Coniothyrium-minitans siderophore transporter (CmSit1) gene as well as preparation method and application thereof

A technology of transport protein and iron shell shell, which is applied in the biological field, can solve the problems such as the unseen CmSit1 gene and antifungal ability research reports, and achieve the goal of improving the ability of resisting plant pathogenic fungi, improving the antifungal ability, and improving the resistance Effect

Inactive Publication Date: 2013-10-23
HUAZHONG AGRI UNIV
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  • Description
  • Claims
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Problems solved by technology

[0005] After searching the domestic and foreign literature of the prior art, there has been no research repo

Method used

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  • Coniothyrium-minitans siderophore transporter (CmSit1) gene as well as preparation method and application thereof
  • Coniothyrium-minitans siderophore transporter (CmSit1) gene as well as preparation method and application thereof
  • Coniothyrium-minitans siderophore transporter (CmSit1) gene as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] A method for preparing the siderophore transporter gene of the shell mold, the steps are:

[0052] A. Screening of the enhanced antifungal ability of C. shield ZS-1N1812:

[0053] According to the mature Agrobacterium tumefaciens (EHA105)-mediated genetic transformation method of C. shieldias established in our laboratory (LiMoxiao, et al. Transformation of Coniothyrium minitans, a parasite of Sclerotias clerotiorum, with Agrobacterium tumefaciens. FEMS Microbiology Letters. -329.), transformed the C. shield ZS-1 strain isolated from the field in our laboratory, and obtained a C. shield insertion mutant library containing more than 45,000 T-DNA markers. Use PDA medium (PDA adopts the conventional production method: 200g peeled potatoes, add appropriate amount of distilled water (500-800ml) to cook, add glucose 20g, agar powder 13g, supplement distilled water to 1000ml) at 20 °C to activate and cultivate shield shells The transformants of mold ZS-1 bacterial strain and ...

Embodiment 2

[0065] Detection of the expression level of CmSit1 in ZS-1N1812

[0066] First connect ZS-1 and ZS-1N1812 mycelium blocks on cellophane, after culturing for 4 days, collect mycelia and add 3ml of sterile water to grind in a mortar, then suck 200μl with a pipette tip and spread it on a flat plate covered with cellophane Above, fresh mycelia were collected for 48h, 72h, 96h and 120h growth respectively. RNA was extracted using Invitrogen’s TRIzol kit, and RT-PCR was performed to detect the expression level of the target gene (primer sequence SIT1-FP: CCACTT CCA ACC CGA CAC; SIT1-RP: CTT ACG CCT CCG ACA AAT), and the ACTIN gene was used as an internal control (Primer sequence ACTIN-FP: ACC GTG AGA AGA TGA CCC; ACTIN-RP: AAG GAC AGA AGG CTG GAA G) For specific steps, please refer to the kit instructions. The test results found that in ZS-1N1812, the expression levels of CmSit1 at the four time points detected were significantly increased, while the expression of CmSit1 could not ...

Embodiment 3

[0068] Inhibitory effect of culture filtrate of ZS-1N1812 activated and expressed by CmSit1 on pathogenicity of Sclerotinia sclerotiorum

[0069] Rapeseed rape (commercial rapeseed varieties are acceptable) for pathogenicity testing is grown in a greenhouse (15-20° C.) for 30 days, and vigorously growing leaves in the middle of each rapeseed plant are taken for the test. The mycelia of C. shield were inoculated on the PDA plate covered with cellophane, and the mycelium was collected after 6 days of culture. The collected mycelium was ground in a mortar with sterile water, added to PDB, placed on a shaker at 20°C, and then shaken for 9 days. Sclerotinia Ep-1PNA367 mycelium blocks were inoculated on the PDA plate, and after 3 days of cultivation, the mycelium blocks at the edge of the colony were picked up and placed in sterile ddH 2 Soak in O, PDB, ZS-1 filtrate, ZS-1N1812 filtrate for 30min, then inoculate in isolated rapeseed leaves or potted rapeseed (5-6 leaf stage), cultur...

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Abstract

The invention discloses a coniothyrium-minitans siderophore transporter (CmSit1) gene as well as a preparation method and application thereof. A coniothyrium-minitans insertion mutant library containing a plurality of T-DNA (transferred deoxyribonucleic acid) markers is obtained by adopting a mature agrobacterium-mediated genetic transformation method of the coniothyrium minitans. Transformants and sclerotinia sclerotiorum Ep-1PNA367 are cultured at the same time in the PDA (potato dextrose agar) culture medium, and as the comparison of a strain ZS-1, a strain ZS-1N1812 with enhanced anti-fungal capacity is selected. The further study shows that the expression of the CmSit1 gene can be improved significantly when T-DNA in the strain ZS-1N1812 is inserted into a promoter area of the CmSit1 gene, and the capacity of resisting the sclerotinia sclerotiorum of a plant can be improved significantly if the CmSit1 gene is transferred into the nicotiana benthamiana. The CmSit1gene can be used for improving the capacity of resisting pathogenic fungi of various biocontrol bacteria and can be also used for the transgenic breeding for disease resistance of crops.

Description

technical field [0001] The invention belongs to the field of biological technology, and more specifically relates to a siderophore transporter gene of the shell mold, and also relates to a preparation method of the siderophore transporter gene of the shell shell mold, and also relates to the use of the gene in transforming bio-control strains and cultivating antibacterial Application in nuclear disease transgenic plants. Background technique [0002] Sclerotinia sclerotiorum (Sclerotinia sclerotiorum) is a very important plant pathogenic fungus, with a wide range of hosts, which can parasitize more than 540 kinds of plants in 64 families. Common important hosts of S. sclerotiorum include rapeseed, sunflower, soybean and other oil crops, lettuce, carrot and a variety of cruciferous and leguminous vegetables. The sclerotinia caused by it has a very important impact on the yield and quality of these crops . Taking rape as an example, Sclerotinia sclerotiorum caused by Sclerot...

Claims

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Application Information

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IPC IPC(8): C12N15/31C07K14/37C12N1/15C12N15/80C12N15/83C12N15/84A01H5/00C12R1/645
Inventor 姜道宏孙西苹付艳苹程家森谢甲涛李博
Owner HUAZHONG AGRI UNIV
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