36results about How to "Solve the current situation of no internal reference genes" patented technology

The invention provides primers for screening internal reference genes under low temperature stress in lotus mist and applications thereof. The primers are: forward primer 5'-CCTGGCCTCTATGATCTTATCC-3', reverse primer 5'-CAGGCATCATGTGCTTTG-3'. The present invention uses qRT-PCR to detect the expression levels of 7 candidate internal reference genes ACT-7, GAPDH, UBQ, α-TUB, CYP20-1, EF-2 and APRT-5 under the development of lotus mist fruit and low temperature stress, through geNorm , NormFinder, BestKeeper and RefFinder software for gene stability evaluation, the results show that the expression of the most stable internal reference gene CYP20-1 under low temperature stress, this not only solves the current situation that there is no internal reference gene in the existing lotus mist quantitative PCR detection, but also provides a basis for the follow-up Laying the groundwork for gene expression work.

The invention provides a passion flower reference gene as well as a screening method and an application thereof, which belongs to the field of molecular biology. The RNA-Seq sequencing data is used for analyzing stable genes with the medium expression level to serve as the candidate reference genes; five genes with medium expression quantity (FPKM is approximately equal to 25) are selected, primerdesigning is carried out, verifying is carried out in mRNA of different tissue parts of passion flower varieties with obvious phenotypic difference, the expression stability of each candidate reference gene is estimated by utilizing software, and finally the proper passion flower reference gene C27182 is screened out. The C27182 can be stably expressed in stems, leaves, flowers and fruits of Tainung No.1 and golden fruit No.2. The reference is provided for subsequent scientific research related to passion flower gene expression.

The invention discloses a fluorescent quantitative internal reference gene and its special primers and applications under drought stress in Changshan, Haizhou. The fluorescent quantitative internal reference genes are Actin, AP‑2 and RAN genes, wherein the gene sequence of the Actin gene is as shown in SEQ ID NO.1 The gene sequence of the AP‑2 gene is shown in SEQ ID NO.13, and the gene sequence of the RAN gene is shown in SEQ ID NO.6. The present invention screens candidate genes with relatively stable expression from transcriptome data and internal reference genes studied by previous studies, and evaluates their stability through software, and discloses three internal reference genes suitable for the most stable expression under drought stress in Changshan, Haizhou , and based on these genes, real-time fluorescent quantitative PCR primers were designed to fill the status quo of no internal reference genes under drought stress in Changshan, Haizhou, and provide strong support for the accurate quantification of related functional genes under drought stress in Changshan, Haizhou, which can improve the stability of research repeatability, repeatability and reliability.

The present invention discloses fluorescent quantitative internal reference genes of different tissues in Changshan, Haizhou, primers and applications thereof. The internal reference genes are Actin, RPL, RAN, AP‑2 and HSP70 genes, wherein the gene sequence of the Actin gene is as shown in SEQ ID NO.1 As shown, the gene sequence of the RPL gene is shown in SEQ ID NO.3, the gene sequence of the RAN gene is shown in SEQ ID NO.6, the gene sequence of the AP-2 gene is shown in SEQ ID NO.13, and the gene sequence of the HSP70 gene is shown in SEQ ID NO.13. The gene sequence is shown in SEQ ID NO.14. The present invention designs an internal reference gene combination suitable for different tissues in Changshan, Haizhou, reveals the sequence of the internal reference gene, and designs real-time fluorescent quantitative primers accordingly, which not only solves the current situation that there is no internal reference gene in the existing detection of Changshan, Haizhou, but also The designed real-time fluorescent quantitative primers have strong primer specificity when used for gene expression analysis in Haizhou Changshan tissues, which can greatly improve the detection efficiency of real-time fluorescent quantitative detection of Haizhou Changshan genes, and improve the credibility of the detection results Spend.

The invention discloses a fluorescent quantitative internal reference gene and its special primers and applications under waterlogging stress in Changshan, Haizhou. The fluorescent quantitative internal reference genes are MDH, UBC‑E2 and UBQ, wherein the gene sequence of the MDH gene is as shown in SEQ ID NO.10 The gene sequence of the UBC-E2 gene is shown in SEQ ID NO.12, and the gene sequence of the UBQ gene is shown in SEQ ID NO.16. The present invention screens candidate genes with relatively stable expression based on the existing published genes, evaluates their stability through software, and obtains three internal reference genes suitable for the most stable expression under the waterlogging stress in Changshan, Haizhou, and uses these genes as a basis to design The real-time fluorescent quantitative PCR primers have filled the current situation that there is no internal reference gene under the waterlogging stress of Changshan in Haizhou, and provided strong support for the accurate quantification of related functional genes under the waterlogging stress of Changshan in Haizhou, which can improve the stability and repeatability of the research and reliability.

The invention discloses internal reference genes suitable for the expression research of lotus flood stress genes. The internal reference genes include: lotus actin gene and ribosomal protein 18sRNA gene. The present invention discloses two internal reference genes for studying the expression of water-flooding stress genes in lotus, and discloses real-time fluorescent quantitative PCR primers designed based on these two genes, solving the problem that there is no internal reference gene in the study of water-flooding stress gene expression in lotus The current situation of the current situation, the internal reference gene of the present invention can be stably expressed when the lotus responds to flooding stress, and the designed real-time fluorescent quantitative PCR primers are used for gene expression analysis when the lotus responds to flooding stress, which can improve the stability, reliability and reliability of the research. repeatability.