Application of REVOLUTA gene to regulation and control on quantity of leaflets in leguminous plant and leaf-stem ratio

A technology of leguminous plants and small leaves, applied in the field of genetic engineering, can solve the problem of low frequency of multiple leaves

Inactive Publication Date: 2017-10-27
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In view of the above-mentioned prior art and the problem that the frequency of multiple leaves of the existing multi-leaf alfalfa varieties is not high, the present invention discovers a gene that regula...

Method used

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  • Application of REVOLUTA gene to regulation and control on quantity of leaflets in leguminous plant and leaf-stem ratio
  • Application of REVOLUTA gene to regulation and control on quantity of leaflets in leguminous plant and leaf-stem ratio
  • Application of REVOLUTA gene to regulation and control on quantity of leaflets in leguminous plant and leaf-stem ratio

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 Cloning of Medicago truncatula gene MtREVOLUTA

[0019] 1. Cloning of MtREVOLUTA gene of Medicago truncatula

[0020] Obtain the sequence of Arabidopsis REVOLUTA through the bioinformatics website NCBI, and then use BLAST to perform sequence comparison search to obtain the genome sequence of MtREVOLUTA. Design the primers shown in SEQ ID No. 2 and SEQ ID No. 3 according to the sequence. The RNA of Medicago truncatula was extracted with TRIzol kit, and the full-length CDS sequence of MtREVOLUTA gene was amplified by RT-PCR method. Use Gateway technology to link the gene sequence into the pEARLEYGATE201 vector, and then perform sequence determination to verify the correctness of the cloned sequence.

[0021] 2. Sequence information and characteristics analysis of MtREVOLUTA gene of Medicago truncatula

[0022] The CDS of the coding region of MtREV1 gene is 2520bp, contains 18 exons and 17 introns, encodes 839 amino acids, and contains the conserved HD-ZIP, MEKHLA and ...

Embodiment 2

[0025] Example 2 Obtaining and identification of MtREVOLUTA mutant of Medicago truncatula

[0026] Using Thermal asymmetric interlaced-PCR (TAIL-PCR) technology, we screened a mutant library labeled with Tnt1 of Medicago truncatula. Among 22,000 mutant lines, the mutant lines with Tnt1 inserted into the MtREVOLUTA gene were screened out ( figure 2 A). Molecular biology identification results showed that the insertion of Tnt1 into the sixth exon of the MtREVOLUTA gene caused a significant decrease in the expression level of this gene ( figure 2 B).

Embodiment 3

[0027] Example 3 Statistics of leaflet number of MtREVOLUTA mutant of Medicago truncatula

[0028] The phenotype of the mutant was analyzed, and the results showed that the number of small leaves of the knockout MtREVOLUTA mutant increased significantly. The wild-type compound leaf contains three leaflets, while about 81.5% (n=300) of the compound leaf of the mutant contains 4 or 5 leaflets ( image 3 A-D). Further analysis showed that the leaf-to-stem ratio of the mutant was increased by about 11% compared with the wild type (n=10) ( image 3 E).

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Abstract

The invention discloses application of an REVOLUTA gene (A nucleotide sequence is shown as SEQ ID No.1) to regulation and control on quantity of leaflets in a leguminous plant and leaf-stem ratio. According to the application disclosed by the invention, a knockout mutant strain of the REVOLUTA gene as a medicago truncatula HD-ZIP III gene family member is obtained by first separation, the quantity of the leaflets in the plant is discovered to be changed from three into four or five (the quantity of the leaflets in 81.5 percent of leaves is increased), and meanwhile, the leaf-stem ratio is significantly improved by about 11 percent; an experiment proves that the gene is involved in the regulation and the control on the quantity of the leaflets in the leguminous plant and the leaf-stem ratio; through gene knockout, the transgenic leguminous plant with higher leaflet quantity and leaf-stem ratio than those of a wild type plant can be obtained; after the implementation of the invention, a novel leguminous forage plant can be created and can be used for subsequent forage quality, thereby being of a great significance in the production of the grass industry in China.

Description

Technical field [0001] The invention relates to the application of the MtREVOLUTA gene of the Medicago truncatula transcription factor HD-ZIPIII gene family in regulating the number of small leaves and the leaf-stem ratio, and belongs to the technical field of genetic engineering. Background technique [0002] Leguminous forage alfalfa is the earliest cultivated, most widely distributed, and the most important perennial high-quality forage in the world. However, domestic alfalfa has low output, poor quality and unstable supply, which has become a factor that cannot be ignored in restricting the development of herbivorous animal husbandry and dairy industry in my country. At present, the commercial production of alfalfa in my country is growing rapidly, but due to the huge domestic demand for high-quality alfalfa, the shortage of domestic alfalfa has not been alleviated. Therefore, the improvement of alfalfa yield and quality is a major urgent need for the development of my count...

Claims

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Application Information

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IPC IPC(8): C12N15/29A01H5/00
CPCC07K14/415C12N15/8213C12N15/8261
Inventor 周传恩韩璐赵阳
Owner SHANDONG UNIV
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