Application of osmads47 gene in regulating rice grain shape
A technology of transgenic rice and rice, which is applied in the field of genetic engineering and genetic breeding, can solve the problem of unclear specific mechanism of Arabidopsis grain size, and achieve the effects of accelerating the breeding process, increasing rice yield, and increasing grain length
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Embodiment 1
[0031] Example 1 Construction and Genetic Transformation of OsMADS47 Gene Plant Expression Vector
[0032] Use TRIzol extract to extract total RNA from Nipponbare rice leaves. The specific operation steps are as follows: Take 100 mg of fresh plant tissue and grind it quickly and fully in liquid nitrogen. During the grinding process, keep liquid nitrogen in the mortar; transfer the ground sample into powder Add 1ml TRIZOL extract to a pre-cooled 1.5ml RNase free centrifuge tube and shake well; after standing at room temperature for 5 minutes, add 0.5ml chloroform, shake the centrifuge tube vigorously for 5 minutes, centrifuge at 12000rpm for 10min at 4°C, and collect the filtrate in Collection tube; the solution is divided into two layers, the lower layer is a phenol-chloroform light red liquid layer, transfer the upper layer liquid into a clean centrifuge tube, add 0.5ml isopropanol at 15-30°C, precipitate for 10min; at 4°C, 12000rpm Centrifuge for 10 min, RNA precipitates the...
Embodiment 2
[0044] Example 2 OsMADS47 Transgenic Rice Grain Length Increase
[0045] To investigate the function of OsMADS47, the grain phenotypes of transgenic and wild-type rice plants were analyzed. First, for the two obtained T 3 The transgenic homozygous lines OE-1 and OE-2 were identified by PCR, and the primers used were forward primer 5'-GATCCTACCCATACGATGTTCC-3' and reverse primer 5'-TCCAGTCCATCGATCTCATCC-3', one of which was located on the vector, and the other One primer is on the OsMADS47 gene ligated into the vector. The results show that each individual plant of the obtained transgenic homozygous lines OE-1 and OE-2 has a fragment of the target size, compared with it, there is no such fragment in the wild-type control ( figure 2 A). This shows that the target vector has been inserted into the genome of the transgenic homozygous lines OE-1 and OE-2, which are positive lines. The grain phenotypes of OE-1 and OE-2 homozygous transgenic lines were further analyzed, and it w...
Embodiment 3
[0046] Example 3 Analysis of OsMADS47 transcript level and protein level expression in transgenic rice
[0047] In order to further confirm that the increased grain length phenotype of the transgenic lines OE-1 and OE-2 was caused by the overexpression of OsMADS47, the transcription and protein levels of OsMADS47 in transgenic rice were further analyzed. Total RNA was extracted from leaves of wild-type Nipponbare and transgenic lines OE-1 and OE-2 using TRIzol extract, the DNA was digested with DNAase, and reverse-transcribed into cDNA as a template. Real-time PCR amplification was performed with the primers in Table 1, and the expression level of OsMADS47 gene in the transgenic lines was analyzed. The result is as image 3 As shown in A, in the transgenic lines OE-1 and OE-2, the relative expression of the OsMADS47 gene was significantly higher than that of the wild-type control, which was 112 times and 193 times that of the wild-type control, respectively. Since the N segm...
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