Cloning and application of maize kernel carotenoid deficient gene scd
A corn and grain technology, applied in the field of plant molecular genetics and plant genetic engineering, can solve the problems of cumbersome operation, high cost and high off-target rate
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Embodiment 1
[0079] Embodiment 1: Location and cloning of scd gene
[0080] 1. Construction of research groups
[0081] When the inventors propagated the Zong3×Yu87-1 recombinant self-cross, they found that a strain of the RIL105 family of the population had a natural mutation ( figure 1 ), the mutant is denoted as M 0 generation mutant, M 0 The grains obtained from the mutants are denoted as M 1 Generation, M 1 The color of some kernels in the generation kernels changed from yellow to light yellow, and the rest remained yellow. m 1 M 1 Progenitor maize plants had albino leaves and died two weeks after emergence. Further to M 1Seeds obtained by selfing the generation of yellow seeds (M 2 Substituting grains) for observation, it was found that part of the M 1 M of generation plant (homozygous) 2 The generation seeds are all yellow, some of them are M 1 M 2 The seeds of the generation were yellow and light yellow, and the yellow and light yellow seeds showed a 3:1 separation (y...
Embodiment 2
[0086] Embodiment 2, the allelism test of SCD gene mutant
[0087] In order to further verify that SCD is a gene controlling double albinism of maize kernels and leaves, three mutants—518D lw2, 512I lw2-vp12 and 523B lw2-UFMu were obtained from the Maize Genetics Cooperation Stock Center (MGCSC) in the United States. -07766, designated scd-1, scd-2 and scd-3 mutants, respectively.
[0088] Through sequence determination, it is found that the 12th exon of the GRMZM2G137409 gene in the scd-1 mutant undergoes a conversion from G to T, and the specific position is the 5809th position of sequence 2 (also the 1584th position of sequence 3 ), so that the amino acid is converted from glutamic acid to a stop codon, causing premature termination of translation; on the first exon of the GRMZM2G137409 gene of the scd-2 mutant (between the 2738th and 2739th positions of sequence 2, Also between the 220th and 221st positions of sequence 3) an 8bp insertion occurred, resulting in a frameshi...
Embodiment 3
[0091] The spatio-temporal expression analysis of embodiment 3, SCD
[0092] Using qRT-PCR to analyze the expression of SCD in different tissues of the wild type at different stages, it was found that SCD has the characteristics of constitutive expression during the development of maize. Transcript level analysis found that the expression of SCD was highest in leaves, followed by stems and roots. The transcript level expression of embryo, endosperm and grain in different development stages after pollination showed that SCD showed a fluctuating expression in three tissues: 15 days and 35 days after pollination There were two small peaks in 1 day expression, and the expression was relatively low at 20 days and 30 days. In the scd mutants with loss of SCD function, the expression levels were significantly down-regulated, but the pattern was similar to that of the wild type ( Figure 4 ).
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