Phaseolus vulgaris alpha-pyrone reductase PvTKPR2 gene as well as encoding protein and application thereof
A technology encoding protein and pyrone is applied to the common bean PvTKPR2 gene and its encoded protein and application fields, and can solve the problems of bruised stigma, incomplete castration, hybridization failure, etc.
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Embodiment 1
[0044] Example 1: Phaseolus vulgaris male sterile mutant slp may be unisexual fruiting
[0045] 1. Utilization 60 Co radiation bean variety Dalong1, obtained a pool of Phaseolus vulgaris mutants, planted M3 generation in Harbin Bungalow (45 ° 70 ′ N, 126 ° 64 ′ E), from which mutant strains capable of producing a large number of succulent seedless pods were slp (Seedless Pod), such as Figure 1 as shown.
[0046] Second, the Dalong1 and SLP mutants were conducted in a positive and negative experiment, and the results showed that with the SLP mutant as the father and Dalong1 as the mother, it was impossible to obtain the pods that were developing and producing seeds; with Dalong1 as the father and the slp mutants as the mother, the pods with normal seed development could be obtained, such as Figure 1 The * logo is indicated. Therefore, the slp mutant is a male sterile mutant of Phaseolus vulgaris.
[0047] Third, observe the dalong1 and slp mutants completely open flowers, found t...
Embodiment 2
[0052] Example 2: Phaseolus vulgaris SLP gene localization
[0053] First, the use of Phaseolus vulgaris PI60234 as the parent and slp mutant as the mother, hybridization, 10 hybrid grains were obtained, 3 grains were planted, and 1346 F1 seeds were harvested after inbringing, of which the ratio of seed pod plants to succulent seedless pod mutants was 3:1, indicating that the phenotype of slp mutant silty seedless pods was controlled by a single gene recessively, as shown in Table 1.
[0054] Table 1 Separation ratios of F2 generation population phenotypes
[0055]
[0056] 2. The genomes of 22 seeded pods, 24 succulent seedless pods, Dalong1 and PI60234 in the F2 generation were extracted by CTAB method, and the genomes of 48 individuals were genotyped by BARCBean6K_3BeadChip. Removing unstable SNP signals, a total of 5398 SNP molecular markers were obtained, of which a total of 1545 SNP molecular markers were polymorphic between Dalong1 and PI60234. The 1545 molecular markers ...
Embodiment 3
[0074] Example 3: PvTKPR2 and PvTKPR2 slp Cloning of genes
[0075] 1. Using Dalong1 and its mutant SLP mutant as material, the total RNA of the flowers was extracted using the operation manual of the OmniPlant RNA Kit (CW25985) purchased from Conway Century.
[0076]2. Take 1 μg of total RNA for the synthesis of cDNA, and the synthesis of cDNA is carried out according to the manual purchased from The TransScript One-Step gDNA Removal and cDNA Sythesis SuperMix Kit (AT311-03) purchased from All-Inclusive Gold to obtain cDNA;
[0077] Third, the cDNA of the obtained Dalong1 and Slp mutants was used as a template to amplify PvTKPR2 and PvTKPR2 by forward primer F and reverse primer R, respectively slp Gene, PCR reaction conditions are as follows: 94 °C predetermination, 94 °C denaturation 30s, 58 °C annealing 30s, 72 °C extension 35s, a total of 38 cycles, and then 72 °C extension 10 min, pcR products are sequenced on the ABI3130 sequencer (ABI Corporation). The sequencing results ...
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