CN-121759513-B - Function of VvAIL gene in promoting grape hairy root regeneration and efficient hairy root transformation method thereof

Abstract

The invention discloses a function of VvAIL gene in promoting grape hairy root regeneration and a high-efficiency hairy root transformation method thereof, and relates to the fields of plant biotechnology and molecular breeding. Through function acquisition and deletion experiments, vvAIL is a key positive control transcription factor for grape hairy root regeneration. The transgenic hairy root transformation efficiency of the low-efficiency variety '5BB' can be obviously improved from 9.92% to 27.78% by over-expression VvAIL 12. The invention also establishes an optimized Agrobacterium rhizogenes mediated hairy root transformation method, realizes the transformation efficiency of up to 45.40% on 'Ma Selan' varieties through the optimal parameter combination of explants, bacterial liquid concentration and co-culture time, and is successfully applied to 12 different genotypes. Based on the transgenic hairy root obtained by the system, a subcellular localization method based on grape primordial living body tissue is developed, and the nuclear localization of VvAIL < 12 > is confirmed in grape self cells for the first time.

Inventors

• LI XIUJIE
• Pang Binning
• LIU JIE
• WU YUSEN
• MA YUJIAO
• HAN YAN
• LIU LI
• LI BO

Assignees

• 山东省葡萄研究院

Dates

Publication Date

20260508

Application Date

20260302

Claims (4)

1. 1. The application of an isolated VvAIL gene or a coded protein thereof in preparing a product for improving the conversion efficiency of hairy roots of grapes is characterized in that the nucleotide sequence of the VvAIL gene is shown as SEQ ID NO. 1; the product is agrobacterium rhizogenes engineering bacteria containing VvAIL gene expression cassettes, or bacterial agents containing the engineering bacteria, or screening culture mediums required by transformation by using the engineering bacteria.
2. 2. A method for improving the conversion efficiency of hairy roots of grape is characterized by comprising the steps of improving the expression level of VvAIL gene or improving the activity of coded protein in grape explants, wherein the nucleotide sequence of VvAIL gene is shown as SEQ ID NO. 1; The increase in expression level or activity is achieved by introducing and expressing VvAIL gene into the grape explant, by an expression vector comprising VvAIL gene; The expression vector is transformed into grape explants through agrobacterium rhizogenes.
3. 3. The method for improving the conversion efficiency of hairy roots of grape according to claim 2, characterized in that it comprises the following steps: (a) Taking grape tissue culture seedling leaves subjected to subculture for 40-50 days as explants; (b) Infecting the explant with an agrobacterium rhizogenes bacterial solution carrying VvAIL gene expression cassettes, wherein the bacterial solution has an OD 600 value of 0.7-0.9; (c) Co-culturing the infected explant with the agrobacterium rhizogenes for 1.5-2.5 days; (d) Transferring the co-cultured explant to 1/2MS screening medium containing antibiotics, and inducing generation of transgenic hairy roots.
4. 4. The method for improving the conversion efficiency of hairy roots of grape according to claim 3, wherein the explant is obtained from a tissue culture seedling subjected to secondary culture for 45 days, the OD 600 value of the bacterial liquid is 0.8, the co-culture time is 2 days, and the concentration of the antibiotic is termatine and is 250-350 mg/L.

Description

Function of VvAIL gene in promoting grape hairy root regeneration and efficient hairy root transformation method thereof Technical Field The invention relates to the fields of plant biotechnology and molecular breeding, in particular to a novel function of a grape specific gene VvAIL in regulating plant regeneration and application of the novel function in improving grape genetic transformation efficiency and the like. Background The disclosure of this background section is only intended to increase some understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art. Grape (VITIS VINIFERA l.) is one of the most widely planted, economically important horticultural fruit crops worldwide. The method has various applications including wine production, fresh eating, juice squeezing and dried fruit processing. Genetic improvement of grape by using genetic engineering and genetic editing technology has important value, and the core for realizing the aim is to establish a high-efficiency genetic transformation system. Currently, genetic transformation of grapes is primarily dependent on somatic embryogenesis and organogenesis pathways. However, these methods have significant limitations in that transformation efficiencies are low (typically less than 5%), periods are lengthy (over a year), and have a strong genotype dependence, with success only in very few varieties such as 'seedless white', 'nepheline' and the like. In addition, the technical system has strong dependence on accurate regulation and control of plant growth regulators, which results in poor experimental repeatability and severely limits the wide application of grape transgenic technology. In agrobacterium-mediated transformation, agrobacterium rhizogenes (Rhizobium rhizogenes) exhibits higher transformation efficiency in most species than commonly used agrobacterium tumefaciens (a. Tumefaciens). The hairy root transformation system mediated by the method has the remarkable advantages that the transformation period is short (transgenic hairy roots can be obtained in 4 weeks generally), the transformed roots can carry out hormone autotrophy and are long in genetic stability, and the method is very suitable for rapid research of gene functions. More importantly, the system has been shown to have the potential to reduce genotype-dependent in different species. For example, agrobacterium rhizogenes-based transformation systems have been successfully established in a variety of crops such as strawberry, poplar, apple, citrus, soybean, cabbage, and red sage, and efficient genetic transformation and gene editing have been achieved. However, the application of the technology in grapes is still slow, and only reports with limited efficiency in individual varieties such as 'roses', 'Crisens seedless' are currently seen. The development of a hairy root transformation system which is efficient, stable and suitable for a plurality of grape varieties is a technical problem to be broken through in the current grape biotechnology field. Breaking the bottleneck of crop regeneration efficiency generally depends on two strategies, namely, optimization of an external tissue culture system and manipulation of an endogenous genetic control network. In the technical aspect, by finely regulating the composition of the culture medium, the carbon source, the proportion of the plant growth regulator and the light temperature condition, the callus induction and organ differentiation can be effectively improved, for example, the PureWheat technology remarkably improves the wheat conversion efficiency, and the N6 culture medium system improves the maize regeneration. At the molecular level, recent forty years of research have revealed that manipulation of core regeneration regulatory genes can essentially enhance plant regeneration capacity. Among them, AINTEGUMENTA-LIKE (AIL) transcription factor family has been demonstrated to be a key to regulating plant cell totipotency, meristem maintenance and organ regeneration. In many species such as arabidopsis, corn, apple, sweet pepper, cabbage, etc., ectopic or overexpression of AIL family members (e.g., BBM, PLT5, etc.) has been demonstrated to significantly improve somatic embryogenesis and organ regeneration efficiency, effectively shorten transformation cycle, and successfully overcome the limitation of difficult-to-transform genotypes. The method provides clear theoretical basis and feasible technical path for establishing a high-efficiency and wide-adaptability genetic transformation system by mining and utilizing AIL key genes of grapes. However, the prior art has obvious gaps that firstly, systematic identification of the members of the grape AIL gene family and functional research thereof in regeneration, especially hairy root regeneration, are blank, and sec