阅读说明：本技术 梨转录因子PbHB．G7．2及其在促进果实成熟中的应用 (Pear transcription factor PbHB.G7.2 and application thereof in promoting fruit ripening ) 是由 张绍铃 谷超 曹苏豪 谢智华 齐开杰 于 2020-07-03 设计创作，主要内容包括：本发明公开了梨转录因子PbHB.G7.2及其在促进果实成熟中的应用,属于植物基因工程技术领域,提供了一种如SEQ ID No.1所示的具有促进果实乙烯的合成,加速果实成熟过程功能的转录因子PbHB.G7.2基因。将所述梨转录因子PbHB.G7.2瞬时转化到梨果实中获得的超表达系梨果实的乙烯释放量明显高于对照系,成熟过程也加快,经生物学功能验证,表明转录因子PbHB.G7.2能够结合并激活PbACS1b启动子,提高PbACS1b基因表达水平,具有提高梨果实乙烯合成量,能够促进梨果实成熟的功能。为促进梨果实成熟的分子育种提供新的基因资源,该遗传资源的开发有利于增加农民收入和降低农业成本。(The invention discloses a pear transcription factor PbHB.G7.2 and application thereof in promoting fruit ripening, belongs to the technical field of plant genetic engineering, and provides a transcription factor PbHB.G7.2 gene which is shown as SEQ ID No.1 and has the functions of promoting fruit ethylene synthesis and accelerating fruit ripening. The ethylene release amount of the over-expression system pear fruit obtained by instantly transforming the pear transcription factor PbHB.G7.2 into the pear fruit is obviously higher than that of a control system, the maturation process is also accelerated, and biological function verification shows that the transcription factor PbHB.G7.2 can combine with and activate a PbACS1b promoter, so that the expression level of the PbACS1b gene is improved, the ethylene synthesis amount of the pear fruit is increased, and the function of promoting the maturation of the pear fruit is realized. Provides a new gene resource for promoting the molecular breeding of pear fruit maturity, and the development of the genetic resource is beneficial to increasing the income of farmers and reducing the agricultural cost.)

1. A pear transcription factor PbHB.G7.2 has a nucleotide sequence shown in SEQ ID No. 1.

2. The pear transcription factor PbHB.G7.2 encoded protein of claim 1, the amino acid sequence of which is shown as SEQ ID No. 2.

3. An expression cassette, a recombinant vector, a transgenic cell line or a transgenic recombinant bacterium containing the pear transcription factor PbHB.G7.2 of claim 1.

4. Primer pair for amplifying the pear transcription factor PbHB.G7.2 as claimed in claim 1, characterized in that it comprises a forward primer F1 and a reverse primer R1; the sequence of the forward primer F1 is shown as SEQ ID No. 5; the sequence of the reverse primer R1 is shown as SEQ ID No. 6.

5. Use of the pear transcription factor pbhb. g7.2 as defined in claim 1, a protein as defined in claim 2 or an expression cassette, a recombinant vector, a transgenic cell line or a transgenic recombinant bacterium as defined in claim 3 for promoting fruit ripening.

6. The use according to claim 5, wherein the fruit transiently over-expressing the transcription factor PbHB.G7.2 of claim 1 is constructed to increase the ethylene synthesis amount of the fruit and promote the fruit ripening.

7. The use according to claim 6, characterized in that the construction of the fruit transiently overexpressing the transcription factor PbHB.G7.2 comprises the following steps:

1) obtaining the pear transcription factor PbHB.G7.2 of claim 1;

2) connecting the transcription factor PbHB.G7.2 with a vector to obtain a recombinant vector;

3) transferring the recombinant vector into agrobacterium tumefaciens to obtain recombinant agrobacterium tumefaciens;

4) infecting the recombinant agrobacterium tumefaciens on fruits to obtain the fruits transiently overexpressing the transcription factor PbHB.G7.2.

8. The use of the pear transcription factor PbHB.G7.2 of claim 1 in pear crossbreeding.

9. A gene PbACS1b regulated by the transcription factor PbHB.G7.2 of claim 1, the nucleotide sequence of which is shown in SEQ ID No. 3.

10. The pear HB transcription factor of the protein that encoded can interact with the protein encoded by the transcription factor PbHB.G7.2 of claim 1, characterized in that the transcription factor is the transcription factor PbHB.G1 and/or the transcription factor PbHB.G2.1, the nucleotide sequence of the transcription factor PbHB.G1 is shown as SEQ ID No.15, and the nucleotide sequence of the transcription factor PbHB.G2.1 is shown as SEQ ID No. 17.

Technical Field

The invention belongs to the field of plant genetic engineering, relates to a pear transcription factor PbHB.G7.2 and application thereof, and particularly relates to a Homeobox family member PbHB.G7.2 gene which is separated and cloned from 'Cuiguan pear' and is related to fruit ripening and application thereof.

Background

Fruit ripening refers to the process of a series of physiological and biochemical changes after the fruit finishes growing development, and the processes are the comprehensive result of expression, transcription and regulation of related genes on molecular level. Physiologically, according to whether the fruit has ethylene synthesis peak and respiration rate is increased significantly during ripening, the fruits can be divided into climacteric fruits and non-climacteric fruits^[1]. The ripening process of climacteric fruit depends greatly on the catalytic and inductive action of ethylene, which is not essential for ripening of non-climacteric fruit^[2-3]. At present, the synthetic pathway of ethylene in plants is well-known, and S-methionine (S-adenosylmethionine) is catalyzed by 1-aminocyclopropane-1-carboxylic acid synthetase (ACS) to produce 1-aminocyclopropane-1-carboxylic Acid (ACC), and then the ACC is oxidized by ACC oxidase (ACO) to produce ethylene^[4]. Of these, ACS is generally considered to be the rate-limiting enzyme in plant synthesis.

The 'Cuiyuan' pear (Pyruspyrifolia Cuiguan) belongs to a respiratory transition type fruit, is an important economic fruit, has great commercial value, is widely planted in southern areas of China, and has big and thin peel and more fruitsSweet juice taste, tender meat quality and the like^[5]. Therefore, the research on the ripening process of 'Cuiguan' pear and the cloning of the gene related to ripening in the pear have very important theoretical and practical significance for prolonging the shelf life of pear fruits, improving the efficiency of crop improvement and breeding and guiding agricultural production.

The Homeobox (Homeobox, HB) family is a transcription factor superfamily in plant cells, encodes a conserved DNA binding protein domain, called Homeodomain (HD)^[6]. To date, HB proteins have been reported to be involved in a number of plant growth and development processes. In Arabidopsis, members of the HB subfamily WOX were found to be involved in the formation and maintenance of organelles, and members of KNOX were involved in meristem activity, leaf development and cell differentiation^[7-10]. AtHB-1 is crucial to regulation of leaf development, and AtHB-2 can regulate the shade-avoiding response of plants^[11-12]. In addition, the formation of the outer cell layer and the embryo meristem of plant organs, the polarity of organs, the formation of apical embryos and the function of meristems are also regulated by the HB protein^[13-14]. It should be noted that, although the HB family has been extensively studied, so far, no HB genes regulating the ripening of pear fruits have been reported.

Disclosure of Invention

The invention aims to provide a transcription factor PbHB.G7.2 capable of promoting the synthesis of pear ethylene and accelerating the maturation process and application of the gene.

Another objective of the invention is to provide a gene PbACS1b regulated by the transcription factor PbHB.G7.2, and two transcription factors PbHB.G1 and PbHB.G2.1 which interact with the transcription factor PbHB.G7.2 protein and can also regulate the PbACS1b gene.

The purpose of the invention can be realized by the following technical scheme:

the invention provides a transcription factor PbHB.G7.2 gene which is separated from 'Cuiguan pear' and has the functions of promoting the synthesis of fruit ethylene and accelerating the fruit ripening process, belongs to Homeobox family members, and the nucleotide sequence of the gene is shown as SEQ ID No. 1. Contains an open reading frame of 912bp and encodes 304 amino acids, and the amino acid sequence of the encoded protein is shown as SEQ ID No. 2.

An expression cassette, a recombinant vector, a transgenic cell line or a transgenic recombinant bacterium containing the pear transcription factor PbHB.G7.2. The recombinant vector is a recombinant expression vector, preferably, the recombinant expression vector containing the PbHB.G7.2 gene takes pSAK277 as a super-expression vector, and the insertion site of the PbHB.G7.2 gene on the pSAK277 vector is between HindIII and XbaI.

The primer pair for amplifying the pear transcription factor PbHB.G7.2 comprises a forward primer F1 and a reverse primer R1; the sequence of the forward primer F1 is shown as SEQ ID No. 5; the sequence of the reverse primer R1 is shown as SEQ ID No. 6.

The invention also provides a gene PbACS1b regulated by a transcription factor PbHB.G7.2, which belongs to ACS family members, the nucleotide sequence of the gene is shown as SEQ ID No.3, the gene comprises 1488bp open reading frame, and the sequence of the promoter of 2000bp at the upstream of the initiation codon of the gene PbACS1b is shown as SEQ ID No. 4.

The invention also provides two pear HB transcription factors PbHB.G1 and PbHB.G2.1, the encoded protein of the two pear HB transcription factors PbHB.G7.2 can form interaction with the protein encoded by the transcription factor PbHB.G7.2, and can be combined with and activate the promoter of the PbACS1b gene, the nucleotide sequence of the transcription factor PbHB.G1 is shown as SEQ ID No.15, and the nucleotide sequence of the transcription factor PbHB.G2.1 is shown as SEQ ID No. 17.

The pear transcription factor PbHB.G7.2 and the protein coded by the pear transcription factor PbHB.G7.2 or the expression cassette, the recombinant vector, the transgenic cell line or the transgenic recombinant bacterium are applied to improving the ethylene synthesis amount of fruits and promoting the fruit ripening.

By the application, the fruit which transiently over-expresses the transcription factor PbHB.G7.2 is constructed, so that the ethylene synthesis amount of the fruit can be increased, and the fruit ripening is promoted. The fruit takes pear as an example, and the construction of the pear fruit of the transient overexpression transcription factor PbHB.G7.2 comprises the following steps:

1) performing PCR amplification by taking pear pulp cDNA as a template to obtain a pear HB gene PbHB.G7.2; the primer pair for amplification comprises a forward primer F1 and a reverse primer R1; the sequence of the forward primer F1 is shown as SEQ ID No. 5; the sequence of the reverse primer R1 is shown as SEQ ID No. 6.

2) Connecting the pear HB gene PbHB.G7.2 with a vector to obtain a recombinant vector;

3) transferring the recombinant vector into agrobacterium tumefaciens to obtain recombinant agrobacterium tumefaciens;

4) and infecting pear fruits by the recombinant agrobacterium tumefaciens to obtain the pear fruits over-expressing the PbHB.G7.2 gene.

The invention relates to application of a pear HB gene PbHB.G7.2 in promoting pear fruit ripening.

The recombinant expression vector disclosed by the invention is applied to promotion of pear fruit ripening.

The invention also provides application of the pear HB gene PbHB.G7.2 in pear crossbreeding.

The invention has the beneficial effects that:

the pear HB gene PbHB.G7.2 provided by the invention has the functions of promoting the synthesis of fruit ethylene and accelerating the fruit ripening process, and can be applied to shortening the fruit ripening time, and the protein coded by the pear HB gene PbHB.G7.2 can combine with and activate the promoter of the PbACS1b gene in cells, and promote the transcription of the PbACS1b gene, thereby promoting the synthesis of fruit ethylene and accelerating the fruit ripening process. According to the records of the implementation part, the pear HB gene PbHB.G7.2 is transferred into the pear fruit to obtain the over-expressed pear fruit, the ethylene release amount is obviously higher than that of a control group, and the maturation process is accelerated. The expression level of the PbACS1b gene in the over-expressed pear fruit is obviously increased compared with that of a control. The expression trend of the PbHB.G7.2 gene in over-expressed pear fruits is consistent. In addition, the invention also provides two pear HB transcription factors PbHB.G1 and PbHB.G2.1, and the proteins coded by PbHB.G1 and PbHB.G2.1 can form interaction with the protein coded by PbHB.G7.2, and also can combine and activate the promoter of PbACS1b gene. The pear HB gene PbHB.G7.2 provided by the invention can also be used as a molecular marker to be applied to pear crossbreeding, so that a new gene resource is provided for promoting the molecular breeding of pear fruit maturity, and the income of farmers is increased, and the agricultural cost is reduced.

Drawings

FIG. 1 is a diagram of the gene expression pattern of the transcription factor PbHB. G7.2 in 11 pear quality mature and immature fruits;

FIG. 2 is a schematic diagram of the overexpression vector pSAK 277-PbHB.G7.2;

FIG. 3 is a fruit phenotype map of transient overexpression of the transcription factor PbHB.G7.2 in pear fruits;

FIG. 4 is a schematic diagram showing the ethylene release amount of fruits transiently overexpressing the transcription factor PbHB.G7.2 in pear fruits;

FIG. 5 is a schematic diagram showing the expression levels of the transient overexpression transcription factors PbHB.G7.2, PbHB.G7.2 and PbACS1b genes in pear fruits;

FIG. 6 is a diagram showing the results of a yeast two-hybrid experiment in which PbHB.G7.2 and PbHB.G1 proteins interact;

FIG. 7 is a diagram showing the results of a yeast two-hybrid experiment in which PbHB.G7.2 and PbHB.G2.1 proteins interact;

FIG. 8 is a graph showing the results of fluorescence complementation experiments of interaction between PbHB.G7.2, PbHB.G1 and PbHB.G2.1 proteins;

FIG. 9 is a schematic diagram of the vector construction of the dual luciferase reporter assay for the transcription factors PbHB.G7.2, PbHB.G1, PbHB.G2.1 and PbACS1b gene promoters;

FIG. 10 is a diagram showing the results of a dual-luciferase experiment using the promoters of the transcription factors PbHB.G7.2, PbHB.G1, PbHB.G2.1 and PbACS1 b;

FIG. 11 is a diagram showing the results of yeast single-hybrid experiments on the promoters of the transcription factors PbHB.G7.2, PbHB.G1, PbHB.G2.1 and PbACS1 b;

FIG. 12 is a diagram showing the sequence display of the promoter of biotin-labeled PbACS1b gene and the results of gel electrophoresis mobility experiments for PbHB.G7.2 protein and PbACS1b gene promoter;

FIG. 13 is a display diagram showing the sequence of the promoter of the biotin-labeled PbACS1b gene and the results of gel electrophoresis mobility experiments of the PbHB.G1 and PbHB.G2.1 proteins with the promoter of PbACS1b gene;

Detailed Description

The present invention will be described in detail with reference to specific examples. From the following description and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.