A kind of soybean Phytophthora root rot resistance gene and its application
A technology for Phytophthora sojae and root rot, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problem of low efficiency of variety screening, no gene of Phytophthora soja root rot found, and unfavorable screening of disease-resistant soybean varieties. And other issues
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Embodiment 1
[0035] 1 Reagents used in the experiment
[0036] 1) Reagents for extracting soybean RNA
[0037] TRIzol Reagent was purchased from Invitrogen Company; M-MLV reverse transcriptase, RNase-free DNaseI, RNasin, Taq enzyme, dNTP, Oligo (dT), and DTT were all purchased from TaKaRa Company; DEPC was purchased from Sigma Company; agarose for electrophoresis ( Agarose) is a reagent imported from Spain; QuantiFast SYBR Green PCR Kit was purchased from QIAGEN.
[0038] 2 Genomic RNA extraction method
[0039] Preparation:
[0040] (1) Preparation of DEPC (0.1%) water: add 1 mL of DEPC to 1000 mL of deionized water, and stir magnetically for 24 hours.
[0041] (2) Put the mortar, tweezers, beaker, measuring cylinder, glass rod, etc. into the oven, and dry heat sterilization at 180°C for more than 6 hours.
[0042] (3) Put the centrifuge tube, pipette tip, etc. into the reagent bottle that has been sterilized by dry heat, fully soak it with the prepared DEPC (0.1%) water, seal the bot...
Embodiment 2
[0080] Real-time quantitative analysis was performed on the control and treated RNA extracted from the different tissues of the resistant variety Suinong 10 and the susceptible variety Hefeng 25 at different times. The expression levels of all have changed (results such as Figure 1-3 shown). Among the resistant varieties: the reaction time of the stem is the longest, and the reaction of the leaf is the most sensitive; among the susceptible varieties: the reaction of the stem is the most sensitive. In the middle leaves of the disease-resistant variety Suinong 10, the expression peak was reached at 6 hours, and the expression level was 12 times, while in the susceptible variety Hefeng 25, there was only a slight up-regulation at 12 hours, and the expression level was not high, about 3 times, such as figure 1 ; In the resistant variety Suinong 10, the roots and stems responded slowly, reaching the peak at 12 hours, and the expression levels reached 14 times and 18 times respec...
Embodiment 3
[0082] Real-time quantitative analysis was performed on the control and treated RNA extracted from the different tissues of the resistant variety Jiusan H05-23 and the susceptible variety Baojiao 06-6046 at different times, and it was found that the GmDRR gene in the resistant variety soybean GmDRR gene in each tissue from 0h to 48h expression levels have changed. In the disease-resistant variety Jiusan H05-23, the response of leaves is the most sensitive, reaching the peak expression at 6 hours, and the expression level is 8 times. The reaction of roots and stems is slower than that of leaves, reaching the peak at 12 hours, and the expression level is 11 times and 7 times, the change trend was consistent with the change trend of the gene in Suinong 10 after being inoculated with Phytophthora soybean No. The amount is 14 times and 4 times respectively ( Figure 4 with Figure 5 ), the expression level in the roots was relatively low, which was slightly different from the cha...
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