Application of cds sequence of rice transcription factor os06g07010 gene
A technology of rice transcription factor and transcription factor, which is applied in the field of genetic engineering, can solve the problems of weakening advantages of hybrid breeding and achieve the effect of improving rice grain traits and grain shape
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Embodiment 1
[0035] Obtaining CDS Sequence of Os06g07010 Gene and Construction of Plant Expression Vector
[0036] Acquisition of CDS Sequence of 1Os06g07010 Gene
[0037] Find the Os06g07010 gene in the plant transcription factor database (http: / / rice.plantbiology.msu.edu / analyses_search_locus.shtml), design PCR amplification primers according to its sequence, forward primer F: 5′-CAAAAAAGCAGGCTTCATGGACAGTAGCCGAGAT-3′ and reverse Primer R: 5'-CAAGAAAGCTGGGTCTCCAGACATTCTTTGCACCA-3'. The complete CDS sequence of the Os06g07010 gene (as shown in SEQ ID No.1) was obtained by PCR using the total cDNA of wild-type Nipponbare 'kitaake' rice as a template, using primers F and R.
[0038] 2 Construction of plant expression vectors
[0039] The CDS sequence of the rice transcription factor Os06g07010 gene was constructed downstream of the four transcription factor repression motif EAR coding genes (as shown in SEQ ID No.4) through the Gateway system.
[0040] 2.1 Cloning the above PCR product in...
Embodiment 2
[0049] The acquisition of embodiment 2 transgenic rice plants
[0050] 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:EAR-Os06g07010 was transferred into the rice callus by the Agrobacterium-mediated method. The AAM culture solution of Agrobacterium was transformed, and the callus soaked in the transformation solution was placed on the co-culture medium for co-cultivation, cultured in the dark at 25°C for 3 days, then placed on the 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 callus that differentiated into green seedlings was tra...
Embodiment 3
[0056] Identification of embodiment 3 transgenic positive strains
[0057] In order to detect the overexpression of the ubi:EAR-Os06g07010 gene in the T2 generation transgenic rice (E1581H-07, E1581H-35) in the EAR-Os06g07010 transgenic rice line obtained in Example 2, in this example, the linker between the vector and the target gene Primers (forward primer: 5′-GTGGGGACAAGTTTGTACAAAAAAGCAGGCTTC-3′ and reverse primer: 5′-GTGGGGACCACTTTGTACAAGAAAGCTGGGTC-3′) were designed for PCR detection, and obvious specific bands were obtained. Depend on image 3 It can be seen that the size of the amplified band is above 2000bp, and the primers are designed at the junction between the vector and the target gene, and the size of the amplified fragment is basically the same as that of the target gene (2346bp). Therefore, it can be explained that the transgenic rice lines E1581H-07 and E1581H-35 obtained in Example 2 contain the EAR-Os06g07010 fusion gene.
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