Gene for controlling early rice chloroplast development and application thereof
A chloroplast and rice technology, applied in the field of basic research, achieves the effects of simple operation, improved purity, and low cost
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Embodiment 1
[0017] Example 1 Rice DNA Extraction
[0018] 1. Operate according to the molecular cloning experiment manual, weigh 0.5g of fresh seedling rice leaves (wild type "Jiahua No. 1"), cut the leaves into 0.5cm long pieces, put them in a pre-cooled mortar, Add liquid nitrogen and grind until powdery, then transfer to a 1.5ml centrifuge tube, add 1ml 60°C preheated 2×CTAB, shake gently, then bathe in 60°C water bath for 45min, shake twice during the period.
[0019] 2. Take out the centrifuge tube, cool it at room temperature, centrifuge at 12,000 rpm for 5 minutes, absorb the supernatant, add an equal volume of chloroform-isoamyl alcohol (24:1) mixture, and mix well.
[0020] 3. Balance the centrifuge tube containing the solution, centrifuge at 12,000 rpm for 5 minutes, absorb the supernatant, add 2 / 3 volume of isopropanol, and mix gently to precipitate the nucleic acid.
[0021] 4. Centrifuge at 12,000 rpm for 5 min to precipitate the nucleic acid, discard the supernatant, wash w...
Embodiment 2
[0023] Embodiment 2PCR amplification and gene detection
[0024] (1) Acquisition of genes controlling early rice chloroplast development
[0025] 1. The 25μL PCR reaction system is as follows: 100mM Tris-HCl pH9.0; 100mM KCl; 20mM MgSO 4 ; 80mM (NH 4 ) 2 SO 4 ; 2.5 mM dNTP; 10 μM primer, 5 U / μl Taq enzyme, 1 μl template DNA / 10 μl.
[0026] The primer sequences are:
[0027] Upstream primer 5'-GGGGTACCCACAATGCATCTCGATTCTTA-3' (the endonuclease used is KpnI)
[0028] Downstream primer 5'-GCTCTAGAGCAGAATGGGGGGATATCTCA-3' (the endonuclease used is XbaI)
[0029] 3. The PCR amplification reaction is carried out on a PCR amplification instrument.
[0030] The temperature and reaction conditions were as follows: pre-denaturation at 94°C for 4min, denaturation at 94°C for 30sec, annealing at 50°C for 30sec, extension at 72°C for 60sec, 35 cycles; final extension at 72°C for 6min.
[0031] The length of the PCR amplification product is 3.7kbp, and the sequencing result is shown...
Embodiment 3
[0048] Example 3 Gene function verification
[0049] The gene sequence of SEQ ID No.1 in rice is destroyed by mutagenesis by gamma rays or other physical methods. After the base deletion in the gene is sequenced, the RNA level decreases, and the early rice plants turn yellow. The result of the functional complementation experiment of this gene showed that the leaves of the offspring plants of the mutant containing the normal gene were green.
[0050] The rice leaf color mutant material was obtained from the japonica rice "Jiahua 1" 60 Co γ-ray irradiation is a yellowish mutant, which has become a variety of stable agronomic traits after many years of self-crossing and selection in Hainan and Shanghai.
[0051] 1. Using SSR and InDel molecular markers to locate the mutant gene, through sequencing and semi-quantitative RT-PCR technology, it was shown that the gene sequence was mutated and its RNA level expression decreased.
[0052] 2. The nucleotide sequence of SEQ ID NO.1 was...
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