING protein: comparison with a homologous human protein[J]. Biochim Biophys Acta. 2005, 2:234-244.
    [169] Berna, A, F. Bernier, K. Scott, et al. Ring up the curtain on DING proteins[J]. FEBS Letters. 2002, 524: 6-10.
    [170] Berna, A, F. Bernier , E. Chabrière, et al. DING proteins; novel members of a prokaryotic phosphate-binding protein superfamily which extends into the eukaryotic kingdom[J]. The International Journal of Biochemistry & Cell Biology. 2008, 2: 170-175
    [171] Pantazaki, A. A., G. P. Tsolkas, and D. A. Kyriakidis. A DING phosphatase in Thermus thermophilus[J]. Amino Acids. 2007,May 14, 0939-4451 (Print) 1438-2199 (Online), DOI 10.1007/s00726-007-0549-5
    [172] Perera, T., A. Berna, and K. Scott. Proteins related to St. John's Wort p27SJ, a suppressor of HIV-1 expression, are ubiquitous in plants[J]. Phytochemistry. 2008, 4: 865-872
    [173] 韩斌,彭建营. cDNA-AFLP 技术及其在植物基因表达研究中的应用[J]. 西北植物学报, 2006, 26(8):1753-1758
    [174] 宋欣, 杨文鹏, 赵德刚. cDNA-AFLP 技术在植物遗传育种研究上的应用[J]. 贵州农业科学, 2007,35(5):143-146
    [175] 林凡云, 胡银岗, 宋国琦等. 干旱复水条件下诱导的糜子特异表达基因的分离与分析[J]. 农业生物技术学报, 2006,14(4):537-541
    [176] 宋国琦, 胡银岗, 林凡云等. YS 型小麦温敏雄性不育系 A3017 育性相关基因的SSH 分析[J]. 西北植物学报, 2006,26(7): 1301-1308
    [177] Glazebrook J. Genes controlling expression of defence responses in Arabidopsis-2001 status[J]. Curr. Opin. Plant Biol., 2001, 4: 301-308.
    [178] 李洁. 植物转录因子与基因调控[J]. 生物学通报, 2004, 39(3): 9-11.
    [179] 王永勤, 余小林, 曹家树. 白菜小孢子发育相关基因 BcMF3 的分离及序列分析[J]. 遗传学报, 2004, 31(11):1302-1308
    [180] 王旭静, 王志兴, 贾士荣. 海岛棉 GbXET 基因的克隆及过量表达对酵母细胞伸长的影响[J]. 农业生物技术学报. 2005,13(3):276-281
    [181] 王会峰, 黄群策, 王松丽. 光温敏核不育水稻育性转换机理研究进展[J]. 河南农业科学,2004,8:11-14
    [182] 刘辉, 徐孟亮, 陈良碧. 光(温)敏核不育水稻生态及生理生化研究概述[J]. 湖南环境生物职业技术学院学报, 2002 ,8(1):1-4
    [183] 杨晓云, 曹寿椿. 温度对不结球白菜波里马胞质雄性不育系育性的影响[J]. 南京农业大学学报, 1997,20(2):22-27
    [184] 朱明月,沈文涛,周鹏. 果实成熟软化机理研究进展[J], 分子植物育种, 2005, 3(3): 421-426
    [185] 赵云峰, 林河通, 林娇芬等. 果实软化的细胞壁降解酶及其调控研究进展[J]. 仲恺农业技术学院学报, 2006, 19(1):65-70
    [186] Faure J.D, Paola Vittorioso, Véronique Santoni,et al. The PASTICCINO genes of Arabidopsis thaliana are involved in the control of cell division and differentiation[J]. Development, 1998: 909-918
    [187] Yae¨l Harrar, Yannick Bellec, Catherine Bellini, et al. Hormonal Control of Cell Proliferation Requires PASTICCINO Genes[J]. Plant Physiology, 2003, 132, 1217–1227
    [188] Costa M.D, Bach .L, Landrieu. I ,et al. Arabidopsis PASTICCINO2 Is an Antiphosphatase Involved in Regulation of Cyclin-Dependent Kinase A[J]. The Plant Cell, 2006,18, 1426–1437.
    [189] Hammond SM, Bernstein E, Beach D, et al. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells [J]. Nature, 2000,404(6775):293-296
    [190] Keting RF, Fischer SE, Bernstein E, et al. Dicer functions in RNA interference and in synthesis of small RN A involved in developmental timing in C. elegans [J]. Genes, 2001,15 (20):2654-265
    [191] Paddison PJ, Caudy AA, Bernstein E, et al. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells[J]. Genes 2002, Dev,16 (8):948-958
    [192] Nicholson RH, Nicholson AW. Molecular characterization of a mouse cDNA encoding Dicer ,a ribonucleaseⅢortholog involved in RNA interference[J]. Mamm Genome.2002,13 (2):67-73
    [193] Zamore PD, Tuschl T, Sharp PA, et al . RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21to 23 nucleotide intervals [J].Cell, 2000.101(1):25-33.
    [194] Engelke T, Gera D,Tatlioglu T. Determination of the frequencies of restorer-and maintainer-alleles involved in CMS1 and CMS2 in German chive varieties[J]. Plant Breeding 2004,123(1):51-59