Application of cds sequence of rice transcription factor os05g10670.1 gene
A rice transcription factor and transcription factor technology, applied in the field of genetic engineering, can solve the problems of weakening of hybrid breeding advantages
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Embodiment 1
[0033] Example 1 Isolation of Os05g10670.1 Gene CDS Sequence and Plant Expression Vector Construction
[0034] 1. Acquisition of CDS sequence of Os05g10670.1 gene
[0035]Find the Os05g10670.1 gene in the plant transcription factor database (http: / / rice.plantbiology.msu.edu / analyses_search_locus.shtml), design PCR amplification primers according to its CDS sequence, and design PCR amplification primers according to its sequence (F: 5′-CAAAAAAGCAGGCTTCATGGTTCGGAAGCGTCG-3′ and reverse primer R: 5′-CAAGAAAGCTGGGTCGTTGATGAGGTCGGATACCC-3′). The CDS sequence of the Os05g10670.1 gene was obtained by PCR using the total cDNA of wild-type Nipponbare rice as a template, and its nucleotide sequence is shown in SEQ ID No.1.
[0036] 2. Construction of plant expression vectors
[0037] The CDS sequence of the rice transcription factor Os05g10670.1 gene was constructed downstream of the 4 transcription factor activation motif VP16 coding gene (SEQ ID No.4) through the Gateway system.
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Embodiment 2
[0047] The acquisition of embodiment 2 transgenic rice plants
[0048] Take the mature seeds of rice 'kitaake', shell them manually or mechanically, select the plump, smooth and spot-free seeds and inoculate them on the induction medium for induction culture after being sterilized. Rice callus with good appearance and good growth was selected as the recipient material, and ubi: VP64-Os05g10670.1 was transferred into the rice callus by Agrobacterium-mediated method, with 100 μM acetosyringone and O.D. 0.7 AAM culture solution of Agrobacterium. For transformation, the callus soaked in the transformation solution was placed on a co-culture medium for co-cultivation, cultured in the dark at 25°C for 3 days, then placed on a screening medium for about 30 days, and subcultured every 10 days. Then, the screened out resistant calli were transferred to the differentiation medium for differentiation for about 20 days, and subcultured every 10 days. The resistant calli that differentia...
Embodiment 3
[0054] Identification of embodiment 3 transgenic positive strains
[0055] In order to detect the overexpression of the ubi:VP64-Os05g10670.1 gene in the VP64-Os05g10670.1 transgenic rice line obtained in Example 2 in the T2 transgenic rice (V1430H-04, V1430H-14), this example was Primers were designed on the vector (forward primer: 5'-GTGGGGACAAGTTTGTACAAAAAGCAGGCTTC-3' and reverse primer: 5'-GTGGGGACCACTTTGTACAAGAAAGCTGGGTC-3') for PCR detection, and obvious specific bands were obtained. Depend on image 3 It can be seen that the transgenic rice lines V1430H-04 and V1430H-14 obtained in Example 2 contain the VP64-Os05g10670.1 fusion gene. The above primers are designed at the junction of the vector and the target gene, image 3 It shows that the size of the amplified fragment is basically consistent with the target gene (1395bp)
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