甜菜M14品系花期特异表达基因cDNA文库的构建及筛选
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摘要
本实验室的郭德栋教授通过远缘杂交方法获得了甜菜无融合生殖单体附加系M14品系(VV+1C,2n=18+1)，以下简称甜菜M14品系。它的染色体组成是栽培甜菜(Beta vulgaris L.,VV,2n=18)染色体组附加了白花甜菜(Beta corolliflora Zoss., CCCC, 2n=36)的第9号染色体。经过连续四年的胚胎学鉴定和细胞遗传学统计，这条附加的染色体平均传递率为96.5％。甜菜M14品系是极其难得的克隆无融合生殖基因的材料。
本实验室已经在甜菜M14品系无融合生殖发育表现出的三个特殊的关键时期获得了甜菜M14品系特异表达基因的3个抑制消减EST库和1个差异显示EST库。在此工作基础之上，本实验利用与EST库取样时期一致的总RNA构建了甜菜M14品系花期特异表达基因的3个ZAP表达载体cDNA文库。对库容量进行检测，结果显示每个初始文库滴度均达到1.25×10~6 pfu／mL以上，插入片段长度主要集中在400～3000bp，达到覆盖遗传信息的标准。从抑制消减EST库中随机挑选了3个EST作为探针，对与之同一时期的cDNA文库Ⅰ进行筛选，得到了3个阳性单克隆，以噬菌体形式保存并已进行了测序。筛选获得的cDNA片段分别命名为Me-c84(797bp)、Me-c86(1182bp)和Me-c264(测序中)。利用RACE技术对Me-c86进行了5′末端扩增，将1％琼脂糖凝胶上检测的特异性条带进行回收测序。通过核酸序列分析软件拼接Me-c86及其5′末端序列获得全长cDNA，命名为M14-86(1331bp)，并进行了RT-PCR验证。
将已经获得的两条cDNA序列与NCBI网站上的GenBank数据库进行同源序列比对，结果显示Me-c84核苷酸序列与瓶子草(Sarracenia purpurea)等植物的26S核糖体RNA部分基因同源性达到98％。M14-86核苷酸序列与大花马齿苋(Portulaca grandiflora)等植物的26S核糖体RNA部分基因同源性达到97％。
The apomictic monosomic addition line of Beta corolliflora, designated as M14(VV+IC, 2n=18+1), was obtained from remote hybridization of B. vulgaris L. and B.corolliflora Zoss. in sugar beet. M14, which constituted of normal 18 B.vulgaris L. (VV,2n=18) chromosomes with the No.9 B. corolliflora Zoss. (CCCC, 2n=36). chromosome,was found having a chromosome transmission frequency of 96.5% by embryologyidentification and cytogenetic analysis. Thus sugar beet M14 was an invaluable materialfor cloning apomictic gene(s).
Three suppression subtractive EST libraries and one differentially displayed ESTlibrary had been constructed in terms of three key developing phases of sugar beet M14.In this experiment, three ZAP differentially expressed cDNA libraries of M14 atflorescence were constructed with total RNA corresponding to the three EST libraries.Each titer of initial cDNA library was above 1.25×10~6 pfu/mL, while the length ofinserts was between 400bp and 3 000bp, which covered all the genetic information.Three EST were chosen at random from a suppression subtractive EST librariy forscreening corresponding cDNA libraryⅠ. Three positive single clones obtained byplaque in situ hybridization, which were preserved and sequenced, were designated asMe-c84 (797bp), Me-c86 (1182bp) and Me-c264 (sequencing) respectively. Then5'-RACE (rapid amplification of cDNA ends, RACE) was played to obtain 5' cDNAsequences of Me-c84, by which a specific DNA fragment was generated. The specificDNA fragment was collected from 1% agarose gel and subsequently sequenced. Thusfull length cDNA (1331bp), designated as M14-86, was obtained by assembling withMe-c86 using nucleotide sequence analysis soft. Then it was identified by RT-PCR.
Then the sequences of Me-c84 and M14-86 were aligned with known sequences inNCBI non-redundant database. The results showed that Me-c84 shared 98% similarities with the 26S ribosomal RNA gene from Sarracenia purpurea, while M14-86 shared97% similarities with the 26S ribosomal RNA gene from Portulaca Grandiflora.
本实验室已经在甜菜M14品系无融合生殖发育表现出的三个特殊的关键时期获得了甜菜M14品系特异表达基因的3个抑制消减EST库和1个差异显示EST库。在此工作基础之上，本实验利用与EST库取样时期一致的总RNA构建了甜菜M14品系花期特异表达基因的3个ZAP表达载体cDNA文库。对库容量进行检测，结果显示每个初始文库滴度均达到1.25×10~6 pfu／mL以上，插入片段长度主要集中在400～3000bp，达到覆盖遗传信息的标准。从抑制消减EST库中随机挑选了3个EST作为探针，对与之同一时期的cDNA文库Ⅰ进行筛选，得到了3个阳性单克隆，以噬菌体形式保存并已进行了测序。筛选获得的cDNA片段分别命名为Me-c84(797bp)、Me-c86(1182bp)和Me-c264(测序中)。利用RACE技术对Me-c86进行了5′末端扩增，将1％琼脂糖凝胶上检测的特异性条带进行回收测序。通过核酸序列分析软件拼接Me-c86及其5′末端序列获得全长cDNA，命名为M14-86(1331bp)，并进行了RT-PCR验证。
将已经获得的两条cDNA序列与NCBI网站上的GenBank数据库进行同源序列比对，结果显示Me-c84核苷酸序列与瓶子草(Sarracenia purpurea)等植物的26S核糖体RNA部分基因同源性达到98％。M14-86核苷酸序列与大花马齿苋(Portulaca grandiflora)等植物的26S核糖体RNA部分基因同源性达到97％。
The apomictic monosomic addition line of Beta corolliflora, designated as M14(VV+IC, 2n=18+1), was obtained from remote hybridization of B. vulgaris L. and B.corolliflora Zoss. in sugar beet. M14, which constituted of normal 18 B.vulgaris L. (VV,2n=18) chromosomes with the No.9 B. corolliflora Zoss. (CCCC, 2n=36). chromosome,was found having a chromosome transmission frequency of 96.5% by embryologyidentification and cytogenetic analysis. Thus sugar beet M14 was an invaluable materialfor cloning apomictic gene(s).
Three suppression subtractive EST libraries and one differentially displayed ESTlibrary had been constructed in terms of three key developing phases of sugar beet M14.In this experiment, three ZAP differentially expressed cDNA libraries of M14 atflorescence were constructed with total RNA corresponding to the three EST libraries.Each titer of initial cDNA library was above 1.25×10~6 pfu/mL, while the length ofinserts was between 400bp and 3 000bp, which covered all the genetic information.Three EST were chosen at random from a suppression subtractive EST librariy forscreening corresponding cDNA libraryⅠ. Three positive single clones obtained byplaque in situ hybridization, which were preserved and sequenced, were designated asMe-c84 (797bp), Me-c86 (1182bp) and Me-c264 (sequencing) respectively. Then5'-RACE (rapid amplification of cDNA ends, RACE) was played to obtain 5' cDNAsequences of Me-c84, by which a specific DNA fragment was generated. The specificDNA fragment was collected from 1% agarose gel and subsequently sequenced. Thusfull length cDNA (1331bp), designated as M14-86, was obtained by assembling withMe-c86 using nucleotide sequence analysis soft. Then it was identified by RT-PCR.
Then the sequences of Me-c84 and M14-86 were aligned with known sequences inNCBI non-redundant database. The results showed that Me-c84 shared 98% similarities with the 26S ribosomal RNA gene from Sarracenia purpurea, while M14-86 shared97% similarities with the 26S ribosomal RNA gene from Portulaca Grandiflora.
引文
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[3] 殷朝珍，王兆龙，葛才林．草地早熟禾无融合生殖及其育种利用研究进展．草原与草坪，2006，1(114)：18～23．
[4] Matzk F, Prodanovic S, BaumLein H, et al. The inheritance of apomixis in Poa pratensis confirms a five locus model with differences in gene expressivity and penetrance[J]. The Plant Cell, 2005, 17 (1): 13～24.
[5] Pessino S C, Espinoza F, Martinez E J, et al. Isolation of cDNA clones differentially expressed in flowers of apomictic and sexual Paspalum notatum. Hereditas[J], 2001, 134 (1): 35～42.
[6] Barcaccia G, Varotto S, Meneghetti S, et al.Analysis of gene expression during flowering in apomeiotic mutants of Medicagossp cloning of EST and candidate genes for 2n eggs[J]. Sex Plant Reprod, 2001, 14 (4): 233～238.
[7] 马三梅，王永飞．植物无融合生殖相关基因的研究进展[J]．种子，2005，24(10)：42～68．
[8] 陈秋芳，黄群策．中国水稻无融合生殖的研究进展[J]．中国农学通报，2005，21(8)：120～124．
[9] GAO D, et al.Monosomic addition lines of Beta corolliflora Zoss. in sugar beet: cytological and molecular-marker analysis[J]. Theor Appl Genet, 2001, 103:240～247.
[10] 申业，申家恒，郭德栋，方晓华，刘丽萍．甜菜无融合生殖单体附加系M14雌配子体的发生与发育[J]．武汉植物学研究，2006，24(2)：106～112．
[11] Christine D, Rita J, Gue D, Thomas S. Painting of Parental Chromatin in Beta Hybrids by Multi-colour Fluorescent in situ Hybridization[J]. Annals of Botany, 2002, 89: 171～181.
[12] 申业，申家恒，郭德栋，方晓华，刘丽萍．甜菜无融合生殖单体附加系M14大孢子发生期间细胞壁胼胝质的变化[J]．作物学报，2006，32(6)：894～898
[13] Fang X, Gu S, Xu Z, Chen F, Guo D, Zhang H B, Wu N. Construction of a binary BAC library for an apomictic monosomic ddition line of Beta corolliflora in sugarbeet and identification of the clones derived from the alien chromosome[J]. Theor Appl Genet, 2004, 108: 1420～1425.
[14] Roche D, Conner J A, Budiman M A, et al. Construction of BAC libraries from two apomictic grasses to study the micro-colinearity of their apospory-specific genomic regions[J]. Theor Appl Genet, 2002, 104: 804～812.
[15] 景志忠．猪带绦虫六钩蚴cDNA表达文库的构建及免疫原基因的筛选、克隆与表达[D]．中国农业科学院博士论文，2003：1～60．
[16] 许兰珍，何永睿，姜国金，刘小丰，陈善春．cDNA文库构建及其在植物抗性研究中的应用[J]．安徽农业科学，2007，35(3)：660～662，664
[17] Shoham N G, Ard T, et al. Differential display and analysis[J]. Biotechniques, 1996, 20(2): 182～184.
[18] Diataheko L, Lau Y F C, Campbell A P, et al. Suppression subtractive hybridization: A method for generating differentially regulated or tissue-specific cDNA probes and libraries[J]. Pro Natl Acad Sci USA, 1996, 93: 6025～6030.
[19] 刘荣梅，李凤兰，王淑菊，胡宝忠．mRNA差异显示技术研究进展[J]．黑龙江农业科学，2007，1：86～89．
[20] 蔡磊，赵青川．差异表达基因的几种筛选方法[J]．第四军医大学学报，2007，28(3)：286～288．
[21] Hou L, Tang J-W, Cui X-N, Bo Wang, Song B, Sun L. Construction and selection of subtracted cDNA library of mouse[J]. World J Gastroenterol, 2004, 10(16): 2318～2322.
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