CN-121975817-A - Application of PbbHLH gene in regulation and control of pear lignin synthesis and/or stone cell formation

CN121975817ACN 121975817 ACN121975817 ACN 121975817ACN-121975817-A

Abstract

The invention belongs to the technical field of agricultural biological genetic engineering, and particularly discloses application of PbbHLH gene in regulating and controlling pear lignin synthesis and/or stone cell formation. The application of the PbbHLH gene in regulating pear lignin synthesis and/or stone cell formation, wherein the nucleotide sequence of the PbbHLH gene is shown as SEQ ID NO. 1. The PbbHLH gene is directly combined with a PbCAD6 promoter region to inhibit transcription of the gene, so that lignin biosynthesis of pear fruits is negatively regulated, and the gene has great application value for improving the quality of the pear fruits.

Inventors

ZHANG ZONGYING
CHEN XUESEN
LI AIPING
ZHU YANSONG
Men Yongge
QI SUMIN
NIE LUPING
CAO RONGJUN
CHEN XIAOLIU
WANG NAN

Assignees

山东农业大学

Dates

Publication Date: 20260505
Application Date: 20260205

Claims (10)

The application of the PbbHLH96 gene in regulating pear lignin synthesis and/or stone cell formation is characterized in that the nucleotide sequence of the PbbHLH gene is shown as SEQ ID NO. 1.
2. Use according to claim 1, characterized in that the pear lignin synthesis is inhibited and/or stone formation is reduced by increasing the expression level of PbbHLH gene.
3. Use according to claim 1, characterized in that pear lignin synthesis and/or stone cell formation is promoted by decreasing the expression level of PbbHLH gene.
4. An over-expression vector for regulating lignin synthesis and stone cell formation of pear fruits, which is characterized in that the over-expression vector comprises PbbHLH gene as set forth in claim 1.
5. A strain that regulates lignin synthesis and stone cell formation in a pear fruit, said strain comprising the overexpression vector of claim 4.
6. A method for controlling lignin synthesis and/or stone cell formation in pear fruits using the PbbHLH gene of claim 1, comprising the steps of: s1, cloning PbbHLH genes; S2, constructing PbbHLH gene overexpression vectors; and S3, the over-expression vector obtained in the S2 is transformed into agrobacterium to obtain transgenic pear callus.
7. The method according to claim 6, wherein in S1, the clone PbbHLH gene is specifically: Extracting total RNA of pear pulp, reversely transcribing the total RNA into cDNA as a template, and carrying out PCR amplification by using primers PbbHLH-pCAMBIA 2300-F and PbbHLH-pCAMBIA 2300-R to obtain PbbHLH96 genes.
8. The method of claim 7, wherein the nucleotide sequence of primer PbbHLH96-pCAMBIA2300-F is shown in SEQ ID NO.3 and the nucleotide sequence of primer PbbHLH-pCAMBIA 2300-R is shown in SEQ ID NO. 4.
9. The method according to claim 6, wherein in S2, the PbbHLH96 gene overexpression vector is specifically constructed by connecting PbbHLH96 gene obtained in S1 to pCAMBIA2300-35S-GFP vector recovered by Scal cleavage, and constructing the overexpression vector pCAMBIA 2300-35S-PbbHLH-GFP.
10. The method according to claim 6, wherein in S3, the over-expression vector obtained in S2 is transformed into agrobacterium, specifically, the over-expression vector pCAMBIA 2300-35S-PbbHLH-GFP obtained in S2 is transformed into agrobacterium, and then the agrobacterium is used for mediating transient injection into pear pulp to obtain transient pear, and the transient pear is stably transformed into pear callus to obtain transgenic pear callus.

Description

Application of PbbHLH gene in regulation and control of pear lignin synthesis and/or stone cell formation Technical Field The invention belongs to the technical field of agricultural biological genetic engineering, and particularly relates to application of PbbHLH gene in regulating and controlling pear lignin synthesis and/or stone cell formation. Background Pear (Pyrus spp.) is a globally important temperate economic fruit whose fruit quality, particularly the pulp texture, directly affects consumer acceptance and commodity value. Fruit substantial roughening is often closely related to excessive accumulation of stone cells in the fruit. Stone cells are a type of thick-wall cells with highly lignified cell walls and hard structures, and the content of the stone cells is a key factor for determining whether the taste of pear fruits is fine or not. Therefore, the analysis of the molecular regulation network of pear stone cell formation and lignin deposition has important theoretical and application values for improving pear substance and cultivating high-quality varieties by molecular breeding or biotechnology means. Lignin is a complex phenolic polymer, a major component of the secondary cell wall of stone cells. The biosynthesis of which is precisely regulated by a series of structural genes and transcription factors. In pears, monolignol synthesis key enzyme genes such as Cinnamyl Alcohol Dehydrogenase (CAD) have been shown to be involved in stone cell development. In recent years, plant bHLH (basic Helix-Loop-Helix) family transcription factors are widely recognized as key regulatory elements for regulating secondary metabolism, including lignin synthesis. For example, in sorghum and rice species, it has been found that specific bHLH transcription factors can affect plant growth or stress resistance by directly targeting the promoter of lignin synthesis pathway genes, either positively or negatively regulating lignin biosynthesis. However, little is currently known about the specific function of bHLH transcription factors in pear fruit development, particularly stone cell lignin deposition. The lack of the regulation link limits the technical path for accurately improving the pear essence by regulating key transcription factors. Disclosure of Invention The invention aims to provide an application of PbbHLH gene in regulating and controlling lignin synthesis and/or stone cell formation of pear fruits, and the PbbHLH gene is directly combined with a PbCAD promoter region to inhibit transcription of the pear fruits, so that lignin biosynthesis of the pear fruits is negatively regulated, and the invention has great application value for improving pear fruit quality. In order to solve the technical problems, the invention adopts the following technical scheme: the application of the PbbHLH gene in regulating pear lignin synthesis and/or stone cell formation, wherein the nucleotide sequence of the PbbHLH gene is shown as SEQ ID NO. 1. Preferably, pear lignin synthesis is inhibited and/or stone cell formation is reduced by increasing the expression level of PbbHLH gene. Preferably, pear lignin synthesis is promoted and/or stone cell formation is increased by decreasing the expression level of PbbHLH gene. The invention also provides an over-expression vector for regulating and controlling lignin synthesis and stone cell formation of pear fruits, wherein the over-expression vector comprises the PbbHLH gene. The invention also provides a strain for regulating and controlling lignin synthesis and stone cell formation of pear fruits, wherein the strain comprises the over-expression vector. The invention also provides a method for regulating and controlling the lignin synthesis and/or stone cell formation of pear fruits by utilizing the PbbHLH gene, which comprises the following steps: s1, cloning PbbHLH genes; S2, constructing PbbHLH gene overexpression vectors; and S3, the over-expression vector obtained in the S2 is transformed into agrobacterium to obtain transgenic pear callus. Preferably, in S1, the clone PbbHLH gene is specifically: Extracting total RNA of pear pulp, reversely transcribing the total RNA into cDNA as a template, and carrying out PCR amplification by using primers PbbHLH-pCAMBIA 2300-F and PbbHLH-pCAMBIA 2300-R to obtain PbbHLH96 genes; Preferably, the nucleotide sequence of the primer PbbHLH96-pCAMBIA2300-F is shown as SEQ ID NO.3, and the nucleotide sequence of the primer PbbHLH96-pCAMBIA2300-R is shown as SEQ ID NO. 4. Preferably, in S2, the construction of PbbHLH gene overexpression vector is specifically that PbbHLH96 gene obtained in S1 is connected to pCAMBIA2300-35S-GFP vector recovered by Scal digestion, and the overexpression vector pCAMBIA 2300-35S-PbbHLH-GFP is constructed. Preferably, in S3, the over-expression vector obtained in S2 is transformed into agrobacterium, specifically, the over-expression vector pCAMBIA 2300-35S-PbbHLH-GFP obtained in S2 is transformed into agrobacteri