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Cysteine protease gene for regulating and controlling root nodule senescence, and preparation method and application thereof

A cysteine ​​protease and gene technology, which is applied in the fields of botanical equipment and methods, biochemical equipment and methods, and applications, can solve the problem of no research results in the stereotyped root nodule L. japonicus, and can prolong the effective nitrogen fixation time, root nodule Great, increase the effect of nitrogen fixation time

Inactive Publication Date: 2013-09-11
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no relevant research result in stereotyped root nodule japonicus

Method used

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  • Cysteine protease gene for regulating and controlling root nodule senescence, and preparation method and application thereof
  • Cysteine protease gene for regulating and controlling root nodule senescence, and preparation method and application thereof
  • Cysteine protease gene for regulating and controlling root nodule senescence, and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] A method for preparing a cysteine ​​protease gene regulating root nodule senescence, the steps of which are:

[0036] A. Cloning of candidate gene LjCyp5:

[0037]The cysteine ​​protease gene AsNODf32 of milk vetch was used (Naito et al., The involvement of a cysteine ​​proteinase in the nodule development of Chinese milk vetch infected with Mesorhizobium huakuii subsp. rengei. Plant Physiol 124: 1087-1095, 2000; Li et al. , A nodule-specific plant cysteine ​​proteinase, AsNODF32, is involved in nodule senescence and nitrogen fixation activity of the green manure legume Astragalus sinicus. New Phytol 180: 185-192, 2008), NCBI protein accession number is BAB13759, in Japan Baimai The root website (http: / / www.kazusa.or.jp / lotus / ) was compared and analyzed to find the homologous clone (No. LjSGA_035265.1) in Lotus japonicus and locate it on the third chromosome LjB03G07 superior. The promoter region was predicted based on genome sequence analysis, and primers PF (5'-ATGG...

Embodiment 2

[0041] Application of a cysteine ​​protease gene regulating nodule senescence in japonicus japonicus, alfalfa, soybean and peanut (legume) to delay nodule senescence, the steps are: using the gene of the present invention in japonicus japonicus For example, the application procedure in alfalfa, soybean and peanut is the same as that in lotus root.

[0042] Construction and transformation of A, LjCyp5 gene promoter expression and suppression expression vector:

[0043] In order to better describe the function of the gene, the applicant fused its 2.0kb promoter to the GUS reporter gene and transformed J. japonicus to detect its activity; at the same time, it suppressed expression in J. japonicus and obtained from transgenic plants phenotype to verify.

[0044] The fusion vector construction method of the promoter and the reporter gene is as follows: Figure 7 Shown: LjCyp5 gene promoter sequence through primers 5'-AAAGTCGACCCACGTGGCTGTCCTTA-3' and 5'-GGGGAATTCGGTAGCTAGTCAGCTAG...

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Abstract

The invention discloses a cysteine protease gene for regulating and controlling root nodule senescence, and a preparation method and application thereof. According to the preparation method disclosed by the invention, a Japan lotaustralin genomic sequence library is compared by utilizing the known cysteine protease gene of astragalus smicus; after the Japan lotaustralin genomic sequence library is analyzed and compared on the internet in a detailed manner, a primer having strong specificity is designed to carry out gene cloning by taking initiation codon as the starting point and termination codon as the finishing point; in addition, due to sequencing analysis and comparing analysis, a cloned fragment proves that the cysteine protease gene for regulating and controlling root nodule senescence is successfully cloned from the lotaustralin genomic sequence and cDNA (complementary deoxyribonucleic acid). The invention discloses application of the cysteine protease gene on leguminous plants; the expression mode of a gene promoter and the functions of the gene in the root nodule senescence process are researched by utilizing a histochemical localization technology. Compared with contrast plants, the results show that the root nodule of an RNAi (ribonucleic acid interference) plant is enlarged; the nitrogen fixation enzyme activity is increased; senescence of the root nodule is delayed; the effective nitrogen fixation time of the root nodule is prolonged; growth of the plant is more vigorous; the cysteine protease gene disclosed by the invention has application prospect on leguminous crops.

Description

technical field [0001] The invention belongs to the field of new plant technology, and more specifically relates to a cysteine ​​protease gene for regulating root nodule aging, and also relates to a preparation method for regulating root nodule aging cysteine ​​protease gene, and also relates to a method for regulating root nodule aging Use of the cysteine ​​protease gene in legumes. Background technique [0002] Cysteine ​​protease is the most important protease family, which is widely involved in various physiological processes of plants, such as seed germination, seedling development, stress response, plant tissue differentiation and senescence. Plant cysteine ​​proteases mainly belong to the papain-like cydteine ​​proteinases (C1A, papain-like cydteine ​​proteinases) and the bean protease family (C13 the legumains), and the former has been studied in the most detail. Many papain enzymes contain signal peptides composed of 10-26 hydrophobic amino acids located in the end...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/57C12N9/50C12N15/10C12N15/82A01H5/00
Inventor 张忠明苟洪兰朱辉袁松丽陈桃
Owner HUAZHONG AGRI UNIV
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