Application of gene Loc_Os01g12810
A gene and gene silencing technology, applied in the field of genes, can solve the problem of little research on chloroplast development in rice
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Embodiment 1
[0032] The construction of embodiment 1 recombinant plasmid
[0033] This embodiment constructs the recombinant plasmid, and the specific steps are as follows:
[0034] The seedling leaf parts of rice variety Lijiang Xintuan Heigu (LTH) were taken, and the total RNA of the leaves was extracted with TriZol Reagent (Invitrogen Company, its article number: 15596026), and the purity of the total RNA was detected by agarose gel electrophoresis and ultraviolet spectrophotometer And the amount, take 1 μg of total RNA to do the initial reverse transcription reaction, the reverse transcriptase used is PrimeScript (TAKARA company), the steps of the reverse transcription reaction refer to the instruction of the reverse transcriptase. Using the reverse transcription product as a template, primer 1 was used for PCR amplification, and the forward and reverse nucleotide sequences of the primer 1 are respectively shown in SEQ ID NO: 4 and SEQ ID NO: 5 in the sequence listing. The polymerase ...
Embodiment 2
[0037] The genetic transformation method mediated by Agrobacterium EHA105 was used to transform the recombinant plasmid obtained in Example 1 into the seeds of normal japonica rice. Transformed primary (T 0 Generation) Through PCR and fluorescent quantitative PCR detection, the expression level of the target gene Loc_Os01g12810 was detected at the RNA level, and the result of the wild line was used as a control. Real-time quantitative PCR was used to identify the expression effect of the Loc_Os01g12810 gene in transgenic plants, and the steps were as follows:
[0038] Take the leaves of three-leaf stage seedlings of different transgenic lines that are positive in PCR detection and carry out total RNA extraction. The reagent used is TriZol Reagent (Invitrogen Company, its article number is: 15596026). Gel electrophoresis and UV spectrophotometer were used to detect the purity and quantity of total RNA, and 1 μg of total RNA was used for initial reverse transcription reaction. ...
Embodiment 3
[0040] Put the 8 T in embodiment 2 0 The seeds of the generation transgenic line and the seeds of the wild line ZH11 were placed in an incubator at 49°C for 72 hours to break the dormancy of the seeds, soaked in water for 24 hours at room temperature, placed at 32°C for 48 hours, and sowed on a plastic container filled with soil after the seeds germinated. In the dish (3 biological repetitions were designed), grown under the cover of plastic film, the transgenic plants interfered with by Loc_Os01g12810 showed albinism in the aerial parts (see image 3 ). Growing under normal conditions with the same temperature conditions, Loc_Os01g12810 interfering transgenic plants showed partial albinism of leaves, and when transferred to a normal growth environment, the degree of albinism would gradually weaken and gradually return to normal green (see Figure 4 ), when growing to the heading stage, the ears also lack chlorophyll, showing albinism, but compared with the wild line, it does...
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