A rice endosperm-specific expression promoter pospyl8
A promoter and promoter region technology, applied in the fields of plant molecular biology and plant genetic engineering, can solve the problem that there are few endosperm-specific high-efficiency expression promoters, it is difficult to fully meet the needs of plant genetic engineering applications, and the number of promoters is limited, etc. question
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Embodiment 1
[0015] Example 1 Rice expression profile database retrieval OsPYL8 gene expression pattern
[0016] plant PYLs The gene family is a class of abscisic acid (abscisic acid, ABA) receptor protein genes, which encode proteins that bind to ABA and mediate the ABA signaling pathway. Different plant PYLs genes can be expressed constitutively or specifically in different tissue parts of plants. In rice, OsPYLs There are 12 homologous genes in the gene family. By analyzing the rice expression profile database (http: / / ricexpro.dna.affrc.go.jp / ), it was found that, OsPYL8 (genbank accession number: Os06g0527800) is highly expressed only in rice endosperm, but basically not expressed in other tissues and organs.
Embodiment 2
[0017] Example 2 Fluorescent quantitative PCR (Real-time PCR) analysis OsPYL8 Expression patterns in different tissue parts of rice
[0018] Trizol (Invitrogen Company) was used to extract total RNA from different tissue parts such as roots, stems, leaves, spikelets, and seeds of rice variety Nipponbare, and the experimental steps were referred to Invitrogen Company’s Trizol instruction manual; after the Trizol method was used to obtain total RNA, DNaseI ( TAKARA Company) digested the extracted total RNA at 37 °C to fully degrade the possible genomic DNA in the sample; subsequently, the total RNA was further extracted and purified with chloroform / isoamyl alcohol (24:1).
[0019] Take the total RNA (~1 μg) extracted from different tissue parts, and use Thermo#K1622 kit (Thermo Company) to reverse-transcribe into single-stranded cDNA (for specific steps, refer to the instructions of the Thermo#K1622 kit); use the obtained cDNA as a template ,right OsPYL8 The expression patte...
Embodiment 3
[0020] Example 3 Rice OsPYL8 promoter cloning
[0021] Based on the genbank public database OsPYL8 For the upstream promoter region sequence of the gene, specific primers pOsPYL8-F: 5′-GTTTCATGTTAGGTTTGTCCGC-3′, pOsPYL8-R: 5′-CTCCGGTACACAGTAGCAGCAG-3′ were designed and synthesized, and the genomic DNA of the rice variety Nipponbare was used as a template for PCR Amplify. The PCR reaction system is 50 μL: TaKaRa LA Taq enzyme (5 U / μL) 0.5 μL, 2×LA PCR Buffer II 5 μL (containing MgCl 2 ), dNTP (2.5 mM) 8 μL, primer F / R (10 μM) 1 μL each, DNA 2 μL, ddH 2 Make up O to 50 μL, and the amplification program is: 94°C, 5min; 94°C for 30min, 60°C for 30S, 72°C for 2.5min, a total of 30 cycles; finally, 72°C for 10min. PCR amplification obtained a 2055 bp band. Further, the amplified band was recovered from the agarose gel with a DNA recovery kit (DP209, Tiangen Company), and sequenced to obtain a DNA fragment whose sequence is shown in SEQ ID NO.1, which is OsPYL8 The 2055 bp s...
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