矢车菊不同颜色花瓣酵母cDNA文库的构建  

Construction of a yeast cDNA library using Centaurea cyanus petals of different colors

作　　者：邓成燕 王佳颖 戴思兰[2] Deng Chengyan;Wang Jiaying;Dai Silan(College of Agriculture and Forestry Science,Linyi University,Linyi 276000,Shandong,China;School of Landscape Architecture,Beijing Forestry University,Beijing 100083,China)

机构地区：[1]临沂大学农林科学学院,山东临沂276000 [2]北京林业大学园林学院,北京100083

出　　处：《北京林业大学学报》2024年第3期115-122,共8页Journal of Beijing Forestry University

基　　金：国家自然科学基金项目(32101579);山东省自然科学基金项目(ZR2021QC143);临沂大学博士人才科研启动项目(LYDX2021BS046)。

摘　　要：【目的】矢车菊花瓣的蓝色呈色和品种间花色变异的分子调控机制尚不明晰。本研究采用Gateway技术构建了矢车菊6个不同花色品种花瓣的酵母cDNA文库,以期进一步通过酵母单杂交或双杂交技术筛选参与调控花瓣呈色的关键互作蛋白。【方法】本研究以白色、粉色、红色、蓝色、紫色和墨色矢车菊花瓣为材料,提取总RNA后分离和纯化mRNA,合成双链cDNA后依次进行BP重组反应和LR重组反应,分别获得初级和次级文库。最后将次级文库质粒转化酵母Y187,获得矢车菊不同颜色花瓣的酵母cDNA文库。【结果】质量鉴定结果显示:初级文库的库容量为1.3×10^(7) CFU,重组率为100%,且插入片段长度均在1000 bp以上;次级文库的库容量为1.6×10^(7) CFU,重组率为100%,且插入片段长度均在1000 bp以上。酵母文库的滴度为3.5×10^(7) CFU/mL,随机挑选的24个单克隆经PCR检测后均扩增出明亮条带,重组率为100%,插入片段长度均大于1000 bp。【结论】本研究构建的矢车菊不同颜色花瓣酵母cDNA文库的质量较高,能满足酵母文库筛选的试验要求,为后续探究矢车菊花瓣的蓝色呈色和品种间花色变异的分子调控机制提供了材料基础。[Objective]The molecular regulation mechanism of both blue petal coloration and petal color variation among cornflower cultivars remains unclear.In order to further screen the key interaction proteins involved in regulating petal coloration by Y1H or Y2H method in the near future,the yeast cDNA library from cornflower petals of six cultivars with different colors was established by Gateway technology in the present study.[Method]The white,pink,red,blue,mauve and black cornflower petals were used to extract total RNA,followed by mRNA isolation and purification.After the synthesis of double-strand cDNA,the BP recombination and LR recombination were performed to obtain the primary and secondary library,respectively.Finally,the plasmids from secondary library were transformed into yeast Y187 competent cells to build the yeast cDNA library from cornflower petals of different colors.[Result]The quality identification of both the primary and the secondary library revealed that the library capacities were 1.3×10^(7) CFU and 1.6×10^(7) CFU,respectively,the recombination rate was both 100%,and the average length of insert fragment was both more than 1000 bp.After transforming into yeast,the obtained cDNA library titer was 3.5×10^(7) CFU/mL.A total of 24 yeast clones were chosen randomly for PCR detection and showed bright bands,i.e.,the recombinant rate was 100%.Moreover,the length of inserted cDNAs was longer than 1000 bp.[Conclusion]A high quality of yeast cDNA library using cornflower petals of different colors is established,satisfying the standard of yeast library screen,which will provide material basis for research the molecular mechanism of both the blue petal coloration and the petal color variation among cultivars in the near future.

关 键 词：矢车菊 花色 酵母 CDNA文库 GATEWAY技术 

分 类 号：S681.9[农业科学—观赏园艺]