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Nicotiana benthamiana TMP14 (Thylakoid Membrane Phosphoprotein 14) and application thereof in plant virus resistance

A technology of TMP14 and Nicotiana benthamiana, applied in the field of molecular biology, can solve the problem of unknown function of TMP14 protein, and achieve the effect of excellent anti-plant virus efficacy and broad application prospects.

Active Publication Date: 2018-02-06
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report about the interaction between TMP14 protein and TSWV NSm protein, and the resistance to plant viruses. The role of N. benthamiana TMP14 protein is still unknown

Method used

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  • Nicotiana benthamiana TMP14 (Thylakoid Membrane Phosphoprotein 14) and application thereof in plant virus resistance
  • Nicotiana benthamiana TMP14 (Thylakoid Membrane Phosphoprotein 14) and application thereof in plant virus resistance
  • Nicotiana benthamiana TMP14 (Thylakoid Membrane Phosphoprotein 14) and application thereof in plant virus resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 There is a direct interaction between TSWV NSm and N. benthamiana TMP14

[0026] Insertion of TSWV NSm into pBT, respectively 3 -STE, NbTMP14 inserted into pPR 3 -N, get pBT 3 -STE-NSm with pPR 3 -N-TMP14 two vectors. These two vectors were simultaneously transferred into the yeast strain NMY51, and at the same time with pBT 3 -STE-NSm and empty vector pPR 3 -N, with pPR 3 -N-TMP14 and empty vector pBT 3 -STE co-transformed yeast was used as two negative controls, and yeast-free plates were painted, and positive clones were identified by bacterial liquid PCR. After the positive clones were shaken, the bacteria were collected, diluted to different concentrations, and further coated with yeast three-deficient plates to observe the growth of the three-deficient plates. The results show that pBT 3 -STE-NSm with pPR 3 -N-TMP14 co-transformed yeast can grow normally on triple-deficient plates, while pBT 3 -STE-NSm with empty vector pPR 3 -N, pPR 3 -N-TM...

Embodiment 2

[0029] Example 2 Inoculation of Nicotiana benthamiana with TSWV leads to down-regulation of TMP14 expression and abnormal development of chloroplasts

[0030] N. benthamiana was rubbed inoculated with virus TSWV, and the symptoms of tobacco were observed. After 10 days of inoculation, the leaves of N. benthamiana system showed obvious yellowing and wilting symptoms. The diseased leaves were taken to extract total RNA, and after confirming the virus infection by RT-PCR, the expression of TMP14 was detected by qPCR, and it was found that the expression of TMP14 was significantly lower than that of healthy tobacco ( image 3 A, C in).

[0031] At the same time, the diseased leaves were taken to observe the cell structure with an electron microscope, and it was found that the starch grains on the chloroplasts of tobacco were significantly larger than those of wild-type tobacco after inoculation of the virus, and the structure of the chloroplasts was destroyed, and the morphology c...

Embodiment 3

[0033] Example 3 Down-regulation of N. benthamiana TMP14 expression is beneficial to TSWV infection, and up-regulation increases its resistance

[0034] 1. Silence of TMP14 expression by TRV VIGS leads to up-regulation of TSWV accumulation

[0035] TRV2-GFP, TRV2-TMP14 were mixed with TRV1 1:1 and then injected into tobacco; 10 days after injection, samples were taken to detect the expression of TMP14 in these two types of tobacco, and it was found that the expression of TMP14 in TRV2-TMP14 was significantly higher than that in TRV2- The expression level of TMP14 in GFP is low. Further, pTRV2-GFP and pTRV2-TMP14 tobacco were inoculated with TSWV virus, and 10 days after virus inoculation, samples were taken to detect the accumulation of TSWV in these two tobaccos, and it was found that the accumulation of virus in pTRV2-TMP14 tobacco was significantly higher than that in pTRV2-GFP tobacco. Virus accumulation is high ( Figure 5 ).

[0036]2. Transgenic overexpression of TMP...

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Abstract

The invention discloses nicotiana benthamiana TMP14 (Thylakoid Membrane Phosphoprotein 14) and application thereof in plant virus resistance. The amino acid sequence of the nicotiana benthamiana TMP14is as shown in the sequence table SEQ ID NO.1. The invention also discloses an optimized mutant of the nicotiana benthamiana TMP14. The nicotiana benthamiana TMP14 disclosed by the invention directlyinteracts with TSWV (Tomato Spotted Wilt Virus) NSm; the over-expression transgenic tobacco plant of the nicotiana benthamiana TMP14 has the function of resisting plant virus; the discovery providestheoretical and practical basis for researching transgenic anti-virus crops, and has a broad application prospect.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a Nicotiana benthamiana TMP14 protein and its application in anti-plant virus. Background technique [0002] TMP14 is a phosphorylated protein (thylakoid membranenephosphoprotein, TMP) located in the thylakoid membrane of chloroplast. Studies on Arabidopsis TMP14 (AtTMP14) have shown that the protein is encoded by nuclear genes, and its precursor contains a chloroplast signal peptide consisting of 45 amino acids. The molecular weight of AtTMP14 is 14kDa. It is presumed that the N-terminal is located in the chloroplast stroma, the middle region contains two transmembrane helices, and the threonine at position 21 or 22 is the phosphorylation site. TMP14 is a unique protein encoded by photosynthetic organisms such as plants and cyanobacteria, but its specific function in photosynthesis is still unclear. The latest research shows that TMP14 is one of the subun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/11A01H5/00A01H6/82
CPCC07K14/415C12N15/8283
Inventor 张永强张超占锦
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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