Ubiquitin ligase gene participating in symbiotic nodulation of leguminous plants, and application thereof

Abstract

The invention discloses a ubiquitin ligase gene participating in symbiotic nodulation of leguminous plants and application thereof. A membrane outer part of crowtoe symbiotic receptor kinase serves as bait; partial sequence of the gene is screened from a crowtoe AD-cDNA library; and full-length sequence is obtained through amplification by gene splicing and reverse transcription-polymerase chain reaction (RT-PCR) methods according to two expression sequence tags Lj-G0027263 and Lj-G0032470 overlapped with the partial sequence. Gene cloning and sequencing comparative analysis proves that the gene is cloned from crowtoe genome and cDNA. Sequence analysis and in vitro ubiquitin activity test prove that the gene is novel ubiquitin ligase. According to the application of the gene to the leguminous plants, the biological function of the gene in a symbiotic nodulation process is researched by RNAi and overexpression methods. The results show that, compared with the control plants, the plants with he gene has the advantages that the number of root nodules is increased obviously after overexpression, and the number of root nodules is reduced obviously after RNAi, so the gene has an application prospect in symbiotic nitrogen fixation of the leguminous plants.

Description

technical field

[0001] The invention belongs to the technical field of new plants, and more specifically relates to a ubiquitin ligase gene involved in nodulation of leguminous plants, and also relates to a preparation method of the ubiquitin ligase gene involved in nodulation of leguminous plants, and also relates to a Application of a ubiquitin ligase gene involved in legume nodulation in legumes. Background technique

[0002] Among various forms of protein post-translational modification, ubiquitination has attracted more and more attention in recent years. Similar to protein phosphorylation signal transduction, ubiquitination plays an important role in the process of cell life. important role. The 2004 Nobel Prize in Chemistry was awarded to scientists who made pioneering contributions to the study of ubiquitin-mediated protein degradation. Ubiquitin is a highly conserved polypeptide composed of 76 amino acid residues, named for its widespread distribution in various c...

Examples

Embodiment 1

[0045] A method for preparing a ubiquitin ligase gene involved in symbiotic nodulation of leguminous plants, the steps of which are:

[0046] A. Cloning of candidate gene LjSIE3:

[0047] Using the outer part of SymRK-EC of the symbiotic receptor kinase SymRK of japonicus japonicus japonicus and pGBKT7 (a well-known and public yeast protein expression vector) as the bait plasmid, the AD- cDNA library, the colony grown on the plate of SD / -Leu-Trp-Ade-His (auxotrophic yeast culture medium) passed the strict rotation verification, and the plasmid was extracted from the positive transformant and subjected to sequencing analysis to obtain The cDNA containing only 663bp of the C-terminal is the 598-1161 nucleotide sequence shown in SEQ NO:1. The specific implementation steps are:

[0048] (1) Preparation of yeast competent cells by LiAc method, yeast transformation, and library screening

[0049] (1) Streak the frozen yeast Y187 (purchased from clontech company) on the YPDA plate...

Embodiment 2

[0147] A kind of ubiquitin ligase gene that participates in leguminous plant symbiotic nodulation in the application of lotus root, milk vetch, alfalfa, soybean and peanut (legume plant) root nodule number, its steps are:

[0148] A, detection of tissue-specific expression of LjSIE3 in japonicus japonicus:

[0149] Using japonicus subspecies MG20 as material, collect the roots, stems, leaves and tumors of japonicus japonicus inoculated and uninoculated at different times, and extract the total RNA of different tissues at different times (Trizol reagent, purchased from Invitrogen company), Then use fluorescent quantitative PCR technology to detect its expression (such as Figure 5 shown). The specific implementation steps are: reverse transcribe the extracted total RNA into cDNA, use the kit PrimeScriptTM RT reagent Kit (Perfect RealTime) for the synthesis of the first strand of cDNA, reverse transcribe 500ng Total RNA in 20 μl system, use Oligo-dT as the primer, and operate ...

technical field

[0001] The invention belongs to the technical field of new plants, and more specifically relates to a ubiquitin ligase gene involved in nodulation of leguminous plants, and also relates to a preparation method of the ubiquitin ligase gene involved in nodulation of leguminous plants, and also relates to a Application of a ubiquitin ligase gene involved in legume nodulation in legumes. Background technique

[0002] Among various forms of protein post-translational modification, ubiquitination has attracted more and more attention in recent years. Similar to protein phosphorylation signal transduction, ubiquitination plays an important role in the process of cell life. important role. The 2004 Nobel Prize in Chemistry was awarded to scientists who made pioneering contributions to the study of ubiquitin-mediated protein degradation. Ubiquitin is a highly conserved polypeptide composed of 76 amino acid residues, named for its widespread distribution in various c...

Examples

Embodiment 1

[0045] A method for preparing a ubiquitin ligase gene involved in symbiotic nodulation of leguminous plants, the steps of which are:

[0046] A. Cloning of candidate gene LjSIE3:

[0047] Using the outer part of SymRK-EC of the symbiotic receptor kinase SymRK of japonicus japonicus japonicus and pGBKT7 (a well-known and public yeast protein expression vector) as the bait plasmid, the AD- cDNA library, the colony grown on the plate of SD / -Leu-Trp-Ade-His (auxotrophic yeast culture medium) passed the strict rotation verification, and the plasmid was extracted from the positive transformant and subjected to sequencing analysis to obtain The cDNA containing only 663bp of the C-terminal is the 598-1161 nucleotide sequence shown in SEQ NO:1. The specific implementation steps are:

[0048] (1) Preparation of yeast competent cells by LiAc method, yeast transformation, and library screening

[0049] (1) Streak the frozen yeast Y187 (purchased from clontech company) on the YPDA plate...

Embodiment 2

[0147] A kind of ubiquitin ligase gene that participates in leguminous plant symbiotic nodulation in the application of lotus root, milk vetch, alfalfa, soybean and peanut (legume plant) root nodule number, its steps are:

[0148] A, detection of tissue-specific expression of LjSIE3 in japonicus japonicus:

[0149] Using japonicus subspecies MG20 as material, collect the roots, stems, leaves and tumors of japonicus japonicus inoculated and uninoculated at different times, and extract the total RNA of different tissues at different times (Trizol reagent, purchased from Invitrogen company), Then use fluorescent quantitative PCR technology to detect its expression (such as Figure 5 shown). The specific implementation steps are: reverse transcribe the extracted total RNA into cDNA, use the kit PrimeScriptTM RT reagent Kit (Perfect RealTime) for the synthesis of the first strand of cDNA, reverse transcribe 500ng Total RNA in 20 μl system, use Oligo-dT as the primer, and operate ...

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