Uncle Hu
Editor|Uncle Hu
preface
As an important bioinformatics analysis method, fluorescence PCR has the characteristics of rapidity, sensitivity, high selectivity and high throughput.
3-phosphate-dehydrogenase, actin, tubulin, transcriptional elongation factor, ubiquitin ligase, etc. are currently one of the most studied genes, and there are a large number of genes in melon, apple, cherry, pear, tea tree, soybean, potato and other crops.
Under ideal conditions, under different varieties, different tissues, different stages of development and other experimental conditions, the internal reference gene can be expressed relatively stably, but its expression amount is not fixed, so it is necessary to select the appropriate internal reference gene for specific conditions.
Gui yuan is a unique tropical and subtropical fruit, with high economic and pharmaceutical value, there are many literature on its quantitative analysis of q-PCR at home and abroad.
It is very important to find stable internal standard genes and use them as the basis for quantification, Lin et al. selected 10 candidate internal standard genes such as 18SRNA, UBQ, EF-1α.
Their expression in longan somatic embryos was analyzed, and finally UBQ and Fe-SOD were the most stable internal standard gene combinations at different temperatures and developmental stages.
Wu et al. performed transcriptome sequencing on 12 candidate reference genes and analyzed their expression in fruit peels and pseudoseed coats at six different developmental stages, and carried out research based on internal standard genes.
On the basis of the previous work, the pulp of three different growth periods of longan variety "pomegranate", medium and late maturing variety "fragrant crisp" and "Lidong" in different periods was compared.
Materials and methods
The test materials were taken from the national fruit germplasm Fuzhou Longan Garden, and the female flowers of Longan 'Shikip' cultivars were selected 100d, 110d, 115d and 120d after opening during the fruit expansion and ripening stages, respectively.
After the female flowers of 'crispy' and 'Lidongben' varieties are opened, the normal growth fruits of 100d, 110d, 120d and 130d are taken, the pulp is peeled and frozen with liquid nitrogen, and then frozen in ultra-low temperature freezers for later use.
Using the Trizol method, it was analyzed by 3% agar gel electrophoresis, using the GenStar reverse transcription kit StarScriptIIFirst-strandcDNASynthesisKitwithgDNARemover, which followed the steps in the instructions, after removing the genomic DNA in the RNA, it was reverse transcribed into cDNA and placed in the -20°C freezer for cryopreservation .
Based on the previous work, this project intends to select five possible internal reference genes (EF-1α-1, EF-1α-2, Actin1, Actin2, GAPDH2) through resequencing and transcriptome analysis of the whole genome of longan and the synthesis of relevant literature at home and abroad.
In addition, the functional identification of IPMS2 and SS7 will be carried out, and the primers will be based on the PrimerPremier 5.0 of Suntech Biologics, and then the method will be validated using CFXConnectFluorescencePCR technology using the TB green composition as a template.
Prepare the reaction system 20 μl: 1.0 μl cDNA template, 0.5 μl forward and reverse primers, 10 μl 2x RealStarGreenFastMixtures, 8 μl ddH2O2.
This study uses a two-stage approach, i.e., 2 min of warm-up at 95 °C, 1 cycle; Deformation at 95 °C for 15 seconds, annealing stretching at 60 °C 40 times, deformation for 15-30 seconds, determination of its melting characteristics.
Using geNorm and NormFinder software, the stability value of the internal reference gene was compared, and the number of genes was screened, and then the expression stability of the internal reference gene was analyzed by using BestKeeper software, and finally ReFinder was used for comprehensive evaluation to select the optimal internal reference gene or combination.
Results & Analysis
The whole RNA was extracted by the Trizol method, and its content was determined, and OD260/OD280 and OD260/OD230 were found to be 1.9 and 2.1, respectively.
On the 3% agarose gel electrophoresis pattern, 28SRNA and 18SRNA2 bands were included, and the bands were very clear. Moreover, they have a high degree of integrity, basically no degradation, and the brightness is between 2:1 and 1:1, which meets the requirements for the purity and concentration of RNA in subsequent experiments.
The solubilization curves of the 5 pairs of primers had significant unimodal properties at various growth stages of the "pomegranate" fruit, and the assay results by agar gel electrophoresis showed that the amplification results of PCR were specific and consistent with the desired size.
The Ct value of the internal reference gene is the main indicator to measure the expression of a certain gene, the larger the Ct value, the lower the gene expression; The higher the opposite.
q-PCR results In various samples, the Ct value range of EF-1α-1 of the internal reference gene is 22~31, the Ct value range of EF-1α-2 is 23~31, the Ct value range of Actin1 is 24~33, the Ct value range of Actin2 is 22~30, and the Ct value range of GAPDH2 is 19~26.
This means that the five internal reference genes were expressed in all longan samples, but the expression of GAPDH2 was higher and the expression of Actin1 was low.
The geNorm software defaults to M=1.5 as the evaluation threshold, the larger the M value, the lower the stability, and vice versa.
The size of the M value of each gene was analyzed by geNorm software, and it was obtained that the stability of the five internal reference genes had certain differences in the fruit development process of different longan cultivars, but the expression of EF-1α-1 and EF-1α-2 was the most stable in the three cultivars.
Vn/(n+1) exceeds 0.15, so the number of suitable internal standard genes cannot be determined by the threshold (0.15) alone, and it needs to be combined with subsequent software results to screen them in depth.
NormFinder software generates stable values for gene expression, with lower values for gene expression to screen for internal reference genes.
NormFinder analysis showed that EFF-alpha-2 performed best during the growth of "white-flowered" fruits, followed by EFF-alpha-1, GAPDH2 performed best in "crisp", followed by EF-1α-2; The expression of EF-1α-1 was the most stable in 'Lidongben', followed by EF-1α-2.
BestKeeper is to evaluate the stability of internal standard genes by analyzing and comparing the average Ct value of the sample, and the smaller the standard deviation and coefficient of variation of the Ct value, the better the stability of the gene.
The results showed that the most stable gene expressed in 'Shikip' and 'Lidongben' was Actin1, while the most stable gene in 'Crisp' was EF-1α-1, which was different from the analysis results of geNorm and NormFinder software.
A comprehensive analysis of five internal reference genes was carried out using ReFinder software, and it was found that the stability of five internal reference genes at different growth stages of fruits of three longan cultivars was: EF-1alpha-1> EF-1alpha-2> Actin2> GAPDH2>Actin1.
The results of these three software were compared, and finally the stable internal reference genes EF-1alpha-1, EF-1alpha-2 and Actin2 were selected.
Through q-PCR technology, the IPMS2 and SS72 key enzymes related to malate synthesis were screened, and the expression of EF-1alpha-1 and Actin2 dual core enzymes in q-PCR was detected.
IPMS2 and SS7 had the same pattern of expression at three different periods, both falling first and then rising, and reaching their highest point after 120 days of female flowering.
Therefore, both EF-1alpha-1 and Actin2 are suitable for the quantitative study of their double internal reference genes.
Summary and discussion
Classical endo-reference proteins and Actin are the most critical molecules in the biosynthesis process, and they have high specificity and stability under both physiological and pathological conditions.
In Rhodiola grandiflora and Dendrobium, GAPDH was expressed stably in both plants, and was well expressed under adverse conditions. ACT, EFF-ALPHA, GAPDH and SAND are well expressed in fruit tissue.
In the selection of internal reference genes for lychee, Wei Yongzan et al. selected one of the most stable internal reference genes β-actin from six common internal reference genes in each stage of development of lychee fruit and under the action of external growth regulators.
Through the analysis of seven internal reference genes, we found that EF-1α and Actin could be overexpressed at each growth stage of lychee, while GAPDH and EF-1α could be overexpressed.
In the preliminary work, the applicant conducted preliminary experiments on EF1α, Actin1, Actin2, GAPDH2 and other internal reference genes in the above literature 17-19, and found that only Actin1 and GAPDH2 obtained band specificity was high, and the size was consistent with expectations, indicating that suitable internal reference genes needed to be screened during the experiment.
Transcriptomics techniques have played a large role in q-PCR analysis of many species, and there are some similarities between genes belonging to the same gene family in African oil palms.
For example, Xia et al. found a housekeeping gene (ACT) that exhibits relative stability under low temperature, drought, and high salt stress from several different transcriptomes.
Among them, ACT1 and ACT2 in the ACT family and EIF1 and EIF2 in the EIF family are relatively stable internal reference genes under low temperature, drought and high salinity stress.
Through the analysis of EF-1a and Actin2 genes, we found 6 EF-1a and Actin2 genes, and obtained two genes with little change in FPKM expression through q-PCR verification.
Therefore, in future research, we can mine more internal reference genes through high-throughput sequencing in multiple time and space.
Because Vn/n+1>0.15 cannot directly determine the number of suitable reference genes, it needs to be fully analyzed in conjunction with other software.
The results of the NormFinder software are similar to geNorm, but the results from BestKeeper show that EF-1alpha-1 and Actin1 are the most stable.
Jiang Tingting et al. came to similar conclusions when looking for the most suitable internal reference genes from different tissues and small bulb leaves at different growth stages.
Finally, the authors used ReFinder software to comprehensively evaluate it and found that the expression of EF-1α-1, EF-1α-2 and Actin2 was the most stable.
Actin2 is also stably expressed in the peel, pulp, flower buds and seeds.
Bibliography:
[1] Qi Xiangyu, Chen Shuangshuang, Feng Jing, Wang Huadi, Deng Yanming, "Screening and Verification of Jasmine Real-time PCR Internal Reference Genes"
[2] Lizhen Zhang, Xiaoyun Han, Jinghua Wu, GefuWANG-PRUSKI, ZHANG Zhizhong, "Screening of internal reference genes in real-time PCR analysis of melon"
[3] Lan Zhou, Liyi Zhang, Caixia Zhang, Guodong Kang, Yi Tian, Peihua Cong, "Screening of Internal Reference Genes in Real-time PCR Analysis of Apples"
[4] Youyin Zhu, Yue Wang, Hong Zhang, Jiao Shao, Yongqiang Li, Weidong Guo, "Screening and Identification of Chinese Cherry Real-time Quantitative PCR (qRT-PCR) Internal Reference Genes"