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Gene related to tomato leaf senescence and applications thereof

A technology of leaf aging and genetics, applied in the fields of application, genetic engineering, plant genetic improvement, etc., to achieve obvious effects, prolong the ornamental period, and increase the chlorophyll content

Inactive Publication Date: 2019-07-23
YANGTZE NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on the inhibitory factors of the leaf senescence process. Excavating the inhibitory factors that affect the plant leaf senescence process can make up for the shortcomings of the current situation.

Method used

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  • Gene related to tomato leaf senescence and applications thereof
  • Gene related to tomato leaf senescence and applications thereof
  • Gene related to tomato leaf senescence and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] tomato gene ARF 6 and ARF Cloning of the promoter of 8 and analysis of GUS histochemical staining

[0031] (1) ARF 6 and ARF Cloning of 8 gene promoters

[0032] Using the CTAB method (RTG2405-01, Zhongke Tairui) to extract tomato genomic DNA, according to the sequence shown in SEQ ID NO.5 and SEQ ID NO.6 pARF 8 and pARF 6 Nucleotide sequence of the promoter, design of primers pARF 8-F, pARF 8-R, pARF 6-F and pARF 6-R, with the extracted genomic DNA as a template, with pARF 8-F (forward primer) and pARF 8-R (reverse primer) as primer for pARF Amplification of 8 promoters to pARF 6-F (forward primer) and pARF 6-R (reverse primer) as primer for pARF 6 Amplification of the promoter.

[0033] The primer sequences are as follows:

[0034] pARF8 -F:5'-TTTACCATGTCCCTACCCTCT-3'

[0035] pARF8 -R:5'-CTTTCTCCAAGACCTCCATT-3'

[0036] pARF6 -F:5'-CGAGATCATTAAGATGTATTTTTGTG-3'

[0037] pARF6 -R:5'-TCCCTCAAATCAACAAACTC-3'

[0038] P CR amplifi...

Embodiment 2

[0060] Example 2 Tomato Gene ARF 6 and ARF Cloning of 8 and construction of expression vector

[0061] (1) ARF 8 and ARF Cloning of 6 genes

[0062] Using Micro Tom tomato leaf tissue as material, RNAiso P lus (9108, Takara) kit and Recombinant DNase I (RNase-free) kit (2270A, Takara), total RNA was extracted according to the instructions, and the concentration of RNA was measured with a micro-spectrophotometer for later use.

[0063] Take about 2.0 μg tomato total RNA, using P crimeScri p t II first-strand cDNA synthesis kit (6210A, Takara), and synthesize the first strand of cDNA according to the instructions.

[0064] according to tomato ARF8 and ARF 6 gene sequences, design specific primers ARF 8-F, ARF 8-R, ARF 6-F and ARF 6-R, In-fusion cloning vector linker sequence and restriction site sequence are introduced into the primer. Using the synthesized cDNA as a template, the ARF 8-F (forward primer) and ARF 8-R (reverse primer) as primer for gene ARF Ampl...

Embodiment 3

[0078] Example 3 Agrobacterium-mediated ARF 8 and ARF 6 Gene transformation and screening of transgenic positive lines

[0079] The recombinant expression vector constructed in Example 2 was made by conventional freeze-thaw method p BI121- 35S : ARF 8-NOS and p BI121- 35S : ARF 6-NOS was transformed into Agrobacterium EHA105 respectively, and then transformed into wild-type tomato (Micro Tom) through the Agrobacterium-mediated method. The specific steps are as follows:

[0080] 1) Cultivation of transformed materials

[0081] After the tomato tissue cultured seedlings were cultivated for 30-40 days under the condition that the culture temperature was 23-25°C, the light was 16 / 8h (day / night), and the light intensity was 10000-12000lux, the leaves of the tissue cultured seedlings with good growth were selected for use. in genetic transformation.

[0082] 2) Conversion

[0083] will contain the recombinant expression vector p BI121- 35S : ARF 8-NOS and p BI121- 35S...

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Abstract

The invention discloses a gene related to tomato leaf senescence and applications thereof. The gene is ARF8 or ARF6. The nucleotide sequences of the ARF8 and the ARF6 are respectively as shown in SEQID NO.1 and SEQ ID NO.3, or are obtained by substituting, deleting or adding one or more nucleotides in the nucleotide sequence as shown in the SEQ ID NO.1 or the SEQ ID NO.3, and are also related tothe tomato leaf senescence. Functional genomics tests prove that the ARF8 and the ARF6 genes are regulating factors for regulating and controlling the senescence process of tomato leaves, and the ARF8or the ARF6 genes are over-expressed in transgenic tomatoes, so that the chlorophyll content of the tomato leaves is increased (more dark green), and the senescence starting time is delayed by more than 30 days compared with that of a wild-type control. Besides, the gene has obvious effects of resisting ethylene-induced senescence, provides an effective technical means for regulating and controlling the senescence of the herb leaves, and particularly has wide application prospect and potential market value in foliage plants in the field of gardening.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to genes related to tomato leaf senescence and applications thereof. Background technique [0002] Senescence is the last stage of leaf development and is an active physiological process. Delaying senescence is closely related to crop yield increase and quality improvement. For food crops and most commercial crops, along with the development of yield organs and the gradual formation of quality traits, assimilated products in functional leaves and nutrients accumulated in aging leaves continue to Transport to producing organs. Premature senescence of crops will definitely affect the yield and quality of products. The early senescence of leaves and functions of some hybrid rice leads to low seed setting rate at the later stage of development, which seriously affects the further development of hybrid rice yield potential. The leaf senescence process of gr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82C07K14/415A01H5/12
CPCC07K14/415C12N15/8249C12N15/825
Inventor 符勇耀杨利平徐文姬雷美艳
Owner YANGTZE NORMAL UNIVERSITY
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