CN-122012521-A - Blackberry flower organ specific expression gene RuMADS1 and application thereof

Abstract

The application belongs to the technical field of plant genetic engineering, and relates to RuMADS gene related to specific expression of blackberry flower organs and application thereof in fruit shape and hardness improvement. The present application successfully isolates and identifies RuMADS gene, its ORF length 846 bp, codes 281 amino acid composition protein. The nucleotide sequence of the cDNA of RuMADS1 related to the development of the flower organ is shown in SEQ ID NO. 1. The space-time expression analysis shows that RuMADS1 has higher expression level in blackberry flowers and pistils. A significant increase in flower pistil was found in both heterologous overexpressed arabidopsis and tomato, and a large fruit shape index and a significant increase in hardness were found in the overexpressed tomato plants. The gene and the protein provided by the application have wide application prospects in the improvement of comprehensive properties related to the appearance of fruits, such as changing the shapes of the fruits, improving the hardness and the like and the quality of fresh foods.

Inventors

• ZHANG CHUNHONG
• YANG HAIYAN
• WU YAQIONG
• HUANG ZHENGJIN
• WU WENLONG
• LV LIANFEI

Assignees

• 江苏省中国科学院植物研究所

Dates

Publication Date

20260512

Application Date

20260206

Claims (9)

1. 1. The cDNA of blackberry flower organ specific expression gene RuMADS is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.
2. 2. The blackberry flower organ specific expression gene RuMADS encodes a protein, and is characterized in that the amino acid sequence is shown as SEQ ID NO. 2.
3. 3. An expression vector comprising the cDNA of claim 1.
4. 4. The expression vector of claim 3, wherein the expression vector employs pGLN.
5. 5. An engineered bacterium comprising the expression vector of claim 4, wherein the engineered bacterium is produced by transferring the expression vector pGLN comprising the cDNA into agrobacterium tumefaciens EHA 105.
6. 6. The cDNA primer for amplifying the cDNA according to claim 1, wherein the forward primer is 5'-ATGGTGGGTAACGATCCAGAAC-3' and the reverse primer is 5'-GTCAGAGGCTACTTGACTCCC-3'.
7. 7. Use of the cDNA of the blackberry flower organ specific expression gene RuMADS1 according to claim 1 for increasing fruit shape index and fruit firmness.
8. 8. Use of the blackberry flower organ specific expression gene RuMADS of the protein encoded by gene RuMADS in increasing fruit shape index and fruit hardness.
9. 9. A tomato with improved fruit quality contains exogenous insert gene shown in SEQ ID NO. 1.

Description

Blackberry flower organ specific expression gene RuMADS1 and application thereof Technical Field The invention belongs to the field of plant genetic engineering, and relates to a method for improving fruit shape index and hardness by utilizing specific expression regulation genes of blackberry flower organs. Background The blackberry is a perennial bush of Rubus of Rosaceae, is rich in vitamin C, vitamin K, folic acid, manganese and other nutrient elements, contains a large amount of dietary fibers and antioxidants, and is an important third-generation small-fruit economic forest fruit tree. Blackberry fruits are usually ripe in summer, and the fruits are soft, juicy and easy to rot and deteriorate under high temperature conditions, so that the shelf life of the commodity is short due to poor storability. Hardness is one of the important fresh food quality traits of blackberry fruits. In the maturation and softening process, fruits undergo a complex series of physiological and biochemical changes, and it is currently believed that changes in firmness are due to changes in cell wall components and key cell wall degrading enzymes. Pectin, a key component constituting the primary layer and the cytoplasm of plant cell walls, plays an important role in maintaining the mechanical strength and structural stability of the cell walls and has a close relationship with the ripe softening of fruits. During fruit ripening, the decrease in firmness is closely related to the change in cell wall structure, and pectin degradation triggers not only a series of changes in cytoplasm, but also weakening of intercellular junctions and increase of cell gaps under the combined action of the changes, thereby causing relaxation of fruit tissues and decrease of firmness. The influence factors of the actual hardness are numerous, and the effective technology for improving the hardness of fruits on the fruit trees still lacks at present. Fruit shape is one of the important agronomic and commodity traits in horticultural plant breeding. The occurrence and formation of fruit shape are closely related to the regulation of fruit growth and development. The fruit shape index refers to the ratio of the longitudinal to transverse diameter of the fruit. Fruits with different fruit shape indexes can meet the requirements of different processing and grading packaging. Therefore, with the increasing comprehensive requirements of people on both the appearance and the quality of fresh foods, there is an urgent need to find an effective method for improving both the shape and the hardness of fruits. MADS-box transcription factors are a class of regulatory factor genes involved in the regulation of floral development. The MADS-box family is divided into two groups, type I and type II, according to the difference of protein domains, wherein the protein encoded by the type II group mainly consists of 4 parts of MADS domain, I domain, K domain and C domain, so also called MIKC type, wherein the K domain is the characteristic sequence of MIKC type MADS-box gene, MIKC type MADS-box gene can be further divided into MIKC C subfamilies and MIKC subfamilies, and MIKC C type gene mainly participates in the specific regulation of flower organ development. At present, the regulation and control effect of MADS-box transcription factors on the development and maturation of fruits is only found to promote the maturation of fruits in tomatoes and strawberries, the functional disclosure of the regulation and control of the fruit development and the improvement of the fruit quality is not visible, and the expression mode and specific regulation and control effect of the MADS-box transcription factors in blackberries are still yet to be further verified. The improvement of fruit shape and hardness by adopting the traditional crossbreeding technology or the artificial directional selection method requires long time and takes effect slowly. At present, the genetic engineering improvement of fruit shapes of fruits realizes the identification and genetic transformation verification of fruit shapes on various fruit types such as tomatoes, grapes, apples and the like, but most of the genes are realized by influencing the movement of fruit cytoskeleton to change cell division and cell growth, and functional genes for regulating the appearance and the internal quality of fruits are not realized yet from the aspect of regulating and controlling the development of flowers and organs. The comprehensive improvement of the appearance and the fresh food quality is possible by a biotechnology method, and no report of successful application of the floral organ development regulating gene to the improvement of the fruit quality is currently seen. Disclosure of Invention The invention aims to solve the defects in the prior art and provides a biotechnological method capable of effectively combining fruit shape change and hardness improvement through over-expression transformation. In ord