组培条件下苹果实时定量PCR内参基因的筛选

Selection of reference genes for Real-Time Quantitative PCR in apples(Malus domestica) in vitro

【目的】筛选在组织培养条件下苹果苗中稳定表达的内参基因。【方法】应用实时荧光定量PCR技术,分析了18S r RNA,GAPDH,TUB,UBQ,ACT,EF-1α六个常用植物内参基因在苹果组培苗不同基因型、不同组织、不同继代时间的m RNA表达差异情况。供试的4个苹果基因型为‘长枝富士’‘短枝富士’‘红珍珠海棠’、砧木‘A8-11’;继代培养的不同组织为茎、叶、整株,生根培养的不同组织为根、茎、叶、整株;不同继代期间为10、20、30、40、50、60、70、80 d。【结果】经Ge Norm软件对6个内参基因的表达稳定性进行评价,ACT和EF-1α在苹果组培苗的不同组织和不同继代时间的基因表达分析中较稳定,UBQ和EF-1α在苹果组培苗不同品种中表达均稳定。【结论】组培条件下,苹果基因表达相关研究的理想内参基因为ACT、UBQ和EF-1α。

【Objective】Quantitative real- time reverse transcriptase polymerase chain reactions（q RTPCR） have become a widely used technique for rapid and reliable quantification of gene expression studies. The stability of the selected reference genes is very important for the accuracy of the q RT-PCR. However the transcription levels of the target genes may be misinterpreted due to the various expressions of the reference gene which was thought to express stably. Meanwhile, only several reference genes for the q RT-PCR analysis were available from the field apple trees. Up to now, there are no related studies about reference genes which adapt to apple shoots in vitro, while the technique of tissue culture and rapid propagation plays a very important role in cultivating virus-free apple seedlings. In order to identify stable and reliable expressed genes of apple shoots in vitro, six traditional and novel candidate reference genes（18S r RNA, GAPDH, TUB, UBQ, ACT, EF-1α） were evaluated for transcript normalization.【Methods】The expression stability of candidate reference genes was analyzed in the following experimental conditions：（1）samples from different cultivars including‘Tianhong 1’‘Tianhong 2’‘Red pearl crabapple’‘A8-11’;（2） samples from different tissues of apple subculture plantlets including stem, leaf and whole plant;（3）samples from different tissues of apple rooted plantlets including root, stem, leaf and whole plants;（4） samples from apple shoots in different cultivation phases including 10, 20, 30, 40, 50, 60, 70, and 80 d after inoculation. Total RNA was isolated from 100 mg apple tissue culture seedling samples using the RNAprep Pure Plant Kit（TIANGEN） according to the manufacturer’s protocol. The total RNA concentration260280ples ranged from 2.0~2.1 and it reflected pure and protein-free RNA. The RNA integrity was assessed using 1.2% agarose gel electrophoresis. Only the RNA whose mean A260/A280 ratio was between 2.0~2.1 and its integrity was perfect was used, all RNA samples were adjusted to the same concentration to homogenize the RNA input in the subsequent reverse-transcription reaction. The first strand c DNA was synthesized using the Hi Fi Script Quick g DNA Removal c DNA Kit according to the manufacturer’s instructions in a final volume of 20 μL. The q RT-PCR reactions were performed in 96-well plates with a Bio-Rad real-time PCR system, the reaction contained 12.5 μL of SYBR Premix Ex TaqⅡ, 8.5 μL of RNase-free Water, 2 μL of c DNA template and 1 μL（10 μmol·L- 1） of each primer to a final volume of 25 μL. The q RT-PCR conditions were as follows： 95 ℃ for 30 s, followed by 40 cycles of 15 s at 95 ℃, 20 s at 60 ℃,and 20 s at 72 ℃. After the cycles, the melting curves were generated to verify the specificity of primers.The amplification efficiency of the primers was estimated using the PCR program. The expression levels of the candidate reference genes were determined by Ct values, and relative expression levels were imported into ge Norm to analyze the gene expression stability.【Results】Agarose gel electrophoresis of RNA samples exhibited obvious bands of 28 S, 18 S and 5.8S r RNA. Ultraviolet spectrophotometer detection showed that the A260/A280 and A260/A230 values of the RNA samples were between 2.0 and 2.1, indicating high quality RNA for c DNA synthesis. The amplification efficiency（E） and correlation coefficient（R2） of the six references genes were generated using the slops of the standard curves obtained by serial dilutions, the R2 values varied from 0.997 to 1.000, which indicated that the primer pairs were suitable for q RT-PCR tests.The Ct values of the six candidate reference genes ranged from 8.37 to 23.74. 18 S r RNA showed the lowest median Ct values, which indicated that it had the highest expression abundance; the Ct values of EF-1α and GAPDH were higher than 18 S r RNA, so they showed more lower expression levels; the genes of ACT and UBQ presented more lower expression levels than the EF-1α gene; finally, the ampification of the TUB produced the highest Ct values, corresponding to the lowest expression levels.【Conclusion】The results suggest that different suitable reference genes should be selected case by case, and the most stable reference genes for apple culture tissue seedlings were different under various experiment conditions. According to the evaluation on the stability of six reference genes using the ge Norm procedure, the ranking of expression stability in different genotypes was UBQ（0.117） 〉 EF- 1α（0.256） 〉 ACT（0.386） 〉 GAPDH（0.503） 〉 TUB（0.955） 〉 18 S r RNA（1.444）, the ranking in different tissues was ACT（0.087） 〉 EF- 1α（0.249） 〉 18 Sr RNA（0.386） 〉 UBQ（0.989） 〉 TUB（1.147） 〉 GAPDH（1.445）, and in different cultivation days was ACT（0.267） 〉 UBQ（0.347） 〉 EF-1α（0.387） 〉 GAPDH（0.585） 〉 18 S r RNA（0.891） 〉 TUB（1.484）.In short, ACT and EF-1α presented stable expressions in different tissues and cultivation days. UBQ and EF-1α both gave steady expression in different genotypes of apple shoots.