miR159a related to barley powdery mildew resistance and application thereof
A powdery mildew and barley resistance technology, which is applied in the fields of application, microbial measurement/inspection, DNA/RNA fragments, etc., can solve problems affecting crop yield and quality, phytotoxicity, etc., to improve and improve germplasm resources, enhance resistance Sexuality, the effect of inhibiting accumulation
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Embodiment 1
[0032] Example 1 Analysis of the expression profile of miR159a during the infection of barley by Erysiphe hordei
[0033] (1) Preparation of plant material. After the seeds of barley variety P01 are sown, wait for about 1 week, cut off the flag leaves, and place them on a petri dish containing a medium (1% agar, 100mg / L bemimidazole), and recover for 24 hours. Physiological race K1 or A6 of powdery mildew of barley were inoculated respectively, and leaf materials were collected at different time points after inoculation.
[0034] (2) Extraction of plant total RNA. Grind 300mg of barley leaves quickly in liquid nitrogen to powder, add 1ml TRizol, shake well and centrifuge at 12,000rpm at 4°C for 10min; transfer the supernatant to a new centrifuge tube, add 1 / 5 TRizol volume of chloroform, and mix well Let it stand for 3 minutes to wait for its separation, and then centrifuge at 12,000g at 4°C for 15 minutes. Transfer the supernatant to a new centrifuge tube, first add 3M sod...
Embodiment 2
[0042] Example 2 Prediction and cloning of miR159a target gene GAMYB gene
[0043] (1) Prediction of miRNA target genes.
[0044] The prediction of miR159a target genes was done online on the psRNATarget website, and the parameters were set as default. (psRNATarget website address: http: / / plantgrn.noble.org / psRNATarget / ) found that the transcription factor GAMYB gene may be the target gene of miR159a (see figure 2 ).
[0045] (2) Cloning of GAMYB gene. 7-day-old seedlings of barley variety P01 were inoculated with Physiological Race K1 of Erysiphe hordei. After 24 hours, the leaf materials were collected, RNA was extracted and reverse transcription was performed to obtain cDNA. The reverse transcription system and process are as follows: RNA 2 μg, Oligo-dT 1 μl, add DEPC-H 2 From 0 to 12 μl, mix the above solution in a centrifuge tube, denature at 70°C for 10 minutes, then cool on ice for 2 minutes, then add 2 μl of M-MLVBuffer, 1 μl of dNTPmix, 0.5 μl of RNase inhibitor,...
Embodiment 3
[0051] Example 3 Verification of miR159a targeting GAMYB gene and negatively regulating its expression
[0052] (1) Cloning miR159a and its target gene GAMYB into expression vectors pKANNIBAL and CTAP, respectively.
[0053] The primers required for PCR amplification of cloned miR159a are as follows:
[0054] Forward: 5'-GTTCCTCGAGCCGTTCCATATCTTCTTAGCCCC-3'; (SEQ ID NO.7)
[0055]Reverse: 5'-CCTCAAGCTTTTGAGGGGAAAACAAAACACCG-3' (SEQ ID NO. 8).
[0056] The primers required for PCR amplification of the cloned GAMYB gene are as follows:
[0057] Forward: 5'-ATGTACCGGGTGAAGAGCGAG-3' (SEQ ID NO.5);
[0058] Reverse: 5'-TTTGAATTCCTCCGACATTTGAC-3' (SEQ ID NO. 6).
[0059] For the corresponding expression frameworks in vectors used to overexpress miR159a and GAMYB genes in tobacco, see image 3 .
[0060] (2) Transformation of Agrobacterium. Agrobacterium strain GV3101 was streak cultured on LB solid medium, and a single colony was picked two days later and cultured overnight i...
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