Method for transforming medicago sativa by aid of galega orientalis gibberellin receptor genes GoGID
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A technology of gibberellin and receptor genes, applied in the field of molecular biology, can solve the problems of long breeding time and ineffective effects
Pending Publication Date: 2017-01-25
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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Conventional breeding methods take a long t
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Embodiment 1
[0086] Example 1 Preparation of GoGID, a gibberellin receptor gene of Galeus orientalis
[0087] According to the data information of Galena orientalis differential hybridization library, the RACE amplification primers GSP1 and GSP2 were designed, as shown in SEQ ID NO:1 and SEQ ID NO:2 in the sequence listing, using SMARTer TM RACE cDNA Amplification Kit (Clontech, USA) performed 5'RACE and 3'RACE cloning on the sequence respectively, and finally obtained the full-length sequence of the gene. The specific method is as follows:
[0088] 1. Extraction of total RNA from Algae orientalis:
[0089] The extraction of total RNA from Galega orientalis adopts the Trizol reagent method, and the specific steps are as follows:
[0090] (1) Add 0.3g Galega orientalis tissue material to 3000μl Trizol reagent, add liquid nitrogen to quickly grind it, and place it in a sterile environment until it becomes homogenized;
[0091] (2) Use a pipette gun to draw 1 mL of the homogenate into a 1...
Embodiment 2
[0129] Embodiment 2 Agrobacterium-mediated transformation of alfalfa plants with GoGID gene
[0130] 1 Materials and reagents
[0131] 1.1 Plant material
[0132] The tested alfalfa variety was Medicago sativa L.cv.Zhongmu No.1, Zhongmu No.1.
[0133] 1.2 Agrobacterium strains and plasmid vectors
[0134] The Agrobacterium strain used was Agrobacterium tumefaciens: LBA4404
[0135] Agrobacterium culture medium:
[0136] Table 1
[0137]
[0138]
[0139] 121°C high pressure steam sterilization for 20min; carrier: PBI121.
[0140] 2 Experimental methods
[0141] 2.1 insert the GoGID gene into the PBI121 vector plasmid DNA, the specific steps are: design the upstream primer of the XbaI restriction site and the downstream primer of the BamHI restriction site at the 5' end, such as SEQ ID NO: 5 and SEQ ID NO: 5 in the sequence listing IDNO:6 shown;
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Abstract
The invention discloses a method for transforming medicago sativa by the aid of galega orientalis gibberellin receptor genes GoGID. The method includes particular steps of inserting the genes GoGID into vector plasmid DNA (deoxyribonucleic acid); introducing vectors with the inserted genes GoGID into agrobacterium tumefaciens LBA4404; culturing the agrobacterium tumefaciens; preparing and transforming medicago sativa explants; verifying, propagating and evaluating medicago sativa transgenic plants. The method has the advantages that after the galega orientalis gibberellin receptor genes GoGID are overexpressed in the medicago sativa, the plant heights of transgenic medicago sativa can be increased by 0.2-0.35 time, biomass in the transgenic medicago sativa can be increased by 14.9-46.3%, and the leaf lengths and the leaf widths of the medicago sativa can be obviously larger than the leaf lengths and the leaf widths of wild type medicago sativa; in addition, the flowering time of the medicago sativa is delayed by a week as compared with the wild type medicago sativa, accordingly, theoretical basis can be provided for passing pasture gibberellin signal pathways, and possible candidate genes and theoretical foundations also can be provided for genetically improving yield traits of the medicago sativa.
Description
technical field [0001] The invention relates to the field of molecular biology, in particular to the application of the gibberellin receptor gene GoGID of Galeus orientalis. Background technique [0002] Gibberellins are a class of tetracyclic diterpenes, one of the five major plant hormones, and play a role in various important growth and development processes of plants. Including seed germination, leaf extension, stem elongation, flowering, and seed development. The gibberellin receptor is an important link in the gibberellin signal transduction pathway, which can bind active GA to form a GID-GA-DELLA complex, and through the ubiquitin E3 enzyme complex (SCF GID2 / SLY1 ) recognition, mediate DELLA protein to form polyubiquitin chains, and then be degraded by 26S proteasome, thus triggering GA response. [0003] Gibberellin Insensitive Dwarf1 (GID1) was first discovered in 2005 from rice gibberellin insensitive dwarf mutants. This gene encodes a soluble protein similar to...
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IPC IPC(8): C12N15/82A01H5/00
CPCC12N15/8205C12N15/8261
Inventor 王学敏李俊武语迪温红雨常鑫高洪文
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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