A gene controlling rice leaf senescence and its encoded protein
A leaf senescence and gene technology, applied in genetic engineering, plant genetic improvement, introduction of foreign genetic material using vectors, etc., can solve the problem of not finding the genes of rice leaf senescence and plant type.
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Embodiment 1
[0035] Embodiment 1: Mutant isolation and genetic analysis test
[0036] (1) Test materials
[0037] The progeria mutant yld1, Yangdao 6 and Shuhui 527 were all obtained from the Laboratory of Heterosis Utilization, Rice Research Institute, Sichuan Agricultural University.
[0038] (2) Test method
[0039] Mutant yld1, Yangdao 6 and Shuhui 527 were all planted in the Wenjiang Experimental Field of the Rice Research Institute of Sichuan Agricultural University, and F 2 Groups, Statistics F 1 and F 2 group separation ratio.
[0040] Phenotype observation results: Compared with the wild type, the leaves of the mutant yld1 appeared yellowing phenotype at the beginning of the tillering stage (see figure 1 ), and the tips of all leaves turned yellow earlier than wild-type Shuhui 527 at the heading stage (see figure 2 ).
[0041] Separation test results: F 1 , F 1 All generation plants had normal leaf phenotype and growth period, F 2 The segregation ratio of the wild-type ...
Embodiment 2
[0045] Embodiment 2: the localization analysis test of mutant yld1 candidate gene
[0046] (1) Test materials and methods
[0047] 1. Construction of near isogenic pool
[0048] F obtained by crossing mutant yld1 with Yangdao 6 2 In the population, randomly select 30 leaves of a single plant with premature senescence phenotype and 10 leaves of a normal single plant, and mix and extract DNA from every 10 leaves in equal amounts to build a pool, including 3 recessive pools and 1 dominant pool A total of 4 near-isogenic pools were used for initial gene mapping. Leaf DNA was extracted using the improved CTAB method.
[0049] 2. Primer synthesis and gene mapping
[0050] First, nearly 300 pairs of SSR primers distributed evenly on the 12 chromosomes of rice were used (see the rice genome website for details http: / / www.gramene.org / bd / markers ), through PCR amplification, 144 polymorphic primers between the mutant yld1 and the genome of Yangdao No. F constructed by Yangdao 6 ...
Embodiment 3
[0073] Embodiment 3: the cloning and sequencing verification of candidate gene
[0074] (1) Experimental materials and methods
[0075] 1. RNA extraction The RNA extraction step was performed according to the instructions of the Trizol reagent.
[0076] 2. Synthesis of the first strand of cDNA: according to TOYOBO ReverTra Ace-a TM Instructions to proceed:
[0077] 3. Gene fragment amplification
[0078] Using Primer 5.0 software, for the candidate gene LOC_Os06g29380 predicted by the MutMap method, according to the reference cDNA sequence on the Rice Genome Annotation Project (http: / / rice.plantbiology.msu.edu / ) website, respectively design primers (see Table 4), Taking the cDNA of Shuhui 527 and mutant yld1 obtained by reverse transcription in the second step as templates, PCR reaction was carried out with P980 primers to obtain PCR amplification products (see Figure 7 ), wherein the reaction conditions are as follows: PCR reaction system for gene amplification (50 μL): ...
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