Application of ataf2 protein and its related biomaterials in the regulation of plant resistance to powdery mildew of Hevea officinalis
A technology of rubber tree powdery mildew and ATAF2, applied in the biological field, can solve problems such as loss of natural rubber yield
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Embodiment 1
[0080] Embodiment 1, ATAF2 gene is induced to up-regulate expression by Hevea powdery mildew
[0081] 1. The discovery that the expression of ATAF2 gene is up-regulated by the powdery mildew of rubber tree
[0082] Oidium heveae HN1106 was inoculated on wild-type Arabidopsis Col-0, and RNA-seq data analysis was performed 4 days after inoculation.
[0083] The results of RNA-seq data analysis showed that: 4 days after inoculation of Oidium heveae HN1106 on wild-type Arabidopsis Col-0, the expression of Arabidopsis NAC family transcription factor ATAF2 was up-regulated by 6 times.
[0084] 2. Verification that the expression of ATAF2 gene is induced by powdery mildew of rubber tree
[0085] 1. Inoculate Oidium heveae HN1106 to wild-type Arabidopsis Col-0 cultured for about 5 weeks.
[0086] 2. Clipping the leaves of wild-type Arabidopsis Col-0 on day 0, day 2, day 4 and day 8 respectively after inoculation.
[0087] 3. Extracting total RNA from leaves of wild-type Arabidopsis...
Embodiment 2
[0095] Embodiment 2, the application of ATAF2 in positively regulating the resistance of Arabidopsis to powdery mildew of rubber tree
[0096] 1. Acquisition of Mutants
[0097] Ataf2 T-DNA insertion homozygous mutants ataf2-1 and ataf2-2 were purchased from ABRC Seed Center.
[0098] Compared with the wild-type Arabidopsis Col-0, only the ATAF2 gene in the mutant ataf2-1 had a T-DNA insertion mutation, and the insertion position was between the 212th and 213th positions of the second exon of the ATAF2 gene between.
[0099] Compared with the wild-type Arabidopsis Col-0, only the ATAF2 gene in the mutant ataf2-2 had a T-DNA insertion mutation, and the insertion position was between the 42nd and 43rd positions of the second intron of the ATAF2 gene between.
[0100] 2. Obtaining of ataf2 complementary Arabidopsis plants
[0101] 1. Construction of recombinant expression vector
[0102] Insert the DNA molecule (1.5Kb promoter region+ATAF2 genomic DNA molecule) shown in sequ...
Embodiment 3
[0113] Example 3, ATAF2 interacts directly with EDS1
[0114] 1. Co-immunoprecipitation assay to verify the interaction between ATAF2 and EDS1
[0115] Construct the ATAF2 protoplast expression vector with FLAG tag (connect the ATAF2 gene sequence into the PUC18 vector driven by the 35S promoter (Beijing Benowei Biotechnology Co., Ltd.), the vector obtained between the Xho I and BstB I restriction sites) and the EDS1 protoplast expression vector with the HA tag (the vector obtained between the Kpn I and Sal I restriction sites of the PUC18 vector driven by the 35S promoter (Beijing Benowei Biotechnology Co., Ltd.) with the EDS1 gene sequence) , after the plasmid was extracted by cesium chloride density gradient centrifugation, Arabidopsis protoplasts were co-transformed, and XopD protein of Xanthomonas spp. was used as a control for co-immunoprecipitation experiments. Specific steps are as follows:
[0116] XopD-FLAG, ATAF2-FLAG and EDS1-HA plasmids were extracted by cesium ...
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