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Ghpdlp6-3 gene encoding plasmodesmata localization protein and use thereof

A plasmodesmata and genetic technology, applied in genetic engineering, plant genetic improvement, application, etc., can solve problems such as preventing mycelium growth, delaying the onset of Verticillium wilt, and achieving the effect of improving resistance

Active Publication Date: 2021-09-07
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Combined with the characteristics of Verticillium dahliae infecting plant cells, we speculate that the expression of PDLP protein may prevent the growth of hyphae between plant cells, thereby delaying the onset of Verticillium wilt in plants

Method used

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  • Ghpdlp6-3 gene encoding plasmodesmata localization protein and use thereof
  • Ghpdlp6-3 gene encoding plasmodesmata localization protein and use thereof
  • Ghpdlp6-3 gene encoding plasmodesmata localization protein and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] [Example 1] Detection of target gene expression

[0051] Quantitative RT-PCR was used to measure the expression of the target gene in various tissues of cotton or leaves treated with salicylic acid, as well as during the screening of transgenic Arabidopsis. The extraction of total RNA adopts the EASYspin plant RNA rapid extraction kit (Aidlab company) method, and then utilizes The RT reagent Kit with gDNA Eraser Kit (TaKaRa Company) was used for reverse transcription of the first strand of cDNA, which was used as a template for quantitative RT-PCR analysis. The relative expression of the target gene was analyzed using iQ SYBR GreenSupermix (BIO-RAD) reagent. If the gene expression in upland cotton is detected, the internal standard gene selects the Ghhis3 gene (AF024716) of upland cotton, and if the gene expression in Arabidopsis is detected, the internal standard gene selects the Atactin2 gene (AT3G18780) of Arabidopsis, and the primers are respectively in shown in ...

Embodiment 2

[0057] [Example 2] Induced expression of GhPDLP2 and GhPDLP6 by salicylic acid

[0058] Preparation of salicylic acid solution: Accurately weigh 138.12 mg of salicylic acid, dissolve in 1 mL of 95% ethanol solution, and prepare 1000× salicylic acid mother solution. The working concentration of salicylic acid in this test is 1mmol / L, and it can be diluted with tap water when used.

[0059] Materials treated with exogenous salicylic acid: wild-type upland cotton with the same growth state was taken as the experimental material, and absorbent paper soaked with 1mmol / L salicylic acid was placed on the leaves for treatment. Leaf RNA was extracted at 6h, 12h, 24h, 48h and 72h, and reverse-transcribed into cDNA to detect the transcription levels of GhPDLP2s and GhPDLP6s.

Embodiment 3

[0060] [Example 3] Cloning and expression vector construction of GhPDLP6-3

[0061]According to the method of Example 1, the total RNA of the ovule 16 days after flowering of upland cotton was extracted and the corresponding cDNA was synthesized. Using the designed and synthesized GhPDLP6-3 cloning primers GhPDLP6-3-up and GhPDLP6-3-dn (Table 2), the gene fragment was amplified using the cDNA synthesized above as a template. The amplification system is as follows: 10X PCR buffer for KOD Plus 5 μL, 25 mmolMgSO4 5 μL, 2 mmol / L dNTPs 2 μL, primer GhPDLP6-3-up (5 μmol / L) 2 μL, primer GhPDLP6-3-dn (5 μmol / L) 2 μL, KOD Plus Polymerase 1U / μL, upland cotton cDNA about 20ng, double distilled water to make up to 50μL. The amplification program is: 94°C, 2min; 94°C, 15sec, 53°C, 30sec, 68°C, 2min, 35 cycles. For the amplification of the GhPDLP6-3-GFP fusion gene, the same method as above was used, except that the GhPDLP6-3-dn primer was replaced with GhPDLP6-3-GFP-dn. After the amplif...

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Abstract

The invention relates to the GhPDLP6-3 gene encoding the plasmodesmata localization protein and its use, and relates to the GhPDLP6-3 gene and its use in improving plant resistance to Verticillium wilt, and a method for improving plant resistance to Verticillium wilt . The GhPDLP6-3 gene derived from the dominant expression of upland cotton fibers and ovules, after heterologous expression in the target plant, not only promotes the elongation of the main root, but also improves the resistance of the target plant to Verticillium wilt, which can be applied to crop disease resistance genetic engineering improvements.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering. It relates to a gene encoding plant plasmodesmata localization protein and its application. Background technique [0002] Verticillium wilt seriously affects the yield and quality of crops, and the global annual loss caused by Verticillium wilt reaches billions of dollars (Pegg et al., 2002). It has been reported that the annual yield of potatoes due to Verticillium wilt is reduced by more than 50%, usually causing a 10-15% yield reduction (Powelson et al., 1993; Rowe et al., 1987, 2002). In lettuce-growing areas, losses due to Verticillium wilt can easily reach 100% (Subbarao et al., 1997). Studies such as Levin (2003) found that the yield reduction of olives due to Verticillium wilt can reach more than 70%. Verticillium wilt is a devastating disease in cotton production, which seriously affects cotton yield and fiber quality. It is one of the important limiting factors for the develo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/82A01H5/00A01H6/20
CPCC07K14/415C12N15/8282
Inventor 侯磊张国庆朱茜杨洋丁博刘芹孟伟张云逸刘浩儒裴炎李先碧赵娟梁爱敏
Owner SOUTHWEST UNIV