Pear transcription factor pbhb.g7.2 and its application in promoting fruit ripening

A technology of transcription factor and pear fruit, applied in application, genetic engineering, plant gene improvement, etc., can solve the problems that HB gene has not been reported yet

Active Publication Date: 2021-10-26
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It must be pointed out that although the HB family has been extensively studied, so far, no HB genes regulating pear fruit ripening have been reported.

Method used

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  • Pear transcription factor pbhb.g7.2 and its application in promoting fruit ripening
  • Pear transcription factor pbhb.g7.2 and its application in promoting fruit ripening
  • Pear transcription factor pbhb.g7.2 and its application in promoting fruit ripening

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Isolation, cloning and expression analysis of PbHB.G7.2 gene

[0039] The present invention provides a pear HB gene PbHB.G7.2, the sequence of the PbHB.G7.2 gene is shown in SEQ ID No.1.

[0040] In the present invention, the pear HB gene PbHB.G7.2 is preferably derived from 'Cuiguan' pear (P. pyrifolia Nakaicv. 'Cuiguan').

[0041] In the present invention, the pear HB gene PbHB.G7.2 is obtained by the following method: extracting Cuiguan pear RNA from Cuiguan pear pulp; reverse transcription of the RNA to obtain cDNA; using the cDNA as a template, The full length of the PbHB.G7.2 gene was obtained by amplifying the forward primer F1 and the reverse primer R1.

[0042] The extraction of the RNA of Cuiguan pear described in the present invention adopts the routine plant tissue RNA extraction method in this field and gets final product, without other special limitation, adopts Plant Total RNA Isolation KitPlus (purchased from Fuji biological Technology Co., L...

Embodiment 2

[0048] Example 2: Transient overexpression of PbHB.G7.2

[0049] Two restriction sites, HindIII and XbaI, were respectively added to both ends of the PbHB.G7.2 gene amplification primer, and the amplification primer was (forward primer: PbHB.G7.2-F3: 5'-TCCAAAGAATTCAAAAAGCTTATGGGTTTTGATCATGAAGCTTG-3' (SEQ ID NO. 9); reverse primer: PbHB.G7.2-R3: 5'-TCATTAAAGCAGGACTCTAGACTAGCAAGCTGTAGATGGATGAGTACT-3' (SEQ ID NO. 10)). The 50 μl amplification system included 100ng Template DNA, 5×Q5 Reaction Buffer (Q5 Reaction Buffer), 10mM dNTPs, 1U Q5 High-Fidelity DNA Polymerase (the above buffer and Q5 High-Fidelity DNA Polymerase were purchased from New England Biolabs), 1.0 μM of the above primers. The PCR reaction was completed on the Roche480 (Applied Biosystem) amplification instrument according to the following procedures: 98°C, 30s; 35 cycles of denaturation at 98°C for 10s, annealing at 60°C for 30s, and extension at 72°C for 60s; after the cycle was completed, extend at 72°C for 5...

Embodiment 3

[0052] Example 3: Yeast two-hybrid experiment

[0053] The invention provides two transcription factors PbHB.G1 and PbHB.G2.1 interacting with pear transcription factor PbHB.G7.2 protein, both of which belong to Homeobox family members. The nucleotide sequence of the PbHB.G1 gene is shown in SEQ ID No.15, including an open reading frame of 1035 bp, encoding 345 amino acids, and its amino acid sequence is shown in SEQ ID No.16. The nucleotide sequence of the PbHB.G2.1 gene is shown in SEQ ID No.17, including an open reading frame of 1107 bp, encoding 369 amino acids, and its amino acid sequence is shown in SEQ ID No.18.

[0054] In the present invention, the two pear HB genes PbHB.G1 and PbHB.G2.1 are preferably derived from 'Cuiguan' pear (P.pyrifolia Nakaicv.'Cuiguan').

[0055] Two restriction sites, EcoRI and BamHI, were respectively added to the two ends of the PbHB.G7.2 gene amplification primer, and the amplification primer was (forward primer: PbHB.G7.2-F4: 5'-atggccat...

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Abstract

The invention discloses a pear transcription factor PbHB.G7.2 and its application in promoting fruit ripening, belongs to the technical field of plant genetic engineering, and provides a pear transcription factor PbHB.G7.2 as shown in SEQ ID No. The transcription factor PbHB.G7.2 gene that functions in the fruit ripening process. The overexpression line obtained by transiently transforming the pear transcription factor PbHB.G7.2 into pear fruit has significantly higher ethylene release than the control line, and the ripening process is also accelerated. The biological function verification shows that the transcription factor PbHB. G7.2 can bind and activate the PbACS1b promoter, increase the expression level of PbACS1b gene, have the functions of increasing the amount of ethylene synthesis in pear fruit, and can promote the ripening of pear fruit. It provides new genetic resources for molecular breeding to promote pear fruit ripening, and the development of the genetic resources is conducive to increasing farmers' income and reducing agricultural costs.

Description

technical field [0001] The present invention belongs to the field of plant genetic engineering, relates to pear transcription factor PbHB.G7.2 and its application, specifically relates to the separation and cloning of a Homeobox family member PbHB.G7.2 gene related to fruit ripening from 'Cuiguan pear' and its application. Background technique [0002] Fruit ripening refers to the process of a series of physiological and biochemical changes after the fruit completes growth and development, which is the result of the expression, transcription and regulation of related genes at the molecular level. From a physiological point of view, according to whether there is a peak of ethylene synthesis and a significant increase in the respiration rate during the ripening process of the fruit, it can be divided into respiration-climate-type fruits and non-respiration-climate-type fruits [1] . The ripening process of climacteric fruits is largely dependent on the catalysis and induction...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/11C12N15/84A01H5/08A01H6/74
CPCC07K14/415C12N15/8249
Inventor 张绍铃谷超曹苏豪谢智华齐开杰
Owner NANJING AGRICULTURAL UNIVERSITY
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