Protein for adjusting green turning process of plant leave as well as coding gene and application thereof
A plant leaf and plant technology, applied in the direction of plant gene improvement, application, plant peptides, etc., can solve a few problems such as
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[0104] As an embodiment of the present invention, the gene encoding the ClpR4 protein is cloned into an appropriate vector by conventional methods, and the recombinant vector with the foreign gene is introduced into the plant cells that can express the ClpR4 protein , making the plant cell express ClpR4 protein. Plants overexpressing ClpR4 protein can be obtained by regenerating said plant cells into plants.
[0105] Preferably, a method for preparing transgenic plants is provided, comprising:
[0106] (1) transferring the gene encoding the exogenous ClpR4 protein into plant cells, tissues, organs or tissues, and obtaining plant cells, tissues, organs or seeds transformed into the gene encoding the ClpR4 protein; and
[0107] (2) Regenerating plant cells, tissues, organs or seeds obtained in step (1) into which the gene encoding the exogenous ClpR4 protein is transferred.
[0108] As a preferred example, the method comprises the steps of:
[0109] (s1) providing an Agrobact...
Embodiment 1
[0128] Embodiment 1, the identification of plant leaf turning green regulation gene
[0129] The inventors used 0.2% (V / V) ethyl methanesulfonate (EMS) to mutate Arabidopsis wild-type Col-0 ecotype (obtained from ABRC (Arabidopsis Biological Resource Center) in the United States), and screened through the M2 generation It was identified with the M3 generation, and a stable line (mutant) with gradually turning green leaf color was obtained. Compared with Col-0, the mutant's new leaves were yellow, while the old leaves turned green throughout the growth process, and its leaf margins were serrated, and the plant growth rate was relatively slow.
[0130] The F1 generation obtained by crossing the wild-type Col-0 as the male parent and the mutant as the female parent, or the F1 generation obtained by crossing the mutant as the male parent and the wild-type Col-0 as the female parent, the plants are the same as the wild type, without Leaf yellowing phenotype, indicating that the ge...
Embodiment 2
[0137] Example 2. Phenotype and Genetic Complementation Identification of Arabidopsis AtClpR4 Function Loss
[0138] 1. Identification of genetic complementation
[0139] In order to confirm that the phenotype of gradually turning green leaves is indeed related to the ClpR4 gene, the inventors carried out genetic complementation analysis. Through PCR amplification, its full-length cDNA was cloned, and a recombinant expression vector driven by cauliflower mosaic virus (CaMV) 35S promoter was constructed.
[0140] Using 5'-CACCATGGAGGTAGCAGCAGCGACT-3' (SEQ ID NO: 5) and 5'-AGTTCTGACATTCAAATGAGTTGTGC-3' (SEQ ID NO: 6) as primers, PCR amplified from the cDNA library of Arabidopsis wild-type Col-0 ecotype The full-length cDNA of the ClpR4 gene was amplified, and the pENTR / SD / D-TOPO cloning vector (purchased from Invitrogen Company) was introduced into the pENTR / SD / D-TOPO cloning vector (purchased from Invitrogen Company) using the Getaway cloning system. PGWB2 (Invitrogen), a rec...
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