大白菜-结球甘蓝易位系实时荧光定量PCR内参基因的筛选

Reference Genes Selection for Quantitative Real-time PCR in Chinese Cabbage-Cabbage Translocation Lines

为了提高实时荧光定量PCR分析大白菜基因表达的准确性,选取添加结球甘蓝4号染色体片段的大白菜-结球甘蓝易位系营养生长阶段的幼苗期、莲座期、包心期、结球期的叶片和不同大小花蕾,及生长素(Indole-3-acetic acid,IAA)和生长素抑制剂(2,3,5-triiodobenzoic acid,TIBA)处理后的叶片为材料,运用ge Norm和Norm Finder这2种分析方法,对Apr、BcTIP41、U34559、EF1α、TUB4、CYP、DNAJ、HIS、TUA5、UKN1、SKIP16、CAC、ACTIN、ACTIN-1、ACTIN-2、GAPDH、UBC30、UBQ、PPR、PP2A、MDH共21个内参基因进行稳定性鉴定。结果表明,不同发育时期、不同组织部位的材料,以及在不同激素处理条件下,大白菜-结球甘蓝易位系qRT-PCR最适内参基因各不相同。在营养生长阶段(从幼苗到结球),内参基因UKN1和TUB4表达最为稳定。在大白菜-结球甘蓝易位系包心期利用生长素IAA处理后,最稳定的内参基因为BcTIP41和ACTIN;生长素抑制剂TIBA处理后,最稳定的内参基因为UKN1和BcTIP41。在花发育的6个等级大小花蕾中,发现DNAJ、ACTIN和PP2A最为稳定。研究结果为大白菜-结球甘蓝易位系基因表达量的精确分析奠定了基础,同时也为芸薹属其他植物不同发育时期及激素处理对内参基因的选择提供了参考。

In order to analyze the gene expression more accurately in Chinese cabbage by quantitative Real-time PCR,Chinese cabbage translocation lines added No.4 chromosome fragments from cabbage under Chinese cabbage background were used as materials in this experiment.The leaves of Chinese cabbage-cabbage translocation lines in different growth stages,including seedling stage,rosette stage,folding stage and heading stage,floral buds of different sizes and leaves of folding stage treated with auxin(Indole-3-acetic acid,IAA)and auxin inhibitor(2,3,5-triiodobenzoic acid,TIBA)were selected to evaluate the expressions stability of genes.Expression stability of twenty one candidate reference genes were detected by qRT-PCR,including Apr,BcTIP41,U34559,EF1α,TUB4,CYP,DNAJ,HIS,TUA5,UKN1,SKIP16,CAC,ACTIN,ACTIN-1,ACTIN-2,GAPDH,UBC30,UBQ,PPR,PP2A,MDH.geNorm and NormFinder were used to analyze above these results,which showed that the qRT-PCR most suitable reference genes of Chinese cabbage-cabbage translocation lines had some differences in different development periods,different tissue of the material,and under condition of different hormone treatment.The UKN1 and TUB4 genes were most stable expression in vegetative growth stage(from seedling to heading)of Chinese cabbage-cabbage translocation lines.In folding stage of Chinese cabbage-cabbage translocation lines,the most stable expression reference genes were BcTIP41 and ACTIN after treatment with auxin IAA,and the most stable expression reference genes were UKN1 and BcTIP41 after treatment with auxin inhibitor TIBA.DNAJ,ACTIN and PP2A genes showed the most stable expression in the floral buds with six size levels of Chinese cabbage-cabbage translocation lines.These results will lay the foundation for the accurate analysis of the gene expression of Chinese cabbage-cabbage translocation lines,and further provided a reference for the selection of the reference gene in other plants of Brassica at different developmental stages and hormone treatments.