[目的]构建不结球白菜Pol胞质雄性不育花酵母双杂交cDNA文库,筛选与细胞色素C还原酶铁硫蛋白(BcRISP1)互作的蛋白.[方法]从不结球白菜Pol胞质雄性不育花中提取总RNA,通过长距离PCR,扩增双链cDNA(ds cDNA).采用SMART建库技术构建不结球白菜Pol胞质雄性不育花全长cDNA文库.利用BcRISP1基因的开放阅读框,构建诱饵表达载体pGBK T-7-BcRISP1转化AH109酵母菌,根据酵母双杂交技术筛选出与BcRISP1互作的片段,并进行质粒共转化验证.[结果]经检测,文库的转化率为每μg pGADT7-Rec 1.80×106转化子,文库滴度为1.21×107 CFU？mL-1,重组率为94%,成功构建可以用于酵母双杂交的cDNA文库.酵母双杂交技术获得了与BcRISP1互作的阳性克隆,试验表明诱饵载体表达产物对酵母无毒性作用,质粒共转化试验初步验证了BcRISP1与CYP81G、PIP2蛋白的互作关系.[结论]cDNA文库的成功构建为后期互作蛋白的顺利筛选奠定了基础,利用酵母双杂交技术成功获得与BcRISP1互作的蛋白CYP81G和PIP2,为明确不结球白菜Pol胞质雄性不育相关基因的信号传递通路提供更多的依据,以期从分子水平上进一步阐明不结球白菜Pol胞质雄性不育的机制.[Objectives]Construction of yeast two hybrid cDNA library of non-heading Chinese cabbage flowers with Pol cytoplasmic male sterility(Pol CMS), and screening interaction proteins with cytochrome C reductase iron-sulfur protein gene which named BcRISP1. [Methods]The total RNA was isolated from Pol CMS flowers of non-heading Chinese cabbage and then obtained was double strand cDNA by using LD-PCR approach. SMART technique was used to construct the yeast two hybrid cDNA library in yeast strain Y187. This experiment used the ORF of BcRISP1 to construct vector pGBK T-7-BcRISP1 as the bait in the yeast two-hybrid system, transformed into yeast strain AH109. Yeast two hybrid(Y2H)technique was used to screen interaction proteins. [Results]The conversion rate of the library was about 1.80×106 recombinants per μg pGADT7-Rec, the capacity of the library was up to 1.21×107 CFU？mL-1, and the recombinant rate was about 94%. Successful construct of yeast two hybrid cDNA library can be used for screening interaction proteins. Then SD/-Trp/-Leu/-Ade/-His plates were used for screening positive clones whose fragments inserted in the pGADT7-cDNA. The experiment showed that the bait protein was non-toxic to the yeast and the cotransformation in yeast was performed to verify the interaction relations between BcRISP1 and CYP81G/PIP2. [Conclusions]The construction of the cDNA library was prepared for screening interaction proteins, yeast two hybrid technology was used and successfully obtained were interaction proteins CYP81G and PIP2 from BcRISP1. From the experiments, more informations was got for clear understanding the signaling pathways of non-heading Chinese cabbage Pol CMS related genes, which may further clarify the mechanism of non-heading Chinese cabbage Pol CMS from the molecular level
References
[1]
邓晓辉,张蜀宁,侯喜林,等. 胞质雄性不育相关基因的克隆及其表达分析[J]. 西北植物学报,2006,26(9):1859-1863 [Deng X H,Zhang S N,Hou X L,et al. Cloning and expression of genes related with cytoplasmic male sterility[J]. Acta Bot Boreal-Occident Sin,2006,26(9):1859-1863(in Chinese with English abstract)]
[2]
袁美,杨光圣,傅廷栋,等. 甘蓝型油菜Pol CMS育性恢复基因对orf224/atp6的转录调控[J]. 遗传学报,2003,30(5):469-473 [Yuan M,Yang G S,Fu T D,et al. Transcriptional control of orf224/atp6 by the pol CMS restorer Rfp gene in Brassica napus L.[J]. Acta Genetica Sinica,2003,30(5):469-473(in Chinese with English abstract)]
[3]
Fields S,Song O. A novel genetic system to detect protein-protein interactions[J]. Nature,1989,340(6230):245-246
[4]
Fukao Y. Protein-protein interactions in plants[J]. Plant Cell Physiology,2012,53(4):617-625
[5]
Costa M,Nobre M S,Becker J D,et al. Expression-based and co-localization detection of arabinogalactan protein 6 and arabinogalactan protein 11 interactors in Arabidopsis pollen and pollen tubes[J]. BMC Plant Biology,2013,13(7):1871-1890
[6]
Singh R,Lee M O,Lee J E,et al. Rice mitogen-activated protein kinase interactome analysis using the yeast two-hybrid system[J]. Plant Physiology,2012,160(1):477-487
[7]
Matsumoto D,Yamane H,Abe K,et al. Identification of a Skp1-like protein interacting with SFB,the pollen S determinant of the gametophytic self-incompatibility in Prunus[J]. Plant physiology,2012,159(3):1252-1262
[8]
Zhu Y Y,Maehleder E M,Chenchik A,et al. Reverse transcriptase template switching:a SMART approach for full-length cDNA library construction[J]. Biotechniques,2001,30(4):892-897
[9]
何晓兰,许玲,徐照龙,等. 大豆高盐胁迫下根部组织酵母双杂交文库的构建与分析[J]. 上海农业学报,2013,29(6):20-23 [He X L,Xu L,Xu Z L,et al. Construction and analysis of a yeast two-hybrid cDNA library of soybean root tissue under high salt stress[J]. Acta Agriculturae Shanghai,2013,29(6):20-23(in Chinese with English abstract)]
[10]
崔红军,张军杰,黄玉碧. 玉米根部酵母双杂交cDNA文库的构建及评价[J]. 分子植物育种,2008,6(1):161-164 [Cui H J,Zhang J J,Huang Y B. Construction and evaluation of yeast two hybrid cDNA library of maize root[J]. Molecular Plant Breeding,2008,6(1):161-164(in Chinese with English abstract)]
[11]
Hommey L,Brown G G. Organization differences between cytoplasmic male sterile and fertile Brassica mitochondrial genomes are confined to a single transposed locus[J]. Nuclelc Acids Resl,1993,21(8):1903-1909
[12]
Hanson M R,Bentolila S. Interactions of mitochondrial and nuclear genes that affect male gametophyte development[J]. Plant Cell,2004,16:154-169
[13]
Chen J,Carter M B,Edwards B S,et al. High throughput flow cytometry based yeast two-hybrid array approach for large-scale analysis of protein-protein interactions[J]. Cytometry A,2012,81(1):90-98
[14]
郑立双,李向楠,孙城涛,等. 酵母双杂交技术及应用的研究进展[J]. 中国畜牧兽医,2013,40(9):105-108 [Zheng L S,Li X N,Sun C T,et al. Research progress on yeast two-hybrid technology and application[J]. China Animal Husbandry and Veterinary Medicine,2013,40(9):105-108(in Chinese with English abstract)]
[15]
Tang Q Y,Zhang Z,Meng X Y,et al. Structural determinants of PIP2 regulation of BK channel activity through the RCK1 Ca2+ coordination site[J]. J Biol Chem,2014,289:18860-18872
[16]
刘永锋,孔文娟,王伟. BK通道功能性复合体的研究进展[J]. 现代生物医学进展,2014,14(9):1759-1762 [Liu Y F,Kong W J,Wang W. Functional complexes of BK channels[J]. Progress in Modern Biomedicine,2014,14(9):1759-1762(in Chinese with English abstract)]
[17]
Simon T,Steg P G,Gilard M,et al. Clinical events as a function of proton pump inhibitor use,clopidogrel use,and Cytochrome P450 2C19 genotype in a large nationwide cohort of acute myocardial infarction[J]. Am Heart Assoc,2011,123(5):474-482
