Application of rice small molecule RNA osa-miRNA530 in regulating rice leaf angle and leaf length
A technology of leaf length and small molecules, applied in the field of plant genetic engineering, can solve problems such as no functional research, and achieve the effect of promoting yield increase and planting density
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Embodiment 1
[0015] Embodiment 1: Cloning of osa-miR530 precursor gene DNA fragment
[0016] Total RNA was extracted from rice variety Nipponbare seedling leaves using TRIZOL reagent (Invitrogen). The specific steps are as follows: put 20 mg leaves into a liquid nitrogen pre-cooled mortar, add liquid nitrogen to quickly grind into powder, put the powder into a 1.5ml centrifuge tube, quickly add 1ml Trizol (Invitrogen) and invert to mix well, stand at room temperature Leave for 5 minutes. Centrifuge at 12,000 rpm for 10 minutes at 4°C, and transfer the supernatant to a new 1.5ml centrifuge tube. Add 200 μl of chloroform, shake vigorously by hand for 15 seconds, and let stand at room temperature for 2-3 minutes. Centrifuge at 12000 rpm for 15 minutes at 4°C. Take the colorless aqueous phase into a new 1.5ml centrifuge tube, add 250μl isopropanol, 250μl high salt solution, invert and mix well, and let stand at room temperature for 10 minutes. Centrifuge at 12,000 rpm for 10 minutes at 4°C...
Embodiment 2
[0018] Example 2: Construction and genetic transformation of osa-miR530 precursor gene plant expression vector
[0019] In order to better analyze the function of osa-miR530, the applicant increased the expression level of osa-miR530 in rice through overexpression technology. The function of the gene was studied according to the agronomic traits of the transgenic plants.
[0020]The construction method of the osa-miR530 precursor gene plant expression vector is as follows: first, the positive clone pMD18-osa-miR530 cDNA obtained in Example 1 was double-digested with BamHI and SpeI, and the insert fragment was recovered; and pCAMBIA1390- Plant expression vector for ubi, recovery of vector fragments. The recovered insert fragment and vector fragment were used for ligation reaction to transform Escherichia coli DH5ɑ. Positive clones were screened by enzyme digestion to obtain the plant expression vector, which was called pCAMBIA1390-ubi-Osa-miR530 (see figure 1 ). pCAMBIA1390...
Embodiment 3
[0073] Example 3: Detection of expression levels of osa-miR530 in transgenic rice plants and wild-type rice
[0074] Using wild-type Nipponbare rice and 5 independent T3 transgenic rice plants as materials, RNA was extracted from rice leaves at the four-leaf stage, and the transcript level of osa-miR530 in rice leaves was detected by RT-PCR. The specific method is as follows: use the Takara MiniBEST Universal RNA Extraction Kit (Takara) kit to extract total RNA from 100 mg of rice leaves at the four-leaf and one-heart stage, and remove genomic DNA contamination through the genomic DNA removal spin column included in the kit. The specific steps are as described in Example 1.
[0075] The first strand of cDNA corresponding to miRNA was synthesized using miRcute miRNA cDNA First Strand Synthesis Kit (TIANGEN). The first strand of cDNA was synthesized according to PrimeScriptTM RT Enzyme Mix I (TaKaRa) instructions. The specific steps are as follows: Add 2 μg of Total RNA, 0.4 μ...
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